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+{"text": "Daily energy expenditure consists of three components: basal metabolic rate, diet-induced thermogenesis and the energy cost of physical activity. Here, data on diet-induced thermogenesis are reviewed in relation to measuring conditions and characteristics of the diet.Measuring conditions include nutritional status of the subject, physical activity and duration of the observation. Diet characteristics are energy content and macronutrient composition.Most studies measure diet-induced thermogenesis as the increase in energy expenditure above basal metabolic rate. Generally, the hierarchy in macronutrient oxidation in the postprandial state is reflected similarly in diet-induced thermogenesis, with the sequence alcohol, protein, carbohydrate, and fat. A mixed diet consumed at energy balance results in a diet induced energy expenditure of 5 to 15 % of daily energy expenditure. Values are higher at a relatively high protein and alcohol consumption and lower at a high fat consumption. Protein induced thermogenesis has an important effect on satiety.In conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein- and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis. Diet induced thermogenesis (DIT) can be defined as the increase in energy expenditure above basal fasting level divided by the energy content of the food ingested and is commonly expressed as a percentage. It is, with basal metabolic rate and activity induced thermogenesis, one of the three components of daily energy expenditure. Although DIT is the smallest component, it could play a role in the development and/or maintenance of obesity. De Jonge and Bray  evaluateMethodological issues include: was the baseline appropriate, what was the energy content and nutrient composition of the test food consumed, what was the duration of the postprandial measurement period, and what was the error associated with the calculation of DIT from the measured energy expenditure. Weststrate et al  investigHere, the focus is on DIT as a function of the energy content and nutrient composition of the test food consumed and the duration of the postprandial measurement period in adult subjects with a normal bodyweight. The review is based on literature published over the last 15 years.The experimental design of most studies on DIT is a measurement of resting energy expenditure before and after a test meal, with a ventilated hood system. The observation is started after an overnight fast, where subjects are refrained from eating after the last meal at 20.00 h at the latest. Thus, with observations starting between 08.00 and 09.00 h the next morning, the fasting interval is at least 12 h. Postprandial measurements are made for several hours where subjects have to remain stationery, most often in a supine position, for the duration of the measurements. In some studies, measurements are 30 min with 15 min intervals allowing i.e. for sanitary activities.The use of a respiration chamber to measure DIT has the advantage of reproducing more physiological conditions over a longer period of time while regular meals are consumed throughout the day ,6. The DStudies on DIT were selected from Medline. Studies were selected when information was presented on energy intake, diet composition with respect to carbohydrate, protein fat and alcohol of the test food, duration of the postprandial measurement, and DIT.Reported intra-individual variability in DIT, determined with ventilated-hood systems, is 6 to 30% ,8. ReporThe mean pattern of DIT throughout the day is presented in figure Fifteen studies on DIT with information on energy intake, on diet composition and on the postprandial measurement period were selected from literature Table . Five stFor a comparison of DIT between studies as a function of the nutrient composition of the test food consumed, the energy content of the test food was divided by the length of the measurement interval after food consumption and expressed in MJ/h. Only three of the 22 studies presented in table The main determinant of DIT is the energy content of the food, followed by the protein fraction of the food. The thermic effect of alcohol is similar to the thermic effect of protein.Diet induced thermogenesis is related to the stimulation of energy-requiring processes during the post-prandial period. The intestinal absorption of nutrients, the initial steps of their metabolism and the storage of the absorbed but not immediately oxidized nutrients . As suchTheoretically, based on the amount of ATP required for the initial steps of metabolism and storage, the DIT is different for each nutrient. Reported DIT values for separate nutrients are 0 to 3% for fat, 5 to 10% for carbohydrate, 20 to 30% for protein , and 10 Of the studies presented in table The higher DIT value of alcohol and protein compared with carbohydrate and fat has implications for the effect of these nutrients on energy balance. However, the main effect on energy balance does not seem to be primarily linked to the lower bioavailability of alcohol-and protein energy than that of fat and carbohydrate. Alcohol energy is largely additive to the normal diet but does not seem to affect energy balance positively . ProteinAlcohol forms a significant component of many diets and it supplements rather than displaces daily energy intake. Alcohol consumption as an aperitif has even been shown to result in a higher subsequent intake with no intake compensation afterwards . Yet, alThe main effect of protein on energy balance is thought to be DIT related satiety. Satiety scores were higher during meals with a high-protein/high-carbohydrate diet, as well as over 24 h, than with a high-fat diet . The obsIn conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein-and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis."}
+{"text": "The macronutrient composition of the diets were covertly modified with drinks containing 2.1 MJ of predominantly carbohydrate (Hi-CHO), protein (Hi-PRO), or fat (Hi-FAT). Total energy expenditure was measured for seven days on two separate occasions .Ghrelin, a peptide secreted by endocrine cells in the gastrointestinal tract, is a hormone purported to have a significant effect on food intake and energy balance in humans. The influence of factors related to energy balance on ghrelin, such as daily energy expenditure, energy intake, and macronutrient intake, have not been reported. Secondly, the effect of ghrelin on food intake has not been quantified under free-living conditions over a prolonged period of time. To investigate these effects, 12 men were provided with an Preprandial ghrelin concentrations were not affected by macronutrient intake, energy expenditure or energy intake . In turn, daily energy intake was significantly influenced by energy expenditure, but not ghrelin.Preprandial ghrelin does not appear to be influenced by macronutrient composition, energy intake, or energy expenditure. Similarly, ghrelin does not appear to affect acute or chronic energy intake under free-living conditions. Ghrelin, a peptide secreted by endocrine cells in the gastrointestinal tract, is thought to play a significant role in the regulation of energy balance due to its effects on the stimulation of food intake ,2 and weDespite the evidence indicating a role in acute food intake, little is known about the factors regulating ghrelin and its effects on long-term energy balance in humans. One hypothesis is that ghrelin secretion is up-regulated in periods of negative energy balance and down-regulated in periods of positive energy balance . Since eThe purpose of the present study was to determine how changes in macronutrient composition, energy intake, and energy expenditure affect preprandial ghrelin concentrations, and ghrelin's subsequent effects on food intake.Ghrelin was negatively related to body fat percentage  and BMI , but not body weight . There were no significant body weight changes during the seven day observation periods (2).The composition of the treatment beverages and their contribution to daily food intake is listed in Table Average 24 hour energy expenditure  was 13.9 \u00b1 1.9 MJ/d compared to 12.6 \u00b1 1.6 MJ/d for total energy expenditure , which is an average over-reporting of energy expenditure of 11%. Thus, our assumption that subjects would likely misreport energy expenditure and the values would require adjustment was valid.-1 for Hi-CHO, 2869.5 \u00b1 817.3 pg\u00b7mL-1 for Hi-PRO, and 2688.2 \u00b1 755.5 pg\u00b7mL-1 for Hi-FAT  and total (including treatment beverages) energy intake (breakfast or entire day), or the interactions between these variables (previous or same day). As a further test, we included energy intake for seven days prior to- and two days after each ghrelin value. None of these days were significant . Individual day and mean 24EE up to the prior 4 four days before each ghrelin measurement was also not significant .Selected and total energy intake for the entire day were significantly influenced by treatment period (P < 0.02), Monday/Friday effect (P < 0.003), Sunday effect (P < 0.03), and 24EE (P < 0.008) Table . Classif2) of the variation in energy intake, still a relatively small percentage of explained variation for a hormone purported to exert a large influence on intake.The partial correlation between breakfast energy intake and ghrelin was 0.07. At 80% power, we could have detected a ghrelin effect if the true partial correlation was a small as 0.36. For powers of 90% and 95%, the true partial correlations would have had to be 0.40 and 0.43, respectively. The partial correlation between total energy intake and ghrelin was even lower than that with breakfast energy intake (r = 0.003). Note that, for a partial correlation of 0.40, ghrelin would have only been explaining about 16% , none appear to play a role in preprandial ghrelin regulation. Similarly, ghrelin did not significantly predict macronutrient or energy intake, despite a power analysis indicating that we would have detected even a moderate effect of ghrelin on intake.Most of the evidence linking food intake and ghrelin comes from single meal, short-term studies. The ingestion of amino acids or a protein meal results in a post-prandial increase in ghrelin -14, wherWren et al. were theAlthough body weight typically increases by \u2248 4.5 kg in men and \u2248 7.3 kg in women over the course of 30 years , the humIn conclusion, it appears that macronutrient and energy intake, and energy expenditure have no effect on preprandial ghrelin. None of the variables measured in this study explain the high daily variability in preprandial ghrelin observed over the course of two-seven day periods. In turn, this study fails to detect the energy intake-stimulating effect of ghrelin, despite carefully measured food intake that lasted more than a week and a study powered to detect even a moderate effect of ghrelin.Twelve healthy, non-smoking men were recruited from the Beltsville, MD area to participate in this study Table . All subad libitum from the menus, and could consume any part or all of a food item, then return the remaining portion to be weighed. BHNRC staff that came into contact with the subjects provided no guidance as to the quantities and/or types of food items chosen. During weekdays, subjects reported to the BHNRC in the morning to eat breakfast, pack selected food items for lunch, then return again in the evening for dinner. Any food taken from the HSF that was subsequently not eaten , was returned the next day, and weighed and recorded. On Friday evenings, subjects were provided with coolers packed with a large amount of food for weekend meals. The weekend coolers provided a wide variety of foods in excess quantities, and subjects were allowed to request additional food items be included. Weekend food could be consumed on either day as long as the subjects logged which day each food item was eaten. All uneaten weekend food was returned on Monday, and weighed and recorded. Although subjects were instructed to consume only food items provided by HSF, they were allowed free access to beverages including caloric, noncaloric and alcoholic beverages. Detailed records of the amount, composition and name brand of beverages was submitted daily. In addition to beverages provided on the menu (milk and juice), both regular and decaffeinated coffee and tea were available at meals.Voluntary food intake was studied continuously for 16 weeks, whereby subjects consumed only foods provided by the Human Studies Facility (HSF) at the Beltsville Human Nutrition Research Center (BHNRC). Subjects choose foods Food items offered in the morning (breakfast and lunch) were presented in a cafeteria-style setting as three different rotating menus, each lasting seven days Table . Some foThe goals of the menu design were to allow detection of macronutrient selection by offering a wide range of carbohydrate, fat- and/or protein-rich foods, and to provide a variety of commonly available foods typical of what many Americans eat. In a research setting it is impossible to duplicate the degree of food choice available in real life. However, more than 300 food items were used to develop menus for this study, and specific requests for food items were incorporated into the menus whenever possible.After each subject selected his desired foods, he presented them to a staff member that recorded the identity and weight of each food item by hand and on a computer (combination of bar code recognition of the food item and hand-entering of the weight). Upon termination of feeding, each subject presented his tray to a staff member that weighed any uneaten food. The accuracy of the food item recording process was verified by comparing the information on the computer with the hand-entered logs. This verification procedure was followed daily, and repeated at the end of the study with all food records. Energy and macronutrient composition were determined by consultation with the USDA Nutrient Database for Standard Reference .ad libitum intake, subjects were randomly assigned to two of three treatments. Each treatment lasted 8 weeks with no break between the periods. The treatments consisted of a daily beverage that contained \u2248 2 MJ/day of predominantly carbohydrate (Hi-CHO), fat (Hi-FAT), or a combination of protein and carbohydrate (Hi-PRO) . The intra- and interassay coefficients of variation (CV) were 5.6% and 7.3%, respectively.Before breakfast and after voiding, body weight was determined weekly on an electronic balance to the nearest 0.01 kg. Body composition was measured by Dual-energy X-ray Absorptiometry .To \"capture\" daily variations in energy expenditure, we combined a self-reported activity log  and doubTwenty-four hour energy expenditure (24EE) was estimated using a daily recording log method, modified from Bouchard et al. . Briefly2 18O (0.16 g/kg body weight) and 2H2O (0.30 g/kg body weight). Urine samples were collected immediately before the dose and on every morning (second void) for the last seven days of the treatment period. The first sample was collected approximately 24 hr after the dose. Enrichments of 2H and 18O in urine samples were measured by infrared spectroscopy and isotope ratio mass spectrometry, respectively. TEE was calculated using the equations of Weir [Total energy expenditure (TEE) was concurrently measured by the doubly labeled water method as described by Speakman , which p of Weir .Individual daily 24EE values were corrected using the ratio adjustment (notation denoting subjects is suppressed),dayx, corrected = 24EEdayx \u00d7 (TEE/24EEday 1\u20137), where24EEdayx is the uncorrected daily energy expenditure value from the activity log for one of the seven days (day X),24EEdayx is one), andTEE is the daily mean energy expenditure estimate using doubly labeled water. Represents a single value during the seven days of measurement , where b0 and b1 are the estimated coefficients of the line fit by regressing the SDs on the means, and yi represents the energy intake data. The other variables were transformed using this same family of transformations. This procedure resulted in homogeneous variances for all variables once transformed, satisfying ANOVA assumptions. We present the data on the original scale in tables and figures for ease of interpretation (unless indicated otherwise).To check the assumption of homogeneous variances necessary for valid F-tests and correct P-values, we used the standard technique of plotting the standard deviations (SD's) against the means for selected energy intake, grouping observations by subject and treatment period. The results of this scatter plot revealed a strong positive linear relationship . The relationships between the SD and mean for macronutrient and energy intake , and 24EE were also positive and significant. This indicated that the SD's (variances) were a function of the mean and that the data needed to be transformed. We followed methods described by Draper and Smith , and useDue to the free-living nature of the subjects, there were three observations (of 168) where (for unknown reasons) a subject's food intake differed greatly from habitual intake due to a skipped meal or meals with low energy intake. For this reason, these observations were not used in the analyses. Additionally, a preliminary sensitivity analysis and residual diagnostics  suggestThe experimental design was an incomplete block crossover design, with two of the three drink treatments given sequentially to each subject. Data were analyzed in the mixed linear models framework, using the Proc Mixed procedure in SAS (version 9.1). SubjectFor models predicting ghrelin concentration, we included 24EE, energy intake, and the interaction between 24EE and energy intake. We also considered prior day (up to 7 days) and subsequent day (up to 2 days) values for energy intake and ghrelin, and their interactions as candidate covariates. Values for up to 4 prior days for 24EE were used to predict ghrelin. For models predicting daily energy intake, we included preprandial ghrelin concentrations, 24EE, the interaction between ghrelin and 24EE, and additionally considered as candidate covariates the prior (seven days) and subsequent (two days) days for these two variables and their interactions. We explored models that included other variables and interactions, but none of those variables appeared useful. Data are presented as total intake (intake including treatment drinks) and/or selected intake (intake without treatment drinks). Values are presented as means \u00b1 SD unless indicated otherwise.Since a preliminary analysis suggested that the effect of ghrelin on energy intake was small or negligible, we conducted a power analysis to determine our ability to detect an effect of ghrelin if the effect was small. This was accomplished by Monte-Carlo simulation (creating simulated data sets based on the data we collected) and, starting with no effect of ghrelin (a true coefficient of zero for ghrelin in a regression context), determining how large the true coefficient needed to be to obtain significance for most of the simulations, at powers of 80%, 90%, and 95%, with 1000 simulations for each coefficient value. These results are most easily interpreted as how large a partial correlation between ghrelin and energy intake  would be necessary for us to detect it. We conducted this analysis for both total energy intake and breakfast energy intake .MK was responsible for statistical analysis and interpretation. DR was responsible for supervising the food intake portion of the study. WR conceived the study, and supervised the data collection and analysis. DP was responsible for ghrelin analysis, data collection, statistical analysis and manuscript preparation. All authors read and approved the final manuscript."}
+{"text": "The sharing of wild plant foods is infrequent in chimpanzees, but in chimpanzee communities that engage in hunting, meat is frequently used as a \u2018social tool\u2019 for nurturing alliances and social bonds. Here we report the only recorded example of regular sharing of plant foods by unrelated, non-provisioned wild chimpanzees, and the contexts in which these sharing behaviours occur. From direct observations, adult chimpanzees at Bossou  very rarely transferred wild plant foods. In contrast, they shared cultivated plant foods much more frequently (58 out of 59 food sharing events). Sharing primarily consists of adult males allowing reproductively cycling females to take food that they possess. We propose that hypotheses focussing on \u2018food-for-sex and -grooming\u2019 and \u2018showing-off\u2019 strategies plausibly account for observed sharing behaviours. A changing human-dominated landscape presents chimpanzees with fresh challenges, and our observations suggest that crop-raiding provides adult male chimpanzees at Bossou with highly desirable food commodities that may be traded for other currencies. Food sharing has important implications for the evolution of cooperation, offering a means to evaluate the \u2018paradox\u2019 of altruism, whereby a recipient gains fitness benefits at the expense of a donor Food sharing is observed throughout the animal kingdom, albeit at varying levels and complexities. Hypotheses proposed to explain food sharing behaviours in chimpanzees Analysis of such social and political components of meat sharing in chimpanzees has led to insights into the evolutionary basis of human food sharing (Ficus exasperata) from an adult male to his mother. Rough self-scratching (RSS), a likely behavioural indicator of anxiety crop-raid: 1.16 bouts/hr, wild food: 0.25 bouts/hr). All three adult males showed increased RSS . Despite this, cultivated plant foods obtained during crop-raids were shared much more frequently than wild plant foods. Crop raiding bouts occurred on average 22 times per month , but crop sharing was much less frequent, occurring on average 1.5 times per month . This is in contrast to reports of hunting at other chimpanzee sites , most of which are followed by sharing Chimpanzee adults at Bossou very rarely transferred wild plant food . This incident concerned the transfer of fig leaves crop-raid: X2(1)\u200a=\u200a21.835, p<0.001; share: X2(1)\u200a=\u200a9.54, p<0.01) and further from forest edge . Adult males showed more RSS when raiding in the presence compared to the absence of local people (X2(1)\u200a=\u200a4.10, p<0.05), yet they were more likely than other age- and sex-classes to raid and share crops when local people were present (crop-raid: X2(1)\u200a=\u200a6.97, p<0.01; share: X2(1)\u200a=\u200a5.99, p<0.05). Males always transported raided foods from the village to the forest before sharingAdult males were significantly more likely than other age- and sex-classes both to crop-raid and to share crops obtained in exposed locations (X2(1) \u200a=\u200a1.63, ns). However, adult males shared crops - mostly papaya (21 out of 25 sharing events) - overwhelmingly with these females , p\u200a=\u200a0.0004), particularly with one cycling female . As reported for meat sharing at other sites Our observations also raise the question of why adult male chimpanzees at Bossou regularly share raided fruits with females. Long-term data linking food sharing to male reproductive success would provide the most direct answer to this question, but in the absence of such data, we propose possible functions that appear consistent with the observations. Begging for crops appears to impose no energetic costs to the possessor; in contrast to begging for meat Manis tricuspis, a species unlikely to inflict injury on its captor. Instead of meat sharing, the regular sharing of cultivated plant foods may be employed by adult males as a \u2018food-for-sex and -grooming\u2019 strategy to enhance affiliation with reproductively valuable females. Although the \u2018food-for-sex and -grooming\u2019 explanation can plausibly account for these observations, it relies principally on one cycling female, and may not be representative of the behaviour as a whole.Meat sharing is infrequent at Bossou due to the scarcity of mammalian prey In humans, the pursuit of certain foods is also strongly sex-biased; it has been proposed that men in hunter-gatherer societies acquire large and risky-to-obtain food packages for social strategising and to garner attention 2 home range of the Pan troglodytes verus community is fragmented and surrounded by cultivated and abandoned orchards and farms. The chimpanzees regularly visit these areas to consume 17 varieties of crops that are located both within the village and forest. During the study period , community size varied from 12 to 22 individuals; the number of adult females ranged from 4 to 8 individuals, and there was always the same 3 adult males Humans and chimpanzees coexist at Bossou , where the 15kmMovie S1An adult male chimpanzee crop-raiding papaya fruit. Adult male chimpanzees are most likely to share cultivated plant foods obtained in exposed locations and in the presence of people.(10.26 MB AVI)Click here for additional data file.Movie S2An adult male chimpanzee shares papaya fruit with an adult female. Cultivar food sharing at Bossou is the only recorded example of regular food sharing between unrelated and non-provisioned wild chimpanzees.(8.76 MB AVI)Click here for additional data file."}
+{"text": "This study experimentally examined the effects of repeated exposure to different meal portion sizes on energy intake.Nineteen employees of a county medical center were given free box lunches for two months, one month each of 1528 and 767 average kcal. Foods were identical in the two conditions, but differed in portion size. Meals averaged 44% calories from fat. Participants self-reported how much of each lunch was eaten. Unannounced 24-hour dietary recalls were also conducted by phone twice per week during each exposure period.Mean energy intake at the lunch meal was 332 kcal/day higher in large lunch than in small lunch periods (p < .001). Mean 24-hour energy intake was 278 kcal/day higher in large versus small lunch periods (p < .001). There was no evidence of compensation over time. Average weight change over the month of large and small lunches was 0.64 \u00b1 1.16 kg and 0.06 \u00b1 1.03 kg, respectively, about what would be expected with the observed differences in energy intake.This study suggests that chronic exposure to large portion size meals can result in sustained increases in energy intake and may contribute to body weight increases over time. Over the last 20 to 30 years, there have been dramatic increases in the prevalence of obesity in all segments of the US population . Exact cIt has been noted that physiologic regulatory mechanisms are much more efficient at signalling under-consumption than over-consumption of energy . Thus, iDespite demonstrated differences in energy consumption, research has shown no substantial variations in reports of hunger or satiety between subjects served standard or large portion sizes . SubjectWhether chronic exposure to larger portion sizes would result in sustained increases in energy intake or contribute to long-term weight gain, however, remains unclear. Rolls and colleagues ,11,12 haTo assess whether chronic exposure to larger portion sizes can cause chronic increases in energy consumption, the present study presented study subjects with meals of different portion sizes in a naturalistic setting for a sustained period of time and observed energy intake at meals, daily energy intake overall, and body weight. It was hypothesized that chronic exposure to large portion sizes compared to small portion sizes would result in higher energy intake at meals, higher average total energy intake per day, and possibly an increase in body weight.Participants for the present study were recruited from employees of a community medical center by posting fliers on bulletin boards, in-person recruitment outside the center cafeteria, e-mail newsletter announcements, and table tents. The sample was restricted to women in an effort to reduce between subject variability and to simplify the logistics of food provision. Eligibility criteria were age 18 to 40, employment at the medical center, self-reported BMI between 18.5\u201340.0, not pregnant or recently having given birth, not actively dieting to control weight, not more than three days a week of regular moderate or vigorous physical activity and willingness to consent to the conditions of study participation. Study participants were offered $200 dollars for completing all study measures, paid in three installments. Women volunteers had a mean age of 33 (\u00b1 5.2) years, 45% were married, 65% reported having at least a 4-year college degree, and 80% were white. Mean BMI at baseline was 28.9 (\u00b1 7.8).The study employed a within-person, randomized crossover design comparing the effects of providing free box lunches of different portion sizes 5 days per week for four consecutive weeks on energy intake and body weight. The study protocol included a baseline assessment, a 2-week run-in period, a 4-week period in which the participants received a free box lunch daily with small or large portion sizes, a 2-week washout period during which weight was assessed again, a second 4-week period with free lunches of the opposite portion size as in the first month, and finally a follow-up assessment. Twenty women in all were recruited for the study. Half were randomized to each order of lunch presentation. The sample size was chosen to detect differences in energy intake and was similar to sample sizes used in similar, though more tightly controlled laboratory feeding studies ,10,12. WThis research was approved by the University of Minnesota and the Minneapolis Medical Research Foundation institutional review boards. Prospective study participants were screened for eligibility by phone and they then attended orientation sessions at which procedures were described, written consent was obtained, and baseline measures were collected. Candidates were told that the study was being conducted to assess factors influencing eating habits and the feasibility of providing daily box lunches. No specific mention was made of portion size or energy intake as study objectives until the final follow-up visit at which time the study purpose was disclosed. Because all participants received both sets of lunches, and because individuals receiving different portion size lunches were not prevented from interacting during the study, many became aware of the portion size manipulation as the study progressed, but most remained unaware of the study's intent. Although blinding to the portion size manipulation was considered, it was not attempted, in part because we thought it could be difficult to do while keeping the study exposures naturalistic, and in part because we thought that any bias related to knowledge of portion size would probably work against rather than for observing a portion size effect on intake.The lunches used in the study were prepared by a local caterer with guidance from study nutrition personnel. A rotation of seven different lunches was used. The contents were typical lunch items that included a main course, side dish, dessert, and a drink. Main courses were sandwiches or salads. Side dishes were fruit or vegetable salad, chips, or bread depending on the main course. Desserts were cookies or bars. Drinks were water, Coke, or Sprite. Lunches of different energy content had the same selection of items and as close as possible to the same relative distribution of calories in the different items. We targeted 750 kcal for the small lunch in the belief from other data sets that it would approximate a normal lunch, about 1/3 of average energy intake per day in the US . FifteenHeight and Weight: Body weight was measured on a calibrated electronic scale to the nearest 0.1 kg at three points in time: baseline, immediately after the first month of free lunches, and at the follow-up evaluation immediately after the second month of free lunches. Height was measured at baseline using a wall-mounted ruler.Two kinds of dietary information were collected during the study. First, dietary intake at lunch was assessed by having study participants complete a self-administered questionnaire after each lunch in which they estimated the proportion of each food item eaten using a visual analogue scale. Participants received these questionnaires along with their lunch boxes and returned them either by interoffice mail or directly to the study office. They also reported any food items eaten at lunch that were not from their lunch box. These data were entered into the NDS nutrient analyses program described above . EstimatPhysical activity was assessed at baseline using an instrument developed by Jacobs , and twiStatistical analyses of the data from this study were done using SAS version 8.7. The analyses of the meal size manipulation on kilocalories consumed and on percent calories from fat at the lunch meal and per day were carried out using a general linear mixed model analysis, controlling for order of lunch presentation and physical activity as fixed effects and participant as a random effect. The weight change outcome was also analyzed using a general linear mixed model without controlling for physical activity.Completion rates in the study were good. One participant had to withdraw from the study very early due to a health problem. In the remaining 19 participants, completion of weight assessments was 100%, of post-lunch meal reports was 91%, and of telephone recalls of diet and activity was 98%.Results of the study with regard to the effects of portion size on energy and fat intake are shown in Figure In theory, a 278-kcal/day elevation in energy intake would result in a 0.72-kg increase in body weight over 20 days if there were no compensation on days not observed (i.e. weekends). We observed average weight gains of 0.06 \u00b1 1.03 kg during the month of small lunches and 0.64 \u00b1 1.16 kg during the month of large lunched, figures remarkably close to theoretical prediction. Due to our small sample size, the within-person difference in weight change during large and small lunch months did not reach conventional levels of statistical significance (p = 0.13).This study showed that chronic exposure to larger portion sizes in free-living populations can induce sustained increases in energy intake and suggests that the effects of portion size may be powerful enough to affect rate of weight gain over time. The study was of too limited a sample size and duration to provide definitive proof that chronic exposure to larger portion sizes increases risk for undesirable weight gain or that people exposed to them would not \"spontaneously\" bring their weight under better control over time. However, it is believed that this demonstration is a meaningful addition to the accumulating body of evidence supporting the idea that offering larger portion sizes as part of food marketing strategies could have deleterious effects on population obesity rates if practiced widely. Further study of portion size effects that exercise a wider range of food products, more diverse populations, and longer time periods could make an important contribution to better understanding potential environment risks for excess energy intake.Aspects of this study that particularly intrigued us were 1) size and stability of the portion size effect on energy intake with very limited control of extraneous factors such as where, when and with whom people ate their lunches; 2) the very weak compensation for single meal overfeeding in our participants over a fairly long period of time; and 3) the relative ease and low cost of this experimental procedure. Overall, we believe that these observations indicate that studies of food exposures in the natural environment are very feasible and could provide important data for informing possible food policies designed to address obesity.Food portion size is a readily modified characteristic of the environment. Therefore, the potential for large food portion sizes to promote sustained increases in energy intake and conversely small food portion sizes to reduce energy intake deserves more study.The author(s) declare that they have no competing interests.RWJ contributed to the design of the study, was the primary writer of the manuscript, and was involved in the analysis and interpretation of the data. SR coordinated the implementation of the project, recruited participants, collected and analyzed data, and wrote up the methods section of the manuscript. CLD contributed to the conceptualization and design of the experiment and the development of measures and diet intervention components. LJH provided assistance with study design, data collection, and data analysis. ASL contributed to the design and rationale of study. PRP provided consultation and assistance with recruitment and data collection. JEB contributed to data analysis and interpretation. All authors contributed to the manuscript writing."}
+{"text": "Obesity shows an increasing prevalence worldwide and a decrease in energy expenditure has been suggested to be one of the risk factors for developing obesity. An increase in resting energy expenditure would have a great impact on total energy expenditure. This study shows that classical music do not influence resting energy expenditure compared to complete silence. Further studies should be performed including other genres of music and other types of stress-inductors than music. Obesity shows an increasing prevalence worldwide  and a de2 and CO2 contents according to the instructions from the manufacturer. The subjects were instructed to limit their physical activity the evening before measurement. All subjects were measured after an overnight fast and they arrived from their home by car or public transport. After 30 minutes rest in the supine position REE was measured during 35 minutes when the subjects were awake. Due to adaptation to the inside-hood environment, the first five minutes were eliminated from the total result. The music was provided through earphones and measurements were performed in an environmental temperature of 20\u201324\u00b0C. After completion of the measurement, the subjects were asked how they perceived each part of the music, as calm or stressful, or something else. Data are presented as mean and standard deviation. To compare REE during silence to the calm and stressful music, two-sided paired Student's t-tests were performed.In this randomized cross-over study, 2 different music CD's were used. Both CD's started with 10 minutes of silence and were followed by 10 minutes of calm classical music and 10 minutes of stressful classical music, presented in Table 2. Mean (SD) REE during silence was 5720 (1063) kJ/day. No significant differences in REE between silence and the two sets of music were found, 5710 (1054) kJ/day during calm music (p = 0.57) and 5740 (1046) kJ/day during stressful music (p = 0.43). Thirty-eight subjects perceived the calm music as calm and 28 subjects the stressful music as stressful. However, analyzing the results regarding to their own perception of the results, did not yield any statistically significant differences in measured REE between silence and the two music periods.Forty subjects, 29 women and 11 men, completed the study. One subject dropped out because of feeling uncomfortable in the ventilated hood, one subject due to technical issues with the indirect calorimeter, and one subject due to problems with the CD-player. Mean (SD) age of the subjects were 35 (14) y, body height 172 (10) cm, body weight 68 (13) kg, and body mass index 23 (3) kg/mThis study could not detect any statistical significant or clinical relevant influences of music on REE, and then theoretically not on total energy expenditure. We chose to compare classical calm music to classical stressful music. This was to limit the confounding effect of the subjects own music preferences. When the stressful music was selected, not only tempo of the music was taken into consideration. The stressful music was also supposed to be irregular, have large differences between high and low frequencies, include many abrupt sounds, and give a sense of unpredictability. Most of the subjects perceived the calm music as calm and the stressful music as stressful, even if some subjects experienced the stressful music as \"other\". Maybe the stressful music was not stressful enough. Further studies should be conducted to investigate other types of music, i.e. pop music vs. heavy metal, and preferably also other types of stress-inductors than music combined with measurements of heart rate and other measures of stress. The results from this study do not support that music during rest could be used in obesity prevention or treatment alone, but music could of course be combined with physical activity to achieve an increase in total energy expenditure.REE \u2013 resting energy expenditureCD \u2013 compact discSD \u2013 standard deviationEC and HH participated in the study design, carried out the data collection and analyzed the results. FS conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Sub-optimally nourished rats show reduced growth, biochemical and physiological changes. However, no one has assessed metabolic rate adaptations in rats subjected to chronic suboptimal nutrition (CSN). In this study energy expenditure  and physical activity  were assessed in rats subjected to three levels of CSN.Body weight gain was diminished (76.7 \u00b1 12.0 and 61.6 \u00b1 11.0 g) in rats fed 70 and 60% of the ad-libitum fed controls which gained more weight (148.5 \u00b1 32.3 g). The rats fed 80% gained weight similarly to controls (136.3 \u00b1 10.5 g). Percent Fat-free body mass was reduced (143.8 \u00b1 8.7 and 142.0 \u00b1 7.6 g) in rats fed 70 and 60% of ad-libitum, but not in those fed 80% (200.8 \u00b1 17.5 g) as compared with controls (201.6 \u00b1 33.4 g). Body fat (g) decreased in rats fed 80% (19.7 \u00b1 5.3), 70% (15.3 \u00b1 3.5) and 60% (9.6 \u00b1 2.7) of ad-libitum in comparison to controls (26.0 \u00b1 6.7). EE and PA were also altered by CSN. The control rats increased their EE and PA during the dark periods by 1.4 \u00b1 0.8 and 1.7 \u00b1 1.1 respectively, as compared with light the period; whereas CSN rats fed 80 and 70% of ad-libitum energy intake had reduced EE and PA during the dark periods as compared with the light period EE, PA and RQ . In contrast, both light (7.1 \u00b1 1.4) and dark period (6.2 \u00b1 1.0) EE and PA (3.4 \u00b1 0.9 and 2.5 \u00b1 0.5 respectively) were reduced in rats fed 60% of ad-libitum energy intake.CSN rats adapt to mild energy restriction by reducing body fat, EE and PA mainly during the dark period while growth proceeds and lean body mass is preserved. At higher levels of energy restrictions there is decreased growth, body fat and lean mass. Moreover EE and PA are also reduced during both light and dark periods. Suboptimal nutrition in children, due to a chronic reduction of energy/nutrient intake over a long period of time, causes a deceleration of growth accompanied by inadequate weight gain . The decThe biochemical and hormonal changes associated with chronic suboptimal nutrition have been studied utilizing a rodent model . Sodium-Forty-four pre-pubertal four-week old male Sprague-Dawley rats were studied at the Miami Children's Hospital Research Institute in Miami Florida. All experimental protocols were reviewed and approved by the Animal Care and Use Committee of that institution. The animals were individually housed in wire-bottom stainless steel cages avoiding coprophagia. The light/dark cycle maintained within the rodent facility was 12/12 hours respectively beginning at 7:00 AM. The rats were divided into four groups. Twenty-two were ad-libitum fed controls while 8, 7 and 7 rats, respectively, were pair-fed at 80, 70 and 60% of the amount of energy consumed by their ad-libitum fed counterparts. They were fed a balanced purified diet containing 1:1 carbohydrate: fat  providing 3.94 kcal/g. The amount of energy from carbohydrate, fat and protein of this diet was 38.3, 38.3 and 23.4%, respectively, for every gram fed. This diet was adjusted for total energy according to the following :1) Diet A \u2013 100% of energy and all nutrients, fed ad-libitum2) Diet B \u2013 80% of energy and 100% of nutrients, pair-fed with rats in group A3) Diet C \u2013 70% of energy and 100% of all the nutrients, pair-fed with rats in group A4) Diet D \u2013 60% of energy and 100% of all nutrients, pair-fed with rats in group AAll rats were fed their experimental diets for four-weeks prior to the start of metabolic measurements. Purified diets were used in order to precisely control the amount of nutrients and to eliminate the variability often associated with commercial rat chows. The composition of the experimental diets is shown in Table All nutrients, except energy, were concentrated to compensate for the energy restriction of each diet. The percentages of metabolisable energy from fat, carbohydrate and protein were adapted from McCargar et al . BecauseFood fed to the restricted rats was calculated based on the amount of food consumed by the respective pair mates in the ad-libitum fed groups. For example, the amount of food given to the rats paired at 60% of energy intake was calculated as follows: [(food consumed by the ad-libitum fed pair-mate during the previous day/weight of this rat in the previous day) \u00d7 (0.6) \u00d7 (current weight of the rat for which the food was estimated)]. Daily body weights were recorded during the light period beginning at 8:00 AM prior to feeding of the experimental diets. Weighing and feeding the rats took approximately two hours each morning. Caloric efficiency  was also calculated daily by multiplying food intake (g) by the caloric content of the diet and dividing this result by daily body weight gain (g). Delta body weight gain was calculated by subtracting the body weight of the rat the day of the metabolic test from that of the first day of the experiment. The daily body gain was calculated by taking body weight gain and dividing by the number of days between the start of the experiment and the day of the metabolic test. After the completion of the metabolic tests all rats were anesthetized with Nembutal (30\u201350 g/kg body weight) and killed by cardiac puncture in the morning at the same time of recording of body weight. Carcasses were frozen at -20 degrees C for measurements of body composition using Folch's analysis method for fat extraction [2/VO2) and an index of physical activity  measured in the rodent Enhanced Metabolic Testing Activity Chamber (EMTAC). The main analytical unit of this instrument was developed to be suitable for various applications in both humans and animals for comprehensive measurements of energy expenditure and physical activity [[Metabolic measurements for each rat started after completion of four-weeks of feeding. On the day of the metabolic study each rat was weighed and had total energy expenditure , respiratory quotient , generated by movements of the rat, were read from the balance and utilized to calculate an index of physical activity expressed as oscillations in weight (g)/minute/kg body weight of the rat. For these calculations the software calculated the body weight of the rat in kilograms. The formulas used to calculate energy expenditure and physical activity index using the EMTAC have been validated and described previously ,21,22. TDue to the limited number of days available for metabolic measurements only 12, out of the 22 ad-libitum fed rats, were randomly selected for metabolic measurements. Metabolic measurements performed on control rats of similar weight in three previous suboptimal nutrition experiments had minimal variations in EE and PA ,10, and , and 10,Body composition analysis included total body water content (TBW), fat-free mass (FFM) and body fat (BF) determinations. This procedure comprised three different steps: 1) carcass homogenization, 2) water content and dry weight determinations and 3) fat content determination by fat extraction. The first step consisted of autoclaving each animal carcass for one hour at 15.3 psi and 120 degrees C in order to facilitate carcass homogenization. Each carcass was individually placed in a large beaker with covered tops with a known amount of distilled water. Each carcass was allowed to cool off overnight and then homogenized using a PowerGen700 blender. Triplicate aliquots of the homogenate were frozen at -20 degrees C for subsequent analysis. The chemical analysis of each homogenized carcass was carried out in triplicate and mean values of these triplicate samples were taken as ultimate values of total body water (TBW) and body fat (BF) content. The second step consisted of drying samples overnight at -380 mm Hg at 40 degrees C in a vacuum oven to determine water content. The difference between petri dish weight before and after overnight water extraction was considered as the dry weight of the carcass sample.The third step consisted of determining BF content by a modified Folch's method  for fat One way ANOVA utilizing Least Significant Difference (LSD) was utilized to determine the differences for EE, RQ, PA, food intake, caloric efficiency, initial and final body weights, delta body weight gain, fat-free and fat mass and percent total body water between the ad-libitum and each of the energy restricted groups. One way ANOVA with LSD was also utilized to determine differences in EE, RQ and PA between the ad-libitum and restricted energy groups during both the light and dark periods. Differences of all metabolic parameters  within each dietary treatment group between the light and dark periods were analyzed utilizing paired t-test. All data are presented as mean \u00b1 standard deviation (SD) unless otherwise noted.All rats appeared healthy and gained weight throughout the experimental period Table . Despitee in those fed 60%  in comparison to the full fed controls Table . However0% Table . This reThere were changes in body composition associated with chronic suboptimal nutrition. Fat-free and fat mass decreased (p < 0.05) in those rats fed at 70 and 60% of ad-libitum energy intake in comparison to ad-libitum fed controls. The fat mass (p < 0.05) also decreased in those rats fed at 80% ad-libitum energy intake but the fat-free mass was preserved Table . Moreove2/VO2) and physical activity index (oscillations/min/kg body weight) are shown in Figures The differences between the light and dark periods within each dietary treatment group for energy expenditure , respiratory quotient VCO/VO2 and 2/VO2), those rats fed 80, 70 and 60% of ad-libitum intake had a greater respiratory quotient during the light period in comparison to control rats  reduced EE in comparison to the controls Figure . During s Figure . The ress Figure .This is the first study of the daily metabolic profile in rodents subjected to various levels of chronic suboptimal nutrition. We utilized the rodent EMTAC to conduct accurate 21-hour measurements of EE and PA in rats restricted to 80, 70 or 60% of ad-libitum energy consumed by controls. This allowed us to determine the effects of chronic minor  levels of energy restriction on the energy expenditure during both the light and dark periods. Rats that were restricted to only 80% of their ad-libitum energy intake grew at a rate comparable to the ad-libitum fed controls. Furthermore, they preserved fat-free mass but had reduced EE and PA, along with increased RQ during the dark period. These data indicate that these rats utilize their body fat and reduced their physical activity to conserve energy for growth and lean body mass maintenance. However, there may be other effects at this level of energy restriction, not measured in this study, such as reduction of oxygen free radicals from oxidation of substrates  and impaFew studies have reported any differences between the light and dark periods in regards to energy metabolism and physical activity -28. RatsThere are other physiological adaptations that might contribute to the maintenance of health and body weight gain during chronic suboptimal nutrition. For example, a reduction of body temperature might be another energy conservation mechanism. Chronic caloric restriction up to 60% of ad-libitum energy intake has been found to reduce body temperature in mice  and ratsIt is possible that other factors might also contribute to the preservation of metabolic homeostasis. For example, there are alterations in erythrocyte sodium-potassium-ATPase activity which accounts for approximately one third of the basal energy metabolism . In a prThere are several potential reasons why sub optimally fed rats in our study had a higher RQ when compared to controls. For example, chronic suboptimal energy intake lasting up to 30 days might cause increased insulin sensitivity. This has been suggested as the cause of the greater RQ found in human anorexia nervosa patients . MoreoveIt has been reported by numerous investigators ,36, and , and 36,We have demonstrated that energy restriction up to 60% of ad-libitum energy intake causes detrimental effects such as reduced lean body mass, energy expenditure and physical activity. We also found that rats only restricted to 80% of their ad-libitum energy intake preserves their fat-free mass and maintenance of normal body weight gain. It appears their main energy needs for growth were obtained by a reduction of their fat mass and physical activity during the dark period. Moreover, we found that most of the alterations in energy metabolism and physical activity occurred during the dark period.CSN = Chronic suboptimal nutritionEMTAC = Enhanced Metabolic Testing Activity ChamberANOVA = Analysis of VarianceLSD = Least Significant DifferenceSD = Standard DeviationBF = Body FatFFM = Fat-free massTBW = Total body waterBW = Body weightEE = Energy expenditureRQ = Respiratory quotientPA = Physical activityPSI = Pounds per square inch2O2 = Hydrogen peroxideHThe author(s) declare that they have no competing interests.Dr. Russell Rising has contributed to the design of the experiment and conducted the data analysis. Furthermore, he either participated in some of the actual data acquisition or supervised pediatric research fellows in this regard. He also assisted in the preparation of the small grants necessary for funding of this project. Finally, he also assisted in the writing and editing of this manuscript.Dr. Fima Lifshitz contributed with ideas for the study, the design of the experiment, the preparation of the manuscript and assisted with data analysis. He also elicited the grants and funding necessary for the financial support of this study. Both authors were involved in the final editing of this manuscript."}
+{"text": "The large inter-individual variation in weight gain during standardized overfeeding together with a weight gain that is often less than theoretically calculated from the energy excess suggest that there are differences between persons in the capacity to regulate energy expenditure and hence metabolic efficiency. Adaptive thermogenesis is defined as the regulated production of heat in response to environmental changes in temperature and diet, resulting in metabolic inefficiency. The question is whether adaptive thermogenesis can be identified in overfeeding experiments. From the numerous human overfeeding experiments we selected those studies that applied suitable protocols and measurement techniques. Five studies claimed to have found evidence for adaptive thermogenesis based on weight gains smaller than expected or unaccounted increases in thermogenesis above obligatory costs. Results from the other 11 studies suggest there is no adaptive thermogenesis as weight gains were proportional to the amount of overfeeding and the increased thermogenesis was associated with theoretical costs of an increased body size and a larger food intake. These results show that in humans, evidence for adaptive thermogenesis is still inconsistent. However, they do not rule out the existence, but emphasize that if present, adaptive changes in energy expenditure may be too small to measure considering measurement errors, errors in assumptions made and small (day-to-day) differences in physical activity. In addition, it is not clear in which component or components of total energy expenditure adaptive changes can occur and whether components can overlap due to measurement limitations. Obesity develops when energy intake (EI) exceeds energy expenditure (EE) for longer periods. However, overfeeding experiments show that weight gain is often less than expected from the energy excess. In part this is the result of an obligatory increase in EE associated with the increased body weight and fat-free mass  and the EE consists of obligatory EE required for the normal functioning of cells and organs, EE for physical activity, and adaptive (or facultative) thermogenesis, which is defined as the regulated production of heat in response to environmental changes in temperature and diet . The oblFor heat production to increase in response to environmental factors like diet the coupling between mitochondrial oxidation and ATP synthesis must be reduced. Alternatively, the use of ATP can be increased without functional results, which can be referred to as wasting of ATP futile cycles . DiffereObesity needs a positive energy balance to develop, a situation that is mimicked in overfeeding experiments. Our selection of human overfeeding studies is summarized in Table The most reliable studies with regard to overfeeding are studies conducted with subjects living in the research institute during the entire study period. Then, however, a disadvantage is the induction of different lifestyles even when physical activity is not limited. In other experiments, subjects are studied as outpatients, who consume one or more meals per day at the research institute, but otherwise stay in their own environments. In both conditions, the gold standard for measuring EE over longer periods is the doubly labeled water (DLW) method. In combination with sleeping or basal metabolic rate (SMR or BMR), activity-induced energy expenditure (AEE) can be determined without restricting the subjects. In addition, physical activity (PA) can be objectively measured with accelerometers, which measure body movements in terms of frequency, duration and intensity . RespiraThe duration of the overfeeding mainly determines the reliability of changes in body weight as a reflection of energy storage. The overfeeding period should be long enough to expect an increase in body weight in excess of changes due to bowel contents and edema (i.e. excessive storage of body fluids that is not the result of an increased lean body mass).When digested foods enter the bloodstream there is an oxidative hierarchy. The macronutrient that is most easily stored (fat) is oxidized last, while macronutrients that can not be stored at all , or that can only be stored under certain circumstances (protein) or in limited amounts (carbohydrate) are oxidized first . AlcoholThe intake of any macronutrient in excess of energy needs will lead to fat storage, but a reduced capacity for fat oxidation could particularly predispose to obesity. Diaz et al.  overfed DIT is increased on a high-protein, high-carbohydrate diet compared to a high-fat diet . In contde novo lipogenesis, the last two studies suggest that costs of weight gain are rather increased when the carbohydrate content is relatively low which could be explained by increased gluconeogenesis. However, it should be noted that comparison between studies is difficult as macronutrient composition and measurement techniques differed substantial. This is also shown in the large range in costs of weight gain of 23 to 54 MJ/kg with 'average/mixed diet' overfeeding.The limited storage capacity for carbohydrates forces an increase in carbohydrate oxidation with carbohydrate overfeeding, which together with a decrease in fat oxidation results in a positive fat balance . HoweverThe component of daily energy expenditure most affected by changes in body weight is the BMR , any adaAnother component, DIT, will increase due to the increased amount of food that has to be digested and absorbed. Yet, several studies did not find a significant increase in DIT, independent of dietary composition and duration of the experiment ,28-30. OThe last component, AEE, is the most variable component of TEE between persons , and thuIt should be noted that the division of energy expenditure into its components may induce over- or underestimations of the separate components. AEE is particularly hard to determine, as measurement errors in TEE, BMR and DIT are accumulated in AEE . SMR migEnergy cannot get lost; energy that is not expended will be stored. As the digestibility of foods is not affected by intake level or subject ,35, enerThe macronutrient composition of the diet can influence energy storage. With carbohydrate overfeeding 75 to 85% of the excess energy was stored and the remaining expended, while with fat overfeeding 90 to 95% of the excess energy was stored, but there was no difference in fat storage after 14 d between the two diets fed isoenergetically to the same subjects  also fouThe composition of the overfeeding-induced body weight gain is fairly constant over different studies. Between 60 to 67% of the weight gain comprises an increase in fat mass (FM); the remaining part is an increase in fat-free mass (FFM) of 33 to 40% ,22,30. Tde novo lipogenesis during carbohydrate overfeeding [de novo lipogenesis is considered a quantitavely negligible process under normal conditions in humans.In addition, there are other ways to store excess energy as fat. The storage of body fat from dietary fat is the most energy efficient (~0.02 MJ per MJ ingested fat), but dietary protein and carbohydrate can also be stored as fat (~0.25 MJ per MJ ingested protein or carbohydrate) . Though rfeeding ,37-39, tOverfeeding studies that have not found evidence for adaptive thermogenesis mainly base their conclusions on the observation that there is no elevation in metabolic rate above obligatory costs, i.e. EE associated with an increased body size and tissue gain ,22,30,36Other studies conclude that adaptive thermogenesis must be present during overfeeding, because weight gain is smaller than expected  and the If adaptive thermogenesis is present and contributes to the etiology of obesity then it is likely that obesity-prone persons have a reduced capacity for adaptive thermogenesis compared to obesity-resistant persons. As the predisposition to obesity in humans is hard to define, if possible at all, one usually compares lean and overweight or obese subjects. Results suggest that the thermogenic response to fat is flexible in lean subjects but that subjects with familial obesity have a reduced response . AlthougIn humans, evidence for adaptive thermogenesis as a mechanism to explain interindividual differences in weight gain on the same overfeeding regimen is still inconsistent. Though most studies did find increases in EE during overfeeding, these were mainly explained by the theoretical energy costs of weight gain and the maintenance of a larger body weight. Changes in EE above these obligatory costs are considered adaptive thermogenesis, but the magnitude is generally no more than a few percent and includes measurement errors, errors in assumptions made and small (day-to-day) differences in physical activity. In addition, results from different overfeeding studies are hard to pool as there are marked differences in macronutrient composition, measurement techniques and availability of data within the papers. The latter causes comparison between studies using one measure  declare that they have no competing interests.AMCPJ drafted the manuscript. KRW provided constructive, critical review of the manuscript."}
+{"text": "An imbalance between energy intake and energy expenditure will lead to a change in body weight (mass) and body composition (fat and lean masses). A quantitative understanding of the processes involved, which currently remains lacking, will be useful in determining the etiology and treatment of obesity and other conditions resulting from prolonged energy imbalance. Here, we show that a mathematical model of the macronutrient flux balances can capture the long-term dynamics of human weight change; all previous models are special cases of this model. We show that the generic dynamic behavior of body composition for a clamped diet can be divided into two classes. In the first class, the body composition and mass are determined uniquely. In the second class, the body composition can exist at an infinite number of possible states. Surprisingly, perturbations of dietary energy intake or energy expenditure can give identical responses in both model classes, and existing data are insufficient to distinguish between these two possibilities. Nevertheless, this distinction has important implications for the efficacy of clinical interventions that alter body composition and mass. Understanding the dynamics of human body weight change has important consequences for conditions such as obesity, starvation, and wasting syndromes. Changes of body weight are known to result from imbalances between the energy derived from food and the energy expended to maintain life and perform physical work. However, quantifying this relationship has proved difficult, in part because the body is composed of multiple components and weight change results from alterations of body composition . Here, we show that mathematical modeling can provide a general description of how body weight will change over time by tracking the flux balances of the macronutrients fat, protein, and carbohydrates. For a fixed food intake rate and physical activity level, the body weight and composition will approach steady state. However, the steady state can correspond to a unique body weight or a continuum of body weights that are all consistent with the same food intake and energy expenditure rates. Interestingly, existing experimental data on human body weight dynamics cannot distinguish between these two possibilities. We propose experiments that could resolve this issue and use computer simulations to demonstrate how such experiments could be performed. Obesity, anorexia nervosa, cachexia, and starvation are conditions that have a profound medical, social and economic impact on our lives. For example, the incidence of obesity and its co-morbidities has increased at a rapid rate over the past two decades To address these issues and improve our understanding of human body weight regulation, mathematical and computational modeling has been attempted many times over the past several decades U is the change in stored energy in the body, \u0394Q is a change in energy input or intake, and \u0394W is a change in energy output or expenditure. The intake is provided by the energy content of the food consumed. Combustion of dietary macronutrients yields chemical energy and Hess's law states that the energy released is the same regardless of whether the process takes place inside a bomb calorimeter or via the complex process of oxidative phosphorylation in the mitochondria. Thus, the energy released from oxidation of food in the body can be precisely measured in the laboratory. However, there is an important caveat. Not all macronutrients in food are completely absorbed by the body. Furthermore, the dietary protein that is absorbed does not undergo complete combustion in the body, but rather produces urea and ammonia. In accounting for these effects, we refer to the metabolizable energy content of dietary carbohydrate, fat, and protein, which is slightly less than the values obtained by bomb calorimetry. The energy expenditure rate includes the work to maintain basic metabolic function (resting metabolic rate), to digest, absorb and transport the nutrients in food (thermic effect of feeding), to synthesize or break down tissue, and to perform physical activity, together with the heat generated. The energy is stored in the form of fat as well as in lean body tissue such as glycogen and protein. The body need not be in equilibrium for Equation 1 to hold. While we are primarily concerned with adult weight change, Equation 1 is also valid for childhood growth.The human body obeys the law of energy conservation U in terms of body mass M we must determine the energy content per unit body mass change, i.e.\u00a0the energy density \u03c1M. We can then set \u0394U\u200a=\u200a\u0394(\u03c1MM). To model the dynamics of body mass change, we divide Equation 1 by some interval of time and take the limit of infinitesimal change to obtain a one dimensional energy flux balance equation:I\u200a=\u200adQ/dt is the rate of metabolizable energy intake and E\u200a=\u200adW/dt is the rate of energy expenditure. It is important to note that \u03c1M is the energy density of body mass change, which need not be a constant but could be a function of body composition and time. Thus, in order to use Equation 2, the dynamics of \u03c1M must also be established.In order to express a change of stored energy \u0394M is to track the dynamics of the components. The extracellular water and bone mineral mass have no metabolizable energy content and change little when body mass changes in adults under normal conditions M are the macronutrients - protein, carbohydrates, and fat, where we distinguish body fat (e.g. free fatty acids and triglycerides) from adipose tissue, which includes water and protein in addition to triglycerides. We then represent Equation 2 in terms of macronutrient flux balance equations for body fat F, glycogen G, and protein P:F\u200a=\u200a39.5 MJ/kg, \u03c1G\u200a=\u200a17.6 MJ/kg, \u03c1P\u200a=\u200a19.7 MJ/kg are the energy densities FI,CI,PI are the intake rates, and Ff, Cf, 1\u2212Ff\u2212Cf are the fractions of the energy expenditure rate obtained from the combustion of fat, carbohydrates (glycogen) and protein respectively. The fractions and energy expenditure rate are functions of body composition and intake rates. They can be estimated from indirect calorimetry, which measures the oxygen consumed and carbon dioxide produced by a subject de novo lipogenesis where carbohydrates are converted to fat or gluconeogenesis where amino acids are converted into carbohydrates can be accounted for in the forms of Ff and Cf. The sum of Equations 3, 4, and 5 recovers the energy flux balance Equation 2, where the body mass M is the sum of the macronutrients F, G, P, with the associated intracellular water, and the inert mass that does not change such as the extracellular water, bones, and minerals, and \u03c1M\u200a=\u200a(\u03c1FF+\u03c1GG+\u03c1PP)/M.When the body changes mass, that change will be composed of water, protein, carbohydrates (in the form of glycogen), fat, bone, and trace amounts of micronutrients, all having their own energy densities. Hence, a means of determining the dynamics of \u03c1The intake and energy expenditure rates are explicit functions of time with fast fluctuations on a time scale of hours to days The three-compartment flux balance model was used by Hall F and lean mass L\u200a=\u200aM\u2212F, where M is the total body mass. The lean mass includes the protein and glycogen with the associated intracellular water along with the mass that does not change appreciably such as the extracellular water and bone. Hence the rate of change in lean mass is given byPh\u200a=\u200a1.6 and Gh\u200a=\u200a2.7 are reasonable estimates of the hydration coefficients for the intracellular water associated with the protein and glycogen respectively dG/dt\u200a=\u200a0, as observed in numerical simulations Cf\u200a=\u200aCI/E, which can be substituted into Equation 5 to giveThe three compartment macronutrient flux balance model Equations 3\u20135 can be reduced to a two dimensional system for fat mass dG/dt\u200a=\u200a0 into Equation 6 leads to the two compartment macronutrient partition modelL\u200a=\u200a\u03c1P/(1+Ph)\u200a=\u200a7.6 MJ/kg, FI and LI\u200a=\u200aPI+CI are the intake rates into the fat and lean compartments respectively, E\u200a=\u200aE is the total energy expenditure rate, and f\u200a=\u200af \u2261 Ff is the fraction of energy expenditure rate attributed to fat utilization.Substituting Equation 7 and dG/dt\u200a=\u200a0 may be violated if the glycogen content is proportional to the protein content, which is plausible because most of the glycogen mass is stored in muscle tissue and may scale with protein mass. We show that this assumption leads to the same two dimensional system. Substitutingk, into Equation 4 gives CEf\u200a=\u200aCI\u2212\u03c1CkdP/dt, which inserted into Equation 5 leads toL\u200a=\u200a(\u03c1P+k\u03c1C)/((1+Ph+k+Gkh). For k\u200a=\u200a0.044\u226a1 as suggested by Snyder et al. L has approximately the same value as before.We note that E linearized in F and L and different f depending on context. Forbes FI and LI zero, and E linear in F and L.Previous studies have considered two dimensional models of body mass change although they were not derived from the three-dimensional macronutrient partition model. Alpert L\u2013F phase plane follow prescribed paths satisfyingF,L) is a continuous function D is a free parameter, and the lean and fat masses are in units of kg. Forbes found that his general relationship (14) was similar whether weight loss is induced by diet or exercise The two-compartment macronutrient partition model can be further simplified by assuming that trajectories in the F vs. L curves, parameterized by an integration constant . Depending on the initial condition, the body composition moves along one of these curves when out of energy balance. Dividing Equation 8 by Equation 9 and imposing Equation 12 results inEquation 12 describes a family of Energy Partition model:p\u200a=\u200ap\u200a=\u200a1/(1+\u03b1) is known as the p-ratio I\u2013E is divided between the compartments according to a function p that defines the fraction assigned to lean body tissue (mostly protein). Most of the previous models in the literature are different versions of the energy partition model Substituting Equation 15 into the macronutrient partition model 8 and 9 leads to the f  by using Equation 15 p to be a constant Despite the ubiquity of the energy partition model, the physiological interpretation of the p-ratio remains obscure and is difficult to measure directly. It can be inferred indirectly from FI,LI,F,L), which is zero at the fixed point . We use this form in numerical examples below. The fasting model of Song and Thomas I\u200a=\u200a0 and \u03c8 was a function of F representing ketone production. Comparing to Equation 8 and Equation 18 givesIt may sometimes be convenient to express the macronutrient partition model with a unique fixed point asL\u2013F plane. Thus a further simplification to a one dimensional model is possible by finding a functional relationship between F and L so that one variable can be eliminated in favor of the other. Such a function exists if Equation 12 has a unique solution, which is guaranteed in some interval of L if \u03b1 and \u2202\u03b1/\u2202F are continuous functions of F and L on a rectangle containing this interval. These are sufficient but not necessary conditions.The dynamics of the energy partition model Equations 16 and 17 move along fixed trajectories in the F\u200a=\u200a\u03c6(L) can be found between F and L. Substituting this relationship into Equation 16 and Equation 17 and adding the two resulting equations yields the one dimensional equationM\u200a=\u200aL+\u03c6(L). If we can invert this relationship uniquely and obtain L as a function of M, then this can be substituted into Equation 21 to obtain a dynamical equation for M.Suppose a relationship p to be a constant, which was used in F\u200a=\u200a\u03b2L+C\u2261 \u03c6(L) where \u03b2\u200a=\u200a\u03c1L(1\u2212p)/(\u03c1Fp) and C is a constant that is specified by the initial body composition. This results inM0 givesM\u200a=\u200a\u03c1F\u03c1L/(\u03c1L+(\u03c1F\u2212\u03c1L)p), \u03bc\u200a=\u200aI\u2212E(F(M0),L(M0)), and As an example, assume L)\u200a=\u200aDexp(L/10.4)) in Equation 21 yieldsM\u200a=\u200aL+Dexp(L/10.4) or M\u200a=\u200aF+10.4log(F/D) cannot be inverted in closed form, an explicit one dimensional differential equation in terms of the mass cannot be derived. However, the dynamics of the mass is easily obtained using either Equation 24 or Equation 25 together with the relevant mass function. For large changes in body composition, the dynamics could differ significantly from the constant p models 22 or 23.If Equations 16 and 17 are constrained to obey the phase plane paths of Forbes's law, then a reduction to a one dimensional equation can also be made. Using Equation 14 . If the models are smooth and continuous then the global dynamics can be inferred from the local dynamics of the model near fixed points (i.e. where the time derivatives of the variables are zero). To simplify the analysis, we consider the intake rates to be clamped to constant values or set to predetermined functions of time. We do not consider the control and variation of food intake rate that may arise due to feedback from the body composition or from exogenous influences. We focus only on what happens to the food once it is ingested, which is a problem independent of the control of intake. We also assume that the averaged energy expenditure rate does not depend on time explicitly. Hence, we do not account for the effects of development, aging or gradual changes in lifestyle, which could lead to an explicit slow time dependence of energy expenditure rate. Thus, our ensuing analysis is mainly applicable to understanding the slow dynamics of body mass and composition for clamped food intake and physical activity over a time course of months to a few years.Dynamics in two dimensions are particularly simple to analyze and can be easily visualized geometrically L\u2013F phase plane. For FI and LI constant, the flux balance model is a two dimensional autonomous system of ordinary differential equations and trajectories will flow to attractors. The only possible attractors are infinity, stable fixed points or stable limit cycles In two dimensions, changes of body composition and mass are represented by trajectories in the L and F to be positive and finite. For differentiable f and E, the possible trajectories for fixed intake rates are completely specified by the dynamics near fixed points of the system. Geometrically, the fixed points are given by the intersections of the nullclines in the L\u2013F plane, which are given by the solutions of FI\u2212fE\u200a=\u200a0 and LI\u200a=\u200a(1\u2212f)E\u200a=\u200a0. Example nullclines and phase plane portraits of the macronutrient model are shown in L\u2013F plane. In this case, there are an infinite number of possible body compositions for a fixed diet. As we will show, the energy partition model implicitly assumes an invariant manifold. If a single fixed point exists but is unstable then a stable limit cycle may exist around it.Physical viability constrains I\u200a=\u200aFI+LI, and we have suppressed the functional dependence on intake rates. These fixed point conditions correspond to a state of flux balance of the lean and fat components. Equation 26 indicates a state of energy balance while Equation 27 indicates that the fraction of fat utilized must equal the fraction of fat in the diet. Stability of a fixed point is determined by the dynamics of small perturbations of body composition away from the fixed point. If the perturbed body composition returns to the original fixed point then the fixed point is deemed stable. We give the stability conditions in The fixed point conditions of Equations 8 and 9 can be expressed in terms of the solutions ofE and f on F and L determine the existence and stability of fixed points. As shown in f is a monotonic increasing function of F and a monotonic decreasing function of L. If one of the fixed point conditions automatically satisfies the other, then instead of a fixed point there will be a continuous curve of fixed points or an invariant manifold. For example, if the energy balance condition 26 automatically satisfies the fat fraction condition 27, then there is an invariant manifold defined by I\u200a=\u200aE. The energy partition model has this property and thus has an invariant manifold rather than an isolated fixed point. This can be seen by observing that for f given by Equation 15, Equation 26 automatically satisfies condition 27. An attracting invariant manifold implies that the body can exist at any of the infinite number of body compositions specified by the curve I\u200a=\u200aE for clamped intake and energy expenditure rates (see M\u200a=\u200aF+L (except for the unlikely case that E is a function of the sum F+L). The body composition is marginally stable along the direction of the invariant manifold. This means that in flux balance, the body composition will remain at rest at any point on the invariant manifold. A transient perturbation along the invariant manifold will simply cause the body composition to move to a new position on the invariant manifold. The one dimensional models have a stable fixed point if the invariant manifold is attracting. We also show in f must be nonmonotonic in L and be finely tuned. The required fine-tuning makes these latter two possibilities much less plausible than a single fixed point or an invariant manifold.The functional dependence of ates see . Each ofE is a monotonically increasing function of F and Lf on F and L is not well established and the form of f depends on multiple interrelated factors. In general, the sensitivity of various tissues to the changing hormonal milieu will have an overall effect on both the supply of macronutrients as well as the substrate preferences of various metabolically active tissues. On the supply side, we know that free fatty acids derived from adipose tissue lipolysis increase with increasing body fat mass which thereby increase the daily fat oxidation fraction, f, as F increases F with weight loss has been demonstrated to decrease ff should be a decreasing function of L. In further support of this relationship, body builders with significantly increased L have a decreased daily fat oxidation fraction versus control subjects with similar FData suggest that We have shown that all two dimensional autonomous models of body composition change generically fall into two classes - those with fixed points and those with invariant manifolds. In the case of a stable fixed point, any temporary perturbation of body weight or composition will be corrected over time . An invariant manifold allows the possibility that a transient perturbation could lead to a permanent change of body composition and mass.F vs. L trajectories obeying Equation 12, an example being Forbes's law (14). As shown in At first glance, these differing properties would appear to point to a simple way of distinguishing between the two classes. However, the traditional means of inducing weight change namely diet or altering energy expenditure through aerobic exercise, turn out to be incapable of revealing the distinction. For an invariant manifold, any change of intake or expenditure rate will only elicit movement along one of the prescribed f. For example, body composition could be altered directly through liposuction and f could be altered by administering compounds such as growth hormone. Resistance exercise may cause an increase in lean muscle tissue at the expense of fat. Exogenous hormones, compounds, or infectious agents that change the propensity for fat versus carbohydrate oxidation , where F is in units of kg). Consider two cases of the model. If \u03c8\u200a=\u200a0 then the model has an invariant manifold and body composition moves along a fixed trajectory in the L\u2013F plane. If \u03c8 is nonzero, then there can be an isolated fixed point. We will show an example where if the intake energy is perturbed, the approach of the body composition to the steady state will be identical for both cases but if body composition is perturbed, the body will arrive at different steady states.We now consider some numerical examples using the macronutrient partition model in the form given by Equations 18 and 19, with a p-ratio consistent with Forbes's law (13) \u200a=\u200a0 defines the fixed trajectory of the invariant manifold model. Using Forbes's law (14), we choose \u03c8\u200a=\u200a0.05(F\u22120.4 exp(L/10.4))/F. We then take a plausible energy expenditure rate of E\u200a=\u200a0.14L+0.05F+1.55, where energy rate has units of MJ/day and mass has units of kg. This expression is based on combining cross-sectional data For every model with an invariant manifold, a model with a fixed point can be found such that trajectories in the F vs. L trajectories of the two model examples given a reduction in energy intake rate from 12 MJ/day to 10 MJ/day starting at the same initial condition. The time courses are identical for body composition and mass. The mass first decreases linearly in time but then saturates to a new stable fixed point. The dashed line represents the same intake rate reduction but with 10 kg of fat removed at day 100. For the invariant manifold model, the fat perturbation permanently alters the final body composition and body mass, whereas in the fixed point model it only has a transient effect. In the fixed point model, the body composition can ultimately exist only at one point given by the intersection of the nullclines . For the invariant manifold, the body composition can exist at any point on the I\u200a=\u200aE curve  has been pointed out many times previously In this paper we have shown that all possible two dimensional autonomous models for lean and fat mass are variants of the macronutrient partition model. The models can be divided into two general classes - models with isolated fixed points (most likely a single stable fixed point) and models with an invariant manifold. There is the possibility of more exotic behavior such as multi-stability and limit cycles but these require fine-tuning and thus are less plausible. Surprisingly, experimentally determining if the body exhibits a fixed point or an invariant manifold is nontrivial. Only perturbations of the body composition itself apart from dietary or energy expenditure interventions or alterations of the fraction of energy utilized as fat can discriminate between the two possibilities. The distinction between the classes is not merely an academic concern since this has direct clinical implications for potential permanence of transient changes of body composition via such procedures as liposuction or temporary administration of therapeutic compounds.Our analysis considers the slow dynamics of the body mass and composition where the fast time dependent hourly or daily fluctuations are averaged out for a clamped average food intake rate. We also do not consider a slow explicit time dependence of the energy expenditure. Such time dependence could arise during development, aging or gradual changes in lifestyle where activity levels differ. Thus our analysis is best suited to modeling changes over time scales of months to a few years in adults. We do not consider any feedback of body composition on food intake, which is an extremely important topic but beyond the scope of this paper.f. Hall f that does not lead to an invariant manifold. However, our analysis and numerical examples show that the body composition could have an invariant manifold but behave indistinguishably from having a fixed point. Also, the decay to the fixed point could take a very long time, possibly as long as a decade giving the appearance of an invariant manifold. Only experiments that perturb the fat or lean compartments independently can tell.Previous efforts to model body weight change have predominantly used energy partition models that implicitly contain an invariant manifold and thus body composition and mass are not fully specified by the diet. If the body does have an invariant manifold then this fact puts a very strong constraint on the fat utilization fraction averaged equations valid on longer time scales.The three compartment macronutrient flux balance Equations 3\u20135 are a system of nonautonomous differential equations since the energy intake and expenditure are explicitly time dependent. Food is ingested over discrete time intervals and physical activity will vary greatly within a day. However, this fast time dependence can be viewed as oscillations or fluctuations on top of a slowly varying background. It is this slower time dependence that governs long-term body mass and composition changes that we are interested in. For example, if an individual had the exact same schedule with the same energy intake and expenditure each day, then averaged over a day, the body composition would be constant. If the daily averaged intake and expenditure were to gradually change on longer time scales of say weeks or months then there would be a corresponding change in the body composition and mass. Given that we are only interested in these slower changes, we remove the short time scale fluctuations by using the method of averaging to produce an autonomous system of t/\u03b5, where \u03b5 is a small parameter that is associated with the slow changes in body composition and let all time dependent quantities be a function of both t and \u03c4. For example, if t is measured in units of days and \u03c4 is measured in units of hours then \u03b5\u223c1/24. Inserting into Equations 3\u20135 and using the chain rule yieldsF1\u232a\u200a=\u200a\u2329P1\u232a\u200a=\u200a\u2329G1\u232a\u200a=\u200a0 for a time average defined by T represents an averaging time scale of a day. The fast time dependence can be either periodic or stochastic. The important thing is that the time average over the fast quantities is of order \u03b5 or higher. We then expand the energy expenditure rate and expenditure fractions to first order in \u03b5:E0\u2261E+O(\u03b52) and i\u2208{F,G,P}. We assume that the expenditure fractions depend on time only through the body compartments. Substituting these expansions into Equations 28\u201330 and taking lowest order in \u03b5 givesWe do so by introducing a second \u201cfast\u201d time variable \u03c4\u200a=\u200aF1/\u2202\u03c4\u232a, \u2329\u2202G1/\u2202\u03c4\u232a, and \u2329\u2202P1/\u2202\u03c4\u232a are of order \u03b5 or higher leads to the averaged equations:T. A difference in the choice of T will only result in a different interpretation of the averaged quantities.Taking the moving time average of Equations 36\u201338 and requiring that \u2329\u2202F0,L0) are determined by expanding fE and (1\u2212f)E to linear order in \u03b4F\u200a=\u200aF\u2212F0 and \u03b4L\u200a=\u200aL\u2212L0t) yields an eigenvalue problem with two eigenvalues given by J<0 and det J>0. In the case of an invariant manifold, detJ\u200a=\u200a0, so the eigenvalues are Tr J and 0. The zero eigenvalue reflects the marginal stability along the invariant manifold, which is an attractor if Tr J<0. An attracting invariant manifold implies a stable fixed point in the corresponding one dimensional model. Unstable fixed points are either unstable nodes, saddle points or unstable spirals. In the case of unstable spirals, a possibility is a limit cycle surrounding the spiral arising from a Hopf bifurcation, where Tr J\u200a=\u200a0 and det J>0. In this case, body composition and mass would oscillate even if the intake rates were held constant. The frequency and amplitude of the oscillations may be estimated near a supercritical Hopf bifurcation by transforming the equations to normal form. Stability of a fixed point puts constraints on the form of f. Physiological considerations and data imply that \u2202E/\u2202L>\u2202E/\u2202F>0 E/\u2202F\u200a=\u200a\u03b4\u2202E/\u2202L where \u03b4 <1 . Then detJ>0 implies thatJ<0 impliesK\u200a=\u200a[\u03b4f+\u03b3 (1\u2212f)](\u2202E/\u2202L)/E>0 and \u03b3\u200a=\u200a\u03c1F/\u03c1L\u22485.2. Hence \u2202f/\u2202F>0 and \u2202f/\u2202L<0 guarantees stability of a fixed point. In other words, if f increases monotonically with F and decreases monotonically with L then there will be a unique stable fixed point. For an invariant manifold, f is given by Equation 15, which immediately satisfies detJ\u200a=\u200a0; TrJ<0 is guaranteed if E is monotonically increasing in F and L. For a Hopf bifurcation, we require \u2202f/\u2202F\u200a=\u200a\u03b3\u2202f/\u2202L\u2212K and Equation 44, implying (\u03b3\u2212\u03b4)\u2202f/\u2202L\u2212K>0. Since \u03b3>\u03b4, f must increase with L for the possibility of a limit cycle. However, to ensure that trajectories remain bounded f must decrease with L for very small and large values of L. Hence, f must be nonmonotonic in L for a limit cycle to exist. This can also be seen from an application of Bendixson's criterion L\u2013F plane ifThe dynamics near a fixed point (ycle see . Similarycle see , for mul"}
+{"text": "Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days  and long-days  acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.During moderate calorie restriction (CR) the heterotherm  Torpor is associated with a profound reduction of energy expenditure to as little as 3% of the euthermic rates at ambient temperature Energy and water conservation through torpor is likely to be essential for surviving prolonged periods of restricted energy availability. Nevertheless more energy conservation may not necessarily be more advantageous. High rates of metabolism were hypothesized to be beneficial for endotherms when resources are abundant vs. metabolically inactive fat mass would represent a significant strategy to lower energy requirements without cessation of activities.The torpor avoidance may be associated to the several reported negative physiological consequences of torpor arousal, such as a possible increase in oxidative stress as we Seasonal heterotherms accumulate and subsequently loose energy stores in a yearly body mass gain-loss cycle Microcebus murinus) is among the rare known heterothermic primates and shows marked seasonal changes in body mass in response to the Malagasy predictable cycle of food allocations. During the summer season, mouse lemurs are in an active reproductive state. After autumnal fattening, animals increase their torpor propensity for energy and water conservation, to cope with the drastic food shortages during the dry and cold season. We investigated the pattern of torpor expression of this small Malagasy prosimian, as a function of food availability The grey mouse lemur  used in this study were born in the laboratory breeding-colony of Brunoy  from a stock originally caught along the southwestern coast of Madagascar 40 years ago. Seasonal Malagasy rhythms were reproduced by alternating 6-month periods of long-days (light\u2236dark 14\u223610) and short-days (light\u2236dark 10\u223614). In M. murinus, an exposure to long-days triggers a summer-like state, i.e. stimulates reproductive and locomotor's activities, increases metabolic rates and reduces body mass. Conversely, an exposure to short-days sets-up a winter-like phenotype in mouse lemurs, by triggering a fattening process, by increasing food intake, and by reducing metabolic rates and locomotor's activities The 34 adult male grey mouse lemurs . Since grey mouse lemurs, and particularly those under winter phenotype, tend to overfeed when isolated and thus gain mass during the ad-libitum period, energy intake was clamped to the level required to stabilize their body mass. This was necessary to avoid significant underestimation of the calorie restriction (CR) needed for the test diets. Each individual was initially fed ad-libitum with banana and the homemade mixture and progressively, daily energy intake was narrowed according to the body mass time-course, as already done in our previous study After a month of acclimatization to their new environment, individual calorie intakes were measured during a 10-day period, in order to calculate subsequent food-restricted energy allotments. Animals were fed, in ad-libitum. Daily food intake was calculated from the difference between provided and remaining food weights and was corrected for dehydration. Energy equivalents of 3.7 kJ/g for the banana and 4.6 kJ/g for the mixture were used to convert grams of food intake to kJ. During the 35-day food restriction period, mouse lemurs in the 40% CR received an energy allotment of either 47.5\u00b11.3 kJ/day  or 45.8\u00b13.3 kJ/day . The 80% food-restricted LD (LD80) and SD animals (SD80) were provided with an energy allocation of 16.5\u00b12.5 kJ/day and 15.5\u00b10.6 kJ/day, respectively. The LD80 mouse lemur group weighed 78\u00b12 g under ad-libitum diet weighed, 52\u00b11 g after 22 days on the 80% restriction diet. According to the data from the Brunoy colony, weights of 50 g are survival threatening for this photoperiod (21). Therefore, these animals were excluded from the study before the end of the food-deprived trial, replaced in the breeding colony and re-fed with an ad-libitum diet. No urine samples were collected or energy measurements were performed in the CR period for this group.Half of the animals in each photoperiod were then provided with 60% (\u200a=\u200a40% CR) or 20% (\u200a=\u200a80% CR) of these individually derived energy requirements, during 35 days. Food-restricted allotments were available every day at the onset of the dark phase. Water was always provided ad-libitum period and again after 35 days of CR. The tests were identical in both conditions and consisted of the measurement of daily energy expenditure (DEE), fat-free mass (FFM), fat mass (FM) and water flux rate (rH20) by the doubly labeled water (DLW) method, resting metabolic rate (RMR) by respirometry, protein turnover by using [15N]-glycine and catecholamine concentrations in a 24-hr pooled urine. Body temperature was continuously recorded by telemetry during the ad-libitum and food-deprived period.Each animal was studied during the 218O  and 0.15 g/(kg estimated TBW) 99.9% 2H2O  and was diluted with 3% NaCl to physiological osmolarity. We assumed a percentage of hydration of 0.60 and 0.55 for LD and SD animals, respectively, to calculate doses. The doses were calculated to ensure an in vivo enrichment of about 250 and 1200\u2030 for 18-oxygen and deuterium, respectively -glycine end-product technique 15N]-glycine solution. 15N-urea and 15N-amonia enrichments were measured in the 24-hr urine pools, as previously described 15N end-products in urine. Synthesis and catabolism rates were then calculated from the following equation Q\u200a=\u200aS + E\u200a=\u200aC + I, where S is the synthesis rate of protein, E is excretion of nitrogen (urine plus feces), C is the catabolism rate, and I is dietary nitrogen intake.During those 24 hours, protein turnover was determined by means of the \u2013[12Faced to a moderate food shortage, mouse lemurs under summer-like long-days would respond to food deprivation like all typical mammals, by decreasing protein synthesis and elevating protein catabolism, resulting in net nitrogen loss M. murinus in summer under a moderate food shortage could fully be accounted by the reduced fat-free mass because the animals do not change their activity levels during food restriction In association with the reduction in metabolism triggered by the decrease in fat-free mass, water turnover was also reduced in mouse lemurs in summer facing a moderate food shortage. Our findings with the summering mouse lemurs are similar to those with the Arabian Oryx ad-libitum, mouse lemurs in winter displayed a significantly reduced rate of protein turnover when compared to the summer state. Although some mechanisms underlying hibernation and torpor differ, similar phenomenon and potential explanations are found in hibernators. Protein synthesis and breakdown are both lower in wintering compared to summering black bears, indicating a lowered metabolism in animals in winter Under Winter-like short-days mouse lemurs under a moderate food restriction were able to maintain their fat-free mass, which is likely to play a role in the torpor mechanism. During the cold and dry season in the Kirindy forest, mouse lemurs arouse from the low torpor state through a two-step process, consisting of an initial passive climb in Tb in relation to the ambient temperature followed by an active rise of Tb from 28\u201330\u00b0C to euthermic level In mouse lemurs in winter under a moderate food deprivation, the enhanced reduction in protein turnover would contribute to decreases in daily energy expenditure and water turnover. Maintenance of protein pools is the result of a balance between protein synthesis and degradation. In some tissues, protein synthesis and degradation account for as much as 20\u201340 and 4%, respectively, of oxygen consumption Conversely to winter animals under moderate food shortage, mouse lemurs facing a severe food deprivation showed a significant chronic negative nitrogen balance and were unable to reduce their nitrogen flux, due to a dramatic rise in their protein catabolism. This increase led to a reduction in their fat-free mass and was similar to a food starvation response in term of protein use M. murinus, especially in the context of global changes in which the frequency and the intensity of such periods of food scarcity are predicted to increase.Under moderate food shortages, mouse lemurs use distinct efficient mechanisms of energy savings depending on the season. Nevertheless, when faced to a severe food scarcity, wintering mouse lemurs reached a critical state of a high stress level of and protein loss. This severe energy shortfall in the long term would affect the fitness and thus the survival of"}
+{"text": "We conducted this controlled randomized dietary trial to test the hypothesis that n-3-PUFA lower body weight and fat mass by reducing appetite and 2) men and women were included, and received either a diet rich in n-3-PUFA from both plant and marine sources or a control diet. Diets were administered in an isocaloric fashion for 2 weeks followed by 12 weeks of ad libitum intake. The n-3-PUFA and control diets were identical in all regards except for the fatty acid composition. All foods were provided to subjects, and leftovers were weighed back to assess actual food intake accurately for each day of the study. This design gave us 80% power to detect a difference in weight change between the n-3-PUFA and control diet groups of 2.25 kg at an \u03b1-error level of 5%.Twenty-six overweight or moderately obese (body mass index 28\u201333 kg/mad libitum for 12 weeks . Consistent with this finding, we also found no differences between the n-3-PUFA and control groups with regard to appetite as measured by visual analogue scale, ad libitum food intake, resting energy expenditure as measured by indirect calorimetry, diurnal plasma leptin concentrations, or fasting ghrelin concentrations.Both groups lost similar amounts of weight when these diets were consumed Our results suggest that dietary n-3-PUFA do not play an important role in the regulation of food intake, energy expenditure, or body weight in humans. Exclusion criteria included the use of tobacco or recreational drugs, alcohol abuse, a history of cardiovascular disease or diabetes mellitus, the presence of any chronic or psychiatric illness, restrictive eating behavior, pregnancy, and the intake of selective serotonin-reuptake inhibitors, lipid-lowering drugs, \u03b2-blockers, glucocorticoids, or anabolic steroids. All subjects underwent informed consent procedures. The study protocol was approved by the University of Washington Institutional Review Board (#03-9082-D05), and was in compliance with the Helsinki Declaration.Subjects were 26 healthy overweight or mildly obese . In this phase, the caloric content of food provided to subjects was 115% of the amount they had received in the previous isoenergetic period. Subjects continued to weigh twice weekly, pick up food and return uneaten food items, allowing an accurate assessment of the calories consumed on each day of the study. During each food pick-up visit, subjects met with a dietitian for about 15 minutes to review the food records and the returned, uneaten foods. This meeting and the comparison of the food records and returned foods were implemented as they provided valuable information about the compliance of the subject. Specifically, meeting with the subject in person twice weekly for the 16-week dietary period created a rapport with the subject and helped us respond in a timely fashion to specific requests such as switching specific meals or foods. Further, any major or repeated deviations from the study protocol would have required a substantial investment of time and effort from the subject to make sure that the amounts of foods returned were always consistent with those recorded on the food diary. To further increase compliance, subjects were allowed to drink up to 30 alcoholic drinks, and to exchange up to 10 study meals for \"meals out\" or meals prepared at home during the first 10 weeks of the ad libitum diet period. We asked subjects to provide detailed information about alcoholic beverages and meals out on the daily food record. Specifically, we asked them to note the amount and type of alcoholic beverage consumed, and to provide recipes for meals prepared at home, including details such as the amounts and sources of dietary fat. If they had a meal at a restaurant, we asked them to provide the names of the restaurant and the dish they had chosen. A dietitian of the CRC Nutrition Research Kitchen would then call the restaurant to obtain more detailed information about the dish. Of note, 18 out of the 26 subjects who completed the study exchanged a study meal for a \"meal out\" on less than the allowed 10 occasions, suggesting an excellent compliance with the dietary regimen. On day 84 of this diet period, subjects were again admitted to the CRC for visit 3.After CRC 2, subjects continued to consume the diets to which they had been randomized for another 12 weeks but now freely chose the amount they wished to eat  scanner to assess body fat mass. At 0700 on day 2 of each CRC admission, we performed indirect calorimetry for 30 minutes to assess resting energy expenditure using a TrueOne 2400 metabolic cart . Throughout the study, subjects completed daily visual analogue scale (VAS) questionnaires to assess hunger and fullness. They were also asked to complete a modified Blair physical activity questionnaire .Plasma leptin and ghrelin were measured in duplicate by commercially available radioimmunoassays . Leptin was measured in all 38 timed diurnal samples drawn during each of the three clinic visits. Ghrelin was measured in fasting samples, and in pools of plasma created from the timed blood samples by combining 50 \u03bcL plasma from each blood sample drawn at 30-minute intervals and 100 \u03bcL plasma from each blood sample drawn at 60-minute intervals ('pool'-sample). Intra- and interassay CV were 2.4% and 3.6%, respectively, for leptin, and 4.3% and 5.3%, respectively, for ghrelin.ad libitum diet period was derived from two previous controlled dietary trials conducted by our group that had employed an identical design , but not different between the two groups  , but decreased when subjects lost weight during the ad libitum period by 450 \u00b1 896 arbitrary units (AU) in the control group, and by 350 \u00b1 705 AU in the n-3-PUFA group , but without any difference between the groups . Lastly, physical activity remained constant in both groups throughout the study [\u03c72(5) = 6.156, p = 0.291 for control and \u03c72(5) = 7.313, p = 0.198 for n-3-PUFA group, Friedman tests; Table No changes were observed in hunger and fullness ratings as assessed by VAS throughout the study (data not shown). Compared to the isoenergetic periods, food intake tended to be lower in the : F1.100,6.409 = 2Our well-controlled 16-week study showed that increasing dietary n-3-PUFA content to 3.6% of total energy did not have an effect on plasma leptin levels, body weight or fat mass in overweight or moderately obese, healthy men and women. We designed the study as a sequential clinical trial, using the power family of two-sided, inner wedge tests (chapter 5 in [ad libitum food intake were not different between the n-3-PUFA and control diets, suggesting that n-3-PUFA do not increase leptin sensitivity as hypothesized. Our findings are consistent with those of Krebs and colleagues [We decided not to administer more than 3.6% of energy in the form of n-3-PUFA, as higher intakes are not realistic over an extended period of time in a free-living population. Endpoints affected by leptin signaling such as resting energy expenditure, appetite, or lleagues  who studThese results are in contrast, however, to several studies in rodents -9, and oStrengths of our study include the fact that all foods were provided for a period of 16 weeks, and that the quality and quantity of food consumed was assessed for each day of the study. The n-3-PUFA and control diets were designed to be identical except for the fatty acid composition. As all subjects were seen at least twice weekly by study staff, and were encouraged to share with us any deviation from the study protocol, we feel confident in our assessment of their actual intake. Nevertheless, the fact that subjects could have been non-compliant is a potential limitation of this study.ad libitum food intake, resting energy expenditure, or body weight and fat mass in overweight or moderately obese men and women.We conclude that dietary n-3-PUFA do not have a significant effect on plasma leptin and ghrelin concentrations, appetite, The authors declare that they have no competing interests.MK and DSW planned and designed the study, and were responsible for all aspects of the project including subject recruitment, data collection and management, laboratory analyses, statistical analyses and interpretation of the data, and writing of the first draft of the manuscript. HSC and CCM were responsible for all diet-related aspects of this study including recipe development, diet composition calculations, diet preparation, and weigh-back of returned foods. PYY was involved in blood sample collections and processing, and was responsible for laboratory measurements of diurnal leptin and fasting ghrelin plasma concentrations. All authors were involved in the interpretation of the study results, and the writing of the manuscript. All authors read and approved the final version of the manuscript."}
+{"text": "Metabolic rate is known to rise above basal levels after eating, especially following protein consumption. Yet, this postprandial rise in metabolism appears to vary among individuals. This study examined changes in energy expenditure in response to ingestion of a high protein, high fat (HPHF) meal versus an isocaloric high protein, low fat (HPLF) meal in underweight, normal weight, or overweight females (n = 21) aged 19\u201328 years.Energy expenditure, measured using indirect calorimetry, was assessed before and every 30 minutes for 3.5 hours following consumption of the meals on two separate occasions. Height and weight were measured using standard techniques. Body composition was measured using bioelectrical impedance analysis.Significant positive correlations were found between body mass index (BMI) and baseline metabolic rate (MR) , between body weight and baseline MR , between BMI and average total change in MR , and between body weight and average total change in MR . Metabolic rate  was significantly higher in the overweight group than the normal weight group, which was significantly higher than the underweight group across all times and treatments. However, when metabolic rate was expressed per kg fat free mass (ffm), no significant difference was found in postprandial energy expenditure between the overweight and normal groups. Changes in MR  from the baseline rate did not significantly differ in the underweight (n = 3) or in the overweight subjects (n = 5) following consumption of either meal at any time. Changes in MR  from baseline were significantly higher in normal weight subjects (n = 11) across all times following consumption of the HPHF meal versus the HPLF meal.There is no diet-induced thermogenic advantage between the HPHF and HPLF meals in overweight and underweight subjects. In contrast, in normal weight subjects, ingestion of a HPHF meal significantly increases MR  versus consumption of a HPLF meal and provides a short-term metabolic advantage. Obesity is a growing epidemic. Nearly two-thirds of American adults (65%) are overweight with one-third being obese (30%) and this number continues to climb . ObesityWhile the causes of obesity are multifactoral, weight gain ultimately results from an imbalance between energy intake and energy expenditure. The main components of energy expenditure are basal/resting needs and physical activity. Diet-induced thermogenesis (the rise in resting energy expenditure associated with food ingestion) is only a minor component representing on average about 10% of basal needs. Yet, the contribution of diet-induced thermogenesis can be much higher if high protein diets are consumed. Metabolic rate may increase up to about 30% after protein consumption [High protein diets, such as the Atkins\u2122 diet and Zone\u2122 diet are popular for weight loss in the United States. For example, a 2002 Consumer's Report survey found that over 30% of dieters reported the use of the Atkins\u2122 diet to help them lose weight . Yet, whWhether high protein diets offer a metabolic advantage through increased diet-induced thermogenesis over other diets that are lower in protein is not clear, since the thermogenic response to food is only one small component of energy expenditure and thus weight balance. Also unclear is whether there are differences in the thermogenic response to different nutrients in individuals of various body sizes. Should differences in responses exist among individuals, these differences over time could promote weight gain or loss.To date, most studies have examined differences in diet-induced thermogenesis between normal weight and obese individuals with some studies showing no differences and others finding a diminished response in obese adults -13. Few Subjects, recruited through posted flyers and oral announcements in nutrition and food science classes, consisted of 21 females aged 19\u201328 years. Subjects had to be healthy, free from nut and chocolate allergies, and have regular menstrual cycles to participate. The study was approved by the Auburn University Institutional Review Board for the Use of Human Subjects in Research.2), normal weight (18.5\u201324.9 kg/m2), or overweight (\u2265 25.0 kg/m2) based on Centers for Disease Control criteria [Subject participation required three meetings. At the first meeting, height and weight were measured using a stadiometer and calibrated beam scale . From the height and weight measurements, each subject's body mass index (BMI) was calculated, and each subject was classified as underweight  for 12 hours, refrained from caffeine use and exercise for at least 12 hours, and refrained from smoking the morning of their trial. Energy expenditure, assessed using indirect calorimetry , was measured at baseline between about 7 and 8 am after subjects sat quietly for 10 minutes. The term baseline rather than resting is being used since subjects drove to the testing site. Energy expenditure measurements took about 10 minutes/measurement and were conducted with subjects in a seated position in a darkened room with a room temperature of 72\u00b0F. Subjects were then given 15\u201320 minutes to consume a high protein , high fat  meal  or a high protein , low fat  meal . Bars were analyzed for macronutrient contents by proximate analysis . Subjects received the two different meals, each providing 440 kcal, in random order on two different occasions, and the researcher was blinded to what bars the subjects received on each of the two visits. Following consumption, energy expenditure was measured every 30 minutes for 3.5 hours. During the time period following meal consumption and in between energy expenditure measurements, subjects were allowed to read or study in a seated position, were allowed one bathroom break per hour, and were allowed to drink water as desired.Statistical analyses were conducted using InStat  and JMP IN  programs. The Restricted or Residual Maximum Likelihood (REML) method was used to determine the effects of BMI classification , meal type , time, and their interactions on energy expenditure. The effect of subject across time was also included in the model with random effects assigned to subjects. The potential effect of meal order was also determined. Differences between group means were determined by either Least Square Means Differences Student's t or orthogonal contrasts. Pearson correlations were used to assess the relationships between BMI and energy expenditure, and between body weight and energy expenditure.Twenty-one females  aged 19\u201328 years participated in the study. Two subjects, however, only completed one trial each. Four other subjects completed body composition measurements in the initial visit, but failed to return. The main reason cited for not completing the study was the lack of time to commit to the study. In addition, a few underweight subjects were not eligible to participate due to abnormal menstrual cycles and/or disordered eating.2. No significant differences were found among the three groups in age, height, or fat free mass, while body fat was significantly greater in the overweight group versus the normal weight and underweight groups. Most subjects (62%) completed the two meal trials within a week time frame; the remaining subjects (38%) completed the two meal trials within 1 to 2.5 months. No effect of meal order was found, and thus, it was not included in the model. The coefficient of variation in metabolic rate for subjects consuming the same diet on different occasions was 2.74%.Mean (\u00b1 SE) age, height, weight, BMI, fat free mass, and body fat for subjects are shown in Table There was a significant (p = 0.0003) effect of BMI classification on absolute  energy expenditure. This was true prior to (at baseline) and after consumption of the meals Figures  and 2. OThere was a significant (p < 0.0001) effect of time on absolute energy expenditure irrespective of BMI and treatment . Overall, energy expenditure significantly increased by 30 minutes after consumption of the meal. The increased level of energy expenditure remained until 180 minutes, when it began to decrease toward baseline. However, at 210 minutes post-consumption, energy expenditure was still significantly greater than baseline.Significant positive correlations were found between BMI and baseline metabolic rate , and between body weight and baseline metabolic rate . Significant positive correlations also were found between BMI and average total change in metabolic rate , and between body weight and average total change in metabolic rate .Table There was also a significant (p < 0.0001) effect of time when expressed as the change in energy expenditure. However, there was not a significant difference in the overall effect of meal type, when the data were expressed as the change in energy expenditure. There was a tendency for a BMI classification and meal type interaction (p = 0.084). While there was not an effect of meal type in the overweight or underweight groups, the change in energy expenditure was significantly (p = 0.0014) greater in the normal weight group following consumption of the high protein, high fat meal versus following consumption of the high protein, low fat meal. Similar significant (p = 0.0007) results were found when examining change in metabolic rate per kg ffm. The difference in the increase in metabolic rate after consumption of the high protein, high fat meal versus the high protein, low fat meal in the normal weight subjects represents about 69.3 kcal per 3.5 hours .High protein diets remain a popular choice for dieting among Americans. Yet, while high protein foods appeal to the taste buds of many individuals and can be part of a weight loss diet, high protein diets can vary tremendously in fat and carbohydrate contents and can be extremely unhealthy if excessively high in fat or extremely low in carbohydrate. These variations in the macronutrient composition of diets may influence diet-induced thermogenesis. Diet-induced thermogenesis also has been shown to differ among individuals of different body sizes. These thermogenic differences over time could lead to changes in energy balance and thus body weight. This study examined short-term changes in energy expenditure among overweight, normal weight, and underweight females after consumption of a high protein, high fat meal versus a high protein, low fat meal.The overweight group had a significantly higher resting absolute energy expenditure as compared to the normal weight and underweight groups. Several studies have examined differences in resting metabolic rate among individuals of different sizes. Similar to the present findings, James et al , HoffmanThe increases in metabolic rate (time effect) that occurred after overweight, normal weight, and underweight subjects consumed the high protein, high fat and the high protein, low fat meals are consistent with changes in metabolic rate associated with diet-induced thermogenesis. Postprandial metabolic rate was significantly different among the BMI groups across all times and treatments with the highest rate in the overweight group, followed by the normal weight, and the underweight groups; however, when expressed per kg ffm, no difference was found between the overweight and the normal weight group, suggesting the difference in metabolic rate between these two groups related to greater fat free mass in the overweight group. It should be noted that even though fat free mass was not statistically greater in the overweight group, it was numerically greater. Moreover, while increases in metabolic rate were exhibited among all groups, the rise was not significantly different from baseline in the underweight subjects. Only one study has examined postprandial changes in metabolic rate in healthy, underweight females. While differences between baseline and postprandial measurements were not published, Scalfi et al  reportedSeveral studies have examined postprandial metabolic rate in normal weight versus overweight/obese individuals. Results of such studies vary with a few reporting significantly higher metabolic rates in normal weight versus overweight/obese individuals ,25 and aNo significant difference in change in metabolic rate was observed when overweight or underweight subjects consumed the high protein, high fat meal versus the high protein, low fat meal. In contrast, changes in metabolic rate  were significantly higher in normal weight subjects across all times measured following consumption of the high protein, high fat meal versus the high protein, low fat meal. While several studies have examined postprandial metabolic rate in subjects of different sizes after consumption of two different meals, these studies did not examine thermogenic responses within a weight group between the two meals ,11,26,28Reasons for the differential thermogenic response in the present study between normal weight and overweight/obese individuals to meals are unclear. Some studies have suggested that the thermogenic response to carbohydrate intake is blunted in overweight/obese individuals ,29-31. IIn the present study, the greater thermogenic response in the normal weight individuals after consumption of the high protein, high fat meal (containing 37 grams of protein) versus the high protein, low fat meal (containing 30.8 grams of protein) likely resulted from the higher protein content of the high protein, high fat meal since the thermogenic response to fat and carbohydrate is thought to be about equal ,32. The Underweight individuals, like the overweight individuals, showed no significant difference in thermogenic response to the ingestion of the two meals. Only one study has previously reported a reduced thermogenic response to meals in underweight females but did not compare differences in response between two meals . The redChanges in metabolic rate in response to high protein, high fat versus high protein, low fat meals do not differ in overweight and in underweight females and thus, there is no metabolic advantage in diet-induced thermogenesis between the two meals. In contrast, in normal weight subjects, ingestion of a high protein, high fat meal significantly increases metabolic rate  versus consumption of a high protein, low fat meal and provides a short-term metabolic advantage. Whether the higher diet-induced thermogenesis would persist with continuous ingestion of a high protein, high fat diet in normal weight individuals and would lead to greater changes in body weight is not known and requires additional studies.The author(s) declare that they have no competing interests.AJR helped design the study, conducted the study, analyzed the data, and wrote the manuscript. BDW assisted with data analysis and figures in the manuscript. SSG contributed to the study design, data analysis, and writing of the manuscript. This research was financially supported in part by Malone-Zallen Research Scholar Fund and the Alabama Agricultural Experimental Station, Project 13-006. All authors read and approved the final manuscript."}
+{"text": "This study examined the capabilities of an ultraendurance athlete to self-regulate their diet during an attempt on the record for the longest period of stationary cycling. The attempt required the athlete to complete at least 20 km/hr, with a 15 minute break allowed every eight hours. Laboratory tests determined a heart rate-oxygen consumption regression equation enabling calculation of energy expenditure from heart rate during the attempt. Energy intake was determined by a non-weighed dietary record collected at the time of consumption. The athlete completed 46.7 hours, covering 1126 km, at a speed of 24 \u00b1 1.6 km/hr. He expended 14486 kcal and consumed 11098 kcal resulting in an energy deficit  and a weight loss (-0.55 kg). The carbohydrate (42 \u00b1 32 g/hr), water (422 \u00b1 441 ml/hr), and sodium (306 \u00b1 465 mg/hr) intake were all below current recommendations. The athlete was unable to self-regulate his diet or exercise intensity to prevent a negative energy balance. Ultraendurance athletes' are challenged in attempting to balance the high energy requirements of their sport with the high energy consumption required to compete successfully. When the energy intake exceeds 4500 kcal/d, the athlete will commonly have to ingest and digest food while actually competing . In ordeFew athletes have the discipline required to successfully self regulate their energy intake during ultraendurance exercise, but one previous casestudy occurred during the 1000 km running race from Sydney to Melbourne in Australia. Greek ultra marathoner, Yiannis Kouros', self regulated diet across the five days was almost entirely comprised of carbohydrate 95%). Energy dense Greek sweets and honey soaked biscuits were snacked on every 30 minutes, enabling Kouros to consume on average an amazing 11,074 kcal/d [%. EnergyThe purpose of this case study was to assess the capabilities of an experienced ultraendurance athlete to appropriately self regulate his energy intake during an attempt on the world record for the longest period of stationary cycling.The subject was a 35 year old male amateur cyclist  with previous experience in ultraendurance events. The subject gave informed consent to collect data during the record attempt.The subject completed an intense program of endurance training spanning 6 months. He was provided with guidelines about the amounts and types of foods/fluids to consume throughout the world record attempt with specific emphasis on carbohydrate, sodium and fluid amounts. The subject was then responsible for planning and preparing the appropriate items for the ride. He began the attempt with access to ample sports drinks, protein bars and energy gels (Endura\u2122).2) in order to estimate energy expenditure during the record attempt [2max), ventilatory threshold, and peak power.The athlete completed two cycle ergometer protocols a week prior to the record attempt. The first comprised four submaximal workloads, commencing at 0 W and increasing by 25 W every 2.5 minutes. This test was conducted to develop a relationship between heart rate and oxygen uptake (VO attempt . The sec2), minute ventilation (VE), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were calculated. Before testing, the gas analysers were calibrated with a known gas mixture and room air. Heart rate was measured by telemetry  and recorded every 15 seconds.All testing was undertaken on a cycle ergometer  with ventilation and expired gas collected and analysed every 15 seconds using a metabolic cart . Oxygen consumption . The distance covered and the speed maintained was calculated by a calibrated bike computer . Throughout the record attempt, the athlete's heart rate  and blood pressure was monitored, and the self regulated fluid and food intake recorded. Heart rate was recorded every minute and energy expenditure calculated from the equation obtained from the laboratory submaximal workload test. Blood pressure was recorded every hour. Body mass was measured every eight hours and urine output was weighed throughout. The total energy, carbohydrate, protein, fat and sodium consumed by the athlete was analysed using a dietary software program .Throughout the attempt the athlete was provided with verbal encouragement and physical assistance to continue eating and drinking and encouragement to continue cycling.2 max from the incremental test was 60.3 ml/kg/min and he achieved a peak power of 315 W. The relationship between heart rate and VO2 determined with the multiple submaximal workloads was represented by the following equation (VO2 (ml/min) = 26.826 * heart rate \u2013 1434.6, r2 = 0.9974). The energy expenditure measured in kcal was calculated by multiplying the estimated VO2 (in L/min) by 4.948. This conversion factor was calculated from the average non-protein respiratory quotient measured during the multiple submaximal workloads [The subject's VOThe athlete completed 46 hours 44 minutes and 20 seconds of continuous stationary cycling during the record attempt. The ambient temperature ranged from 16.9 to 27.9\u00b0C and the relative humidity from 44 to 65%. The total distance covered was 1126 km at an average speed of 24 \u00b1 1.6 km/hr and heart rate of 91 \u00b1 15 bpm , less than the 17430 kcal (72.9 MJ) reported during the 2004 XXAlps ultraendurance cycle race  or the 2Based on an estimated basal metabolic rate (BMR) for a 70 kg male of 1640 kcal/day , the firThe athlete averaged a fluid intake of 422 ml/h, nearly identical to the 417 ml/h recorded during a 24 hour road race in Britain , but subThe total volume of fluid consumed, plus the water contained within food, was 13 litres greater than the volume of urine produced. This positive balance did not result in an increase in unexplained body mass as the intensity of exercise maintained has been shown to produce sweat at a rate of 233 ml/hour , and insThe minimal sweat rate maintained throughout the attempt would also have enabled the athlete to reabsorb, within the sweat duct, the majority of the sodium secreted by the gland. In more humid and hot conditions, and in unacclimatised individuals, the increased risk of exercise associated hyponatremia  resultinThe current recommendations for CHO supplementation during ultraendurance exercise range from 30 \u2013 90 g/h ,27,29. TDespite being provided with appropriate guidelines, the athlete did not pre-plan or prepare his energy intake for the record attempt. He supplemented his intake of sports drinks, protein bars, and energy gels (Endura\u2122) with impulse selections from nearby commercial food outlets. These impulse selections included fried rice, McDonalds\u2122, and bacon and eggs. In any ultraendurance event, requiring the athlete to eat while competing, it would be strongly recommended that the athlete commence the event with all meals and fluids planned and prepared according to nutritional recommendations, likes and dislikes, and ability to store items to be consumed. Alternatives also need to be prepared for when food and flavour fatigue occurs, as is often the case during ultraendurance events . Poor planning and preparation, as well as other common factors such as gastrointestinal discomfort  and lossIt is unknown from the current data exactly why the athlete was unable to complete the record attempt. An energy deficit situation, and therefore loss of body mass, is common in ultraendurance events ,20,27,31The initial eight hour period particularly highlights the inadequacies of self-regulation with a lower than required total energy, and particularly CHO, intake, and an elevated energy expenditure. Overcoming such a large initial energy deficit, while the athlete is still competing, is difficult due to the limited digestive capacity of the gastrointestinal tract . The inaIBS conducted the laboratory testing, physiological data analysis and drafted the manuscript.KLS conducted the nutritional data analysis and contributed to the manuscript.All authors read and approved the final manuscript."}
+{"text": "The purpose of this study was to examine the acute metabolic effects of a high-energy drink in healthy, physically-active women.2) and heart rate (HR) were determined every 5 min during the first 30 min and every 10 min during the next 150 min. Blood pressure (BP) was determined every 15 min during the first 30 min and every 30 min thereafter. Area under the curve (AUC) analysis was computed for VO2, whereas a 3-hour average and hourly averages were calculated for respiratory quotient (RQ), total kcal, HR, BP, and profile of mood states (POMS).Ten women  underwent two testing sessions administered in a randomized and double-blind fashion. Subjects reported to the laboratory in a 3-hr post-absorptive state and were provided either 140 ml of the high-energy drink  or placebo (P). Subjects consumed two 70 ml doses of SUP or P, separated by 30 min and rested in a semi-recumbent position for 3 hours. Resting oxygen consumption  and third hours . A difference (p = 0.03) in energy expenditure was seen between SUP  and P  for the 3-hour period. Although no difference in energy expenditure was seen in the first hour, significant differences between SUP and P were observed in the second  and third hour . Average systolic BP was significantly higher (p = 0.007) for SUP (110.0 \u00b1 3.9 mmHg) compared to P (107.3 \u00b1 4.4 mmHg). No differences were seen in HR, diastolic BP, or POMS at any time point.AUC analysis revealed a 10.8% difference (p = 0.03) in VOResults showed a significant increase in energy expenditure in young, healthy women following an acute ingestion of a high-energy drink. The overweight and obese population in America has been steadily increasing for the past 20 years ,2. This Energy drinks have been shown, or have been suggested, to increase alertness, energy, and to counteract fatigue ,9. In adEnergy drink supplements often contain several herbal and botanical compounds that work synergistically to enhance energy expenditure, reduce body fat, and possibly enhance mood . CaffeinDespite the high frequency of energy drink consumption, limited studies are available to ascertain the efficacy of these drinks in young adult populations. In addition, with the vast amount of energy drinks that are available on the market containing varying ingredients it becomes difficult to extrapolate the results of a few studies on the benefits of all energy drinks. Thus, it becomes imperative to examine specific combinations of ingredients that are presently available in these drinks and determine the specific efficacy as it relates to the products contents. The purpose of this study is to examine the acute effects of a ready to drink (RTD) supplement marketed as Meltdown RTD\u2122 on resting oxygen consumption, respiratory quotient, caloric expenditure, heart rate, blood pressure and mood in healthy, physically active women.2) underwent two testing sessions administered in a randomized and double-blind fashion. Subjects were recruited from The College of New Jersey through announcements in the Health and Exercise Science Department. Following an explanation of all procedures, risks, and benefits associated with the experimental protocol, each subject gave her written consent prior to participating in this study. Subjects completed a medical history and physical activity questionnaire to determine eligibility. Subjects who were pregnant, smokers, or taking regular medication except birth control pills were excluded from the study. Subjects with any known metabolic or cardiovascular disease, or psychiatric disorder were also excluded. Subjects were also required to have been free of any nutritional supplements or ergogenic aids for 6 weeks preceding the study, and were asked to refrain from taking any additional supplement during the course of the study.Ten healthy, physically active women  or an equal amount of placebo (P) as suggested by the manufacturer's serving recommendation (two 70 ml servings separated by 30 minutes). On the subject's second visit to the laboratory they were provided with the opposite treatment. Measurement began immediately after the initial 70 ml consumption of SUP or P. A half hour following initial ingestion, measurement was paused as the subjects consumed the remaining 70 ml, and restarted immediately following consumption. Subjects remained resting in a semi-recumbent position during the entire measurement period. They were permitted to read, study, and/or listen to music following consumption and refrained from speaking during the study period. The total time of measurement was 3 hours. Oxygen consumption (VO2) and heart rate (HR) were determined every 5 min during the first 30 min and every 10 min during the next 150 min. Blood pressure (BP) was determined every 15 min during the first 30 min and every 30 min thereafter. The profile of mood states (POMS), and a questionnaire focusing on alertness, focus and fatigue were assessed every 30 min.The study followed a double-blind, crossover design. Subjects reported to the Human Performance Laboratory at the same time of day on two separate days. Each testing session was separated by an average of 7 days (7.0 \u00b1 2.8 d). Subjects were instructed to refrain from consuming any caffeine products on the day of each testing session and from performing any strenuous physical activity for the previous 12 hours. In addition, subjects were instructed to be at least 3 hours post-absorptive state prior to each trial. During each visit to the laboratory, upon completing a 30 min resting period, subjects were randomly provided either (140 ml) of the energy drink supplement (SUP), commercially marketed as Meltdown RTD2 and respiratory quotient (RQ) through open-circuit spirometry using a metabolic measurement cart . Machine calibration was performed prior to each session using a 3-liter syringe and calibration gases of known concentration of oxygen and carbon dioxide. Measures of VO2, RQ, energy expenditure, and fat oxidation rate were obtained continuously for the entire 3-hour trial beginning one minute following supplement or placebo consumption. The average for every 5 min was recorded for the first 30 min, and every 10 min thereafter until 180 min post consumption. HR was also measured at these time points using a wireless HR monitor , which updated HR display every 5 s. BP was measured using a sphygmomanometer and ausculatory method at 15 min and 30 min post ingestion, and then for every 30 min until data collection concluded.Immediately following supplement ingestion subjects were fitted with a Medgraphics preVent\u2122 pneumotach  to measure VOThe POMS was administered 7 times during each testing session. The initial POMS administration was given as the subject reported to the Human Performance Laboratory, and every half hour for the three hour period following supplement ingestion. All questionnaires were performed under controlled conditions  and the same researcher performed all test administrations.The POMS consists of 65 words or phrases in a Likert format questionnaire which provides measures of specific mood states. It provides measures of tension, depression, anger, vigor, fatigue and confusion. A total mood score is computed by subtracting vigor from the sum of the five other negative measures and adding 100 to avoid a negative result. McNair et al. , has repSubjects were also asked to complete an energy level questionnaire containing four questions using a 5-point rating scale. Subjects were asked to rate their energy level, fatigue level, feelings of alertness and feelings of focus for task using the following verbal anchors: 1 = very low; 2 = low; 3 = average; 4 = high; 5 = very high. The same researcher performed all test administrations and tests were conducted under controlled conditions (a quiet room).\u00ae or a placebo. Meltdown RTD\u00ae contains 230 mg of anhydrous caffeine, and the following herbal and botanical compounds; methyl tetradecylthioacetic acid, yerba mate extract, methyl-synephrine, methylphenylethylene, 11-hydroxy yohimbine, yohimbine HCL, alpha-yohimbine, and methyl-hordenine HCL. The placebo was similar in appearance and taste to Meltdown RTD\u00ae, but contained only an inert substance.On each visit subjects ingested either 140 ml of Meltdown RTD2 was calculated by using a standard trapezoidal technique. RQ, HR, BP and POMS were averaged over each hour and the entire 180-minute period. Statistical analysis of the data was accomplished using a repeated measures analysis of variance. In the event of a significant F- ratio, LSD post-hoc tests were used for pairwise comparisons. AUC analysis and 180-minute comparisons were analyzed using dependent T-tests. A criterion alpha level of p \u2264 0.05 was used to determine statistical significance. All data are reported as mean \u00b1 SD. A sample size of 10 subjects provided 88% statistical power at an \u03b1 level of 0.05 (two-tailed).The area under curve (AUC) for VO2 between SUP and P for the three hour study period. No significant differences in resting oxygen consumption were seen in the first hour following ingestion of the supplement. However, oxygen consumption was significantly elevated within the second hour  and third hour  following ingestion  and P  for the 3-hour period. Although energy expenditure was not significantly different between SUP and P in the first hour, significant differences between the groups were seen in the second hour  and third hour . No significant change from resting values of RQ were seen between either SUP (0.86 \u00b1 0.05) or P (0.88 \u00b1 0.06) in the 3-hour study period.The AUC analysis revealed a 10.8% difference (p = 0.03) in VOBlood pressure and heart rate responses to the study protocol are displayed in Table No significant differences were seen between SUP and P in any of the mood states measured during the study Figure . In addiResults of this study support previous research indicating that the high-energy supplement Meltdown\u2122 can enhance metabolic rate in healthy, college-aged students -20. PrioCitrus aurantium, and yohimbine [Previous studies have shown increases in resting energy expenditure over 2 to 3-hour time periods following caffeine ingestion in combination with additional ingredients including ephedra, black tea, green tea extract, ohimbine -23. The ohimbine ,23.The combination of caffeine and other herbal ingredients examined in this study resulted in a 10.8% increase in resting energy expenditure over the 3-hour period. The increase in energy expenditure for SUP was observed primarily during the second and third hour after consumption. Due to the manufacturer's serving recommendation, subjects consumed half (70 ml) of the supplement at baseline, and the remaining 70 ml after 30 minutes. It is likely that the thermogenic effect of the energy drink was delayed as a result of the ingestion pattern employed. These results also confirm previous findings by others that showed a delayed thermogenic effect from energy drink consumption for 30 - 60 minutes following ingestion, and then an ability to increase energy expenditure for the subsequent 120 minutes . It is pCitrus aurantium, has an adrenergic effect that may result in appetite suppression, increased metabolic rate, and lipolysis [The increase in the metabolic rate is consistent with other investigations examining caffeine and additional thermogenic agents -21,24-26ipolysis . Alpha aipolysis . Similaripolysis  reportedThe findings of this study do not support previous research from our laboratory that indicated that the combination of yohimbine, yerba mate extract and tetradecylthioacetic acid can enhance utilization of stored fat . Others The addition of phenylethylamine as an ingredient was thought to enhance the mood of subjects using this supplement. Prior research has shown that phenylethylamine can produce relief of depression among a clinical population, even in those that were unresponsive to standard treatments . The mecIn conclusion, the results of this study indicate that acute ingestion of Meltdown RTD\u2122 increases energy expenditure in young, healthy women. In addition, ingestion of this supplement increases systolic blood pressure for three hours following ingestion. However, BP values stayed will within the normal range. No changes in mood, subjective feelings of alertness, focus, or energy were seen. Future research should examine the potential role that chronic intake of this supplement has on fat loss and body composition changes.Vital Pharmaceuticals.  provided funding for this project. All researchers involved independently collected, analyzed, and interpreted the results from this study and have no financial interests concerning the outcome of this investigation. Publication of these findings should not be viewed as endorsement by the investigators, The College of New Jersey or the editorial board of the Lipids in Health and Disease.JRH obtained grant funds for project, designed study, supervised all study recruitment, data/specimen analysis, statistical analysis and manuscript preparation. SLR was the lead author, performed data collection and data analysis. JK, NAR, AC and ADF were co-authors, oversaw all aspects of study including recruitment, data/specimen analysis, and manuscript preparation. All authors have read and approved the final manuscript."}
+{"text": "Some studies in adults indicate a positive correlation between eating later in the day and overall energy intake as well as body weight status. Thus, the time of food intake may be a risk factor in childhood obesity. This study was designed to describe the proportion of energy consumed in the time from 4 pm to midnight measured in two-hour increments and to determine a potential association between the time of proportion of energy consumed and body weight status.Dietary, anthropometric, and socio-demographic data of 2\u201318 year olds  of the National Health and Nutrition Examination Survey (NHANES) 1999\u20132004 was examined to describe the proportion of total energy consumed within two-hour time periods between 4 pm and midnight. To examine the potential association between eating later in the day and body weight status, generalized estimating equations (GEE) models were used to quantify the effect of time trends  on body weight status. Analysis was conducted in the total sample and in subgroups stratified by sex, ethnic group  and age group . Complex sample survey analysis were used to assess differences at a significance level of p-value < 0.05.Proportion of energy consumed varied by sex, ethnic group, and age groups between 4 pm and 11.59 pm. Compared to healthy weight children, overweight school-age children consumed significantly higher while overweight adolescents consumed significantly lower proportions of total daily energy with each advancing two-hour time increment.The association between the circadian rhythm of eating and body weight status needs to be investigated further to examine the effect of time of consumption on the risk of childhood obesity. Especially longitudinal studies in diverse child populations would help elucidate the importance of time of eating on obesity. According to most recent prevalence estimates , adult oStudies on childhood obesity include the investigation of the relationship between non-modifiable characteristics such as age, ethnicity, and sex as well as modifiable factors such as diet quality and nutrient composition . The timAs a prospective study with seven-year follow up in healthy 8\u201312 years old girls showed, eating later in the day (defined as intake after 5 pm and before 6 am on the following day) was associated with a small but significant positive effect on body-mass-index (BMI) z-score . The finTo date, there is lack of data on a potential association between children's body weight status and the time of the day when energy-contributing foods are consumed. The examination of this relationship is complicated by known variations of energy intake, such as the fact that males consume larger total amounts of energy compared to females or that total daily energy intakes increase, as children get older. To accommodate these differences, the exploration of the association between the time of energy consumption and body weight status must be examined by sex and age groups.In an effort to contribute to childhood obesity prevention efforts, the present study focused on a nationally representative sample of the U.S. pediatric population to examine the association between the proportion of total energy consumed during two-hour time periods in the afternoon and evening hours and childrens' body weight status. Regression models were fitted to the data stratified by sex and age to explore whether consuming a greater proportion of total energy intake later in the day was associated with an increased likelihood of being classified as overweight or obese. The hypothesis tested was that eating a higher proportion of total energy later in the day was associated with being classified as overweight or obese. Due to the ethnic diversity of the sample, we expected to find some energy intake reports for times not traditionally considered meal or snack times for children, such as late-night dinners in the Hispanic subpopulation.), an ongoing survey using a multistage, stratified area design to obtain a sample of respondents that is representative of the civilian non-institutionalized U.S. population. Certain groups are over-sampled . Although the data are released in 2-year increments, they were designed to be merged to multi-year data sets [The Centers for Disease Control and Prevention (CDC) conducts the National Health and Nutrition Examination Survey , or not Hispanic. In an effort to capture the cultural differences in food intake behaviors of children living in the U.S., the race and ethnic background variables were used to define five mutual exclusive ethnic groups: Non-Hispanic white, Non-Hispanic black, Mexican American, Other Hispanic, and Other Race Including Multi-Racial.NHANES dietary data were collected with interviewer administered 24-hour recalls. In the survey waves of NHANES 1999\u20132000 and 2001\u20132002, dietary intake data was collected for one single day. In the NHANES 2003\u20132004, two days of 24-hour recalls were collected but only the first day was considered for this study. Data from all three NHANES waves were combined and used in the analysis.To collect 24-hour dietary recall data, respondents were asked to report all consumption of foods and beverages during the past 24 hours using a multiple-pass approach methodology . Intake A standardized protocol of dietary intake data collection ensures the high quality and consistency across dietary interviewers in the NHANES data . For eacFor this study, dietary intake records were used to calculate childrens' total energy intake  as well as the proportion of total energy consumed within two-hour time increments . To focus on the hypothesis of this study that eating higher proportions of total energy later in the day is associated with being classified as overweight or obese, the analysis was limited to the latter hours of the day (between 4 pm and 11.59 pm).th percentile), healthy weight , and obese (\u2265 95th percentile) [Measured height and weight as well as calculated body mass index  are available in the NHANES data set. Weight (kilograms) was obtained using a digital scale; standing height was measured with an electronic stadiometer and recorded in meters. CDC's BMI-for-age and sex-specific growth charts were used to create four distinct groups: underweight (less than 5centile) .As expected, the anthropometric data was not normally distributed, thus, the method by Cole et al. to construct a smoothed curve using the calculated power (L), mean (M), and coefficient of variation (S) to provided standards in terms of centiles was employed to determine children's normalized growth centile standards . UnderweAll analysis was conducted using complex sample survey routines . A four-level discreet time trend variable was created to indicate dietary energy intakes at time point zero (4 pm to 5.59 pm), one (6 pm to 7.59 pm), two (8 pm to 9.59 pm), and three (10 pm to 11.59 pm). GEE models included covariates for sex, body weight categories, ethnic group category, and age group. The Institutional Review Board (IRB) in the Office of Research Protection (ORP) at Pennsylvania State University granted approval for this study based on the use of secondary data with no person identifiers.The study included 11,072 children two through 18 years old who provided a total of 52,506 food records. Approximately 50% of the sample was boys and 60% were non-Hispanic white . Student t-test indicated that compared to their healthy weight peers, average energy consumption in overweight children was 23.0 kcal and in obese children 27.4 kcal higher between 4 pm and 8 pm . No statistical difference was found in the amount of energy consumed between 8 pm and midnight among the three body weight groups.Visual inspection of the consumption patterns does not indicate statistical significance between the patterns of energy intake in the three age groups but serves purely as illustration of the differences in energy intake habits in the latter part of the day.As the line plots of Figure To determine statistical significance of the association between body weight status and the time of energy consumption GEE fixed effects models were fitted to the data in the total sample as well as grouped by sex, age, and ethnic group. The model fitted for the total sample included the sex (dichotomous) and age (continuous) variables as well as the 4-level dummy variables for linear time trend and the variable for children's body weight status . Ethnic group was not a significant contributor to the model in the full sample. The stratified models for the sex, age, and ethnic groups only included the time trend and body weight status variables. Table The tests of the regression coefficients from the GEE model in the total population indicated that there was a significant negative linear trend in energy consumption in the afternoon and evening hours  but this decline of proportion of energy consumed with advancing time was not significantly associated with body weight status. As expected, increasing age was associated with higher energy intake  and females consumed significantly less energy  than their male counterparts .When examining the GEE models grouped by sex, increasing age was significantly associated with higher energy intakes . A statistically significant negative time trend effect on energy intake was observed in boys  but there was no association with body weight status.The GEE model fitting the data of each of the five ethnic subsamples indicated significant lower energy intakes in girls than in boys  in all ethnic groups but the \"other Hispanic\" but higher energy intakes with increasing age . In the models of the Non-Hispanic white and Non-Hispanic black children, a significant negative time trend of energy intake was observed .The GEE model fitted in each one of the three age groups showed that 6\u201311 year old and 12\u201318 year old girls consumed significantly less energy than boys but increasing age was associated with higher energy intakes in all three age groups . In children ages 6\u201311 and 12\u201318 years old statistically negative associations between energy intake and time trends were found . The association between body weight status and energy intake was positive in the 6\u201311 year old overweight children, who consumed 17 kilocalories more than their healthy weight counterparts  per two-hour time period while 12\u201318 year old overweight children consumed 16 kilocalories less per two hour time period than their healthy weight peers . Examination of the beta-coefficient and p-values of each category in the time trend variable indicated differences in the proportion of total energy intake consumed between time period zero and one, (4 pm \u2013 5.59 pm compared to 6 pm \u2013 7.59 pm). There were no significant changes in the proportion of energy consumed after 8 pm in either age group.Some studies conducted in adults indicated that consuming larger amounts of food in the evening hours was associated with higher body weight. In particular, researchers found that consumption of a late dinner was associated with larger overall energy intake throughout the day; conversely, individuals with larger energy intake in the morning hours tended to have smaller total energy intake . TherefoIn an effort to improve the understanding of a potential relationship between the time of energy consumption and the current childhood obesity epidemic in the U.S., we examined the association of the relative amount of childrens' total daily energy intake consumed in the latter part of the day and their body weight status in a nationally representative sample of 2\u201318 year old children. As the results of this study indicated, total energy intake varied by age and sex of the children. The proportion of total daily energy consumed in the latter part of the day was associated positively with being classified as overweight in the 6\u201311 year olds but negatively in the overweight 12\u201318 year olds. No significant differences were found between the proportion of energy consumed between 8 pm and midnight and body weight status. This lack of association however, may have been due to the limited number of individuals consuming food so late in the day , which may have affected the statistical power to detect significant differences in the proportion of energy consumed between the time periods.As reported previously, healthy weight children consume larger amounts of energy during breakfast whereas overweight and especially obese children eat less during that meal ,12. Our Our study indicates a significant association between body weight status and meal or snack consumption in the latter part of the day only in school-age children and adolescents. Surprisingly, children ages 6\u201311 years old who were classified as overweight consumed larger proportions of total energy in the later in the day whereas overweight adolescents consumed less energy in the same time period. This apparent paradox is not easily explained. The positive association between food consumption later in the day and higher body weight status in the 6\u201311 year olds is more intuitive and confirm results from studies on the Night Eating Syndrome (NES), which indicate that adults who consume food during the night have higher body weights than individuals who do not eat during night time . One of A recent study investigating the characteristics of individuals with NES elucidated that the increased risk for higher body weight status may not be attributable to the NES but to the underlying psychological factors leading to NES . For insResults of this study are limited by the use of only one-day dietary intake estimates rather than multiple-day food records to estimate average energy intake. However, since the focus of this paper was to examine dietary intake patterns and report on the association between the time of energy consumption and body weight status in the U.S. pediatric population, the use of a single 24-hour recall was appropriate. In addition, the dietary intake data of the sample is limited by the use of a proxy for the diets of young children, which were reported by their caretakers due to the cognitive limitations in individuals less than six years old. Although this procedure to obtain dietary intake data on young children is widely accepted as the most appropriate method, it is limited by the potential for under- or over-reporting of intakes based on the caretakers' characteristics and the likelihood that the caretaker did not personally observe all food intake occasions of the young children.This study was designed to increase the understanding of the potential importance of the circadian rhythm of food intake on childhood obesity. Heretofore, no data was available on the effect of the time of energy consumption on childhood obesity in sex, age, and ethnic subgroups of the U.S. pediatric population. Although this study was of purely observational nature, results aid in the generation of hypothesis for future studies on childhood obesity treatment and prevention efforts.Increasing age, sex, and belonging to certain ethnic groups affects food consumption patterns in children 2\u201318 years old. Results of this study indicate a need for further in-depth exploration of the effect of the proportion of total energy consumed during the day and especially in the afternoon and evening hours on childhood obesity development. Longitudinal studies using large samples of children from various ethnic backgrounds and age groups would contribute substantially to the body of knowledge on the potentially important effects of the time of food consumption on chronic disease development.The authors declare that they have no competing interests.SK conceived of this study and led the analysis and manuscript preparation. DAW contributed to the manuscript by programming the statistical analyses and provided data analytic advice to SK. SE, an undergraduate student of the Schreyer's Honors College participated in the interpretation of results and preparation of the manuscript in fulfillment of the requirement for her honor's thesis."}
+{"text": "Until now no technology has been available to study energy metabolism in monkeys. The objective of this study was to determine daily energy expenditures (EE) and respiratory quotients (RQ) in female monkeys of various body weights and ages.2 and 11.7 \u00b1 4.6 years] were placed in the primate Enhanced Metabolic Testing Activity Chamber  for 22-hour measurements of EE  and RQ (VCO2/VO2). All were fed monkey chow  ad-libitum under a 12/12 hour light/dark cycle. Metabolic data were corrected for differences in body weight. Results were divided into day (8-hours), dark (12 hours) and morning (2-hours) periods. Data analysis was conducted utilizing SPSS (Version 13).16 socially reared Bonnet Macaque female monkeys [5.5 \u00b1 1.4 kg body weight, modified BMI (length measurement from head to base of the tail) = 28.8 \u00b1 6.7 kg/crown-rump length, mModified BMI negatively correlated with 22-hour energy expenditure in all monkeys . The large variability of daily energy intake  necessitated division into two groups, non-eaters  and eaters . There were reductions (p < 0.05) in both 22-hour and dark period RQs in the \"non-eaters\" in comparison to those who were \"eaters\". Monkeys were also classified as \"lean\" (modified BMI < 25) or \"obese\" (modified BMI > 30). The obese group had lower EE (p < 0.05) during each time period and over the entire 22-hours (p < 0.05), in comparison to their lean counterparts.The EMTAC proved to be a valuable tool for metabolic measurements in monkeys. The accuracy and sensitivity of the instrument allowed detection of subtle metabolic changes in relation to energy intake. Moreover, there is an association between a reduction of energy expenditure and a gain in body weight. The Enhanced Metabolic Testing Activity Chamber (EMTAC) was developed for accurate and reliable measurements of energy expenditure and respiratory quotients in humans -3 and roThere have been a few attempts to utilize indirect calorimetry in non-human primates to study changes of energy expenditure in relation to energy intake. In one study, the energetic cost of bipedal versus quadrupedal walking in Japanese macaques was determined by carbon dioxide measurements while exercising on a treadmill within a respiratory chamber . In anotNon-human primates are especially relevant for the study of metabolism in regards to many metabolic disorders in humans due to their genetic and physiological similarities ,12. The M. radiata) (5.5 \u00b1 1.4 kg and 11.7 \u00b1 4.6 years of age) socially reared in the Primate Behavioral Laboratory of the State University of New York, Downstate, Brooklyn NY. Their modified BMI  ranged from 0.25 to 0.59, with an average of 0.40 \u00b1 0.11 m2. Following weaning, all monkeys were maintained on a commercial laboratory diet  containing 4.02 kcal/g and comprising of 69% carbohydrate, 18% protein and 13% fat. Normal energy intake of healthy female Bonnet Macaque monkeys in this colony at the time of this study was 40\u201350 kcal/kg/day depending on their body weight, age and housing situation. Their diet was occasionally supplemented with fresh fruit and/or vegetables provided once in the mid afternoon. The monkeys used in this study were housed under three different conditions. Six of them had been housed in a large group pen with three to five other female monkeys, while eight were housed in a smaller group pen along with two other female monkeys. Finally, two had been housed alone in regulation monkey cages designed for single occupancy; however these cages resided next to each other in a large room.The monkeys tested were 16 adult female Bonnet macaques . The instrument was considered accurate and precise since the respiratory quotient of the propane being combusted was maintained between 0.58 and 0.60 (0.58 \u00b1 0.08) during the entire 22-hour test . FurtherPrior to each metabolic measurement the EMTAC was calibrated with standard gases with known certified concentrations of oxygen and carbon dioxide . The results from the EMTAC had to be within one percent of the concentration of oxygen and carbon dioxide of the certified calibration gases. This is an acceptable accuracy for metabolic tests using indirect calorimetry . After a2/VO2) were continuously calculated during metabolic testing from the oxygen and carbon dioxide concentrations within the enclosure according to the method of Jequier [Metabolic measurements were performed for 22-hours from 11:00 AM till 9:00 AM the following day. Two hours were allowed for cleaning of the enclosure and instrument calibrations. Energy expenditure  and the respiratory quotient  were also calculated over the course of each time period .To correct the continuous metabolic data for differences in body weight, each five minute data summary period  was divided by kg body weight and expressed as kcal/min/kg. Next, the corrected continuous metabolic data for each female monkey were than divided into three separate periods consisting of eight, 12 and two hours each, respectively. The light period ranged from the time the metabolic test started (11:00 AM) till when the lights went off at 7:00 PM. The dark period started from the time the lights went off till they were turned back on at 7:00 AM the next day. The final two hours of the 22-hour metabolic test (7:00\u20139:00 AM) was defined as the morning period. The sum of the energy expenditure of all three time periods equals the 22-hour energy expenditure. The criteria used for the division of the metabolic data into the specific time periods are similar to that used for metabolic studies in infants . MoreoveThe relationships between modified BMI and 22-hour energy expenditure were determined by Partial Pearson Correlations, controlling for the effects of increasing age.2), respectively. This classification scheme was based on the human BMI criteria for obesity [Two different analyses of the data were performed. First, the female monkeys were classified on the bases of their energy intake. Those who consumed less than 13, or greater than 23 kcal/kg/day were considered as \"non-eaters\" and \"eaters\", respectively. These limits were chosen since mean energy intake in the \"eaters\" group was similar  to that found for female Bonnet Macaque monkeys within the colony. The energy intake for the \"non-eaters\" was significantly less than that found for similar monkeys within the colony. Therefore, we utilized these differences as the basis for our sub-groups of \"non-eaters\" and \"eaters\". Differences between the two groups in regards to anthropometrics and metabolic parameters were determined by Independent t-test. Secondly, the continuous metabolic data for the monkeys were then classified as either \"lean\" or \"obese\" based on their modified BMIs being < 25 or > 30  and expressed as Mean \u00b1 Standard Deviation unless otherwise noted.Figure The anthropometric and metabolic data, corrected for differences in body weight, for all 16 monkeys are shown in Table 2) (Table 2/VO2) were significantly lower (p < 0.05) in the \"non-eaters\" in comparison to those in the \"eaters\" group , with a range from 4.48 to 101.9 kcal/kg/day, the monkeys were first classified as either \"non-eaters\" or \"eaters\", based on their energy intake. No differences were observed between the \"non-eaters\" and \"eaters\" in regards to age (years), body weight (kg) or modified BMI  by 18.6 and 21.8%, respectively, in both the \"lean\" and \"obese\" groups in comparison to energy expended during the day period. However, energy expenditure increased 18.6 and 16.0%, respectively, during the morning period, in comparison to the dark period, in both the \"lean\" and \"obese\" groups  might have affected their energy intake while in the EMTAC. Five of the six female monkeys in the \"non-eaters\" group were normally housed in a small pen with two to three others while one was housed in a large pen with a greater number of fellow monkeys. Since all of the female monkeys were tested one at a time in a primate metabolic cage within the EMTAC, it is possible that being alone, after normally being housed with other monkeys, might have affected their energy intake. This change in food intake in regards to companionship has been found in other animals and humans. For example, adult male rats ate less when fed alone versus being paired with another rodent while feeding . MoreoveIn this study the modified BMI  is the cSince female monkeys were studied, it is possible that their menstrual cycle might have masked some of the metabolic changes associated with body weight gain. In a previous study in woman of childbearing age , there w2H2) in body fat [There have been attempts to utilize the doubly-labeled water technique to obtain accurate daily energy expenditure measurements in socially reared non-human primates. For example, the doubly labeled water technique was utilized in the study of the effects of dietary restriction on energy expenditure in Rhesus monkeys . Some ofbody fat  and chanbody fat . Moreovebody fat . Our newbody fat , our sysbody fat  that accbody fat ,15. ThisWe have shown that the new primate EMTAC is a sensitive and reliable instrument for metabolic measurements. This instrument might be suitable for future studies of obesity, malnutrition or any other physiological disorder that affects energy metabolism in non-human primates. Being able to accurately determine both sides of the energy balance equation (intake and expenditure) under well controlled conditions will lead to new information as to the specific metabolic changes associated with various physiological disorders, such as obesity.2: Volume of carbon dioxide; VO2: Volume of oxygen; Kg: Kilograms; AIDS: Acquired immune deficiency syndrome; m2: Square meters; C: Centigrade; STPD: Standard temperature pressure dry; Min: Minutes; 2H2: deuterium.ANOVA: Analysis of Variance; SD: Standard deviation; EE: Energy expenditure; RQ: Respiratory quotient; BMI: Body mass index; EMTAC: Enhanced metabolic testing activity chamber; VCOThe authors declare that they have no competing interests.RR has contributed to the design of the experiment and conducted the data analysis. Furthermore, he either participated in some of the actual data acquisition or supervised pediatric research fellows in this regard. He also assisted in the preparation of the small grants necessary for funding of this project. Finally, he also assisted in the writing and editing of this manuscript. FL contributed to the preparation of the manuscript and assisted with data analysis. He also edited some of the grant proposals necessary for the financial support of this study.MS, LR and JGK were collaborators on this project and provided the expertise in regards to designing and conducting the study utilizing non-human primates. Moreover, they reviewed the data analysis and assisted with the preparation of this manuscript. All authors were involved in the final editing of this manuscript."}
+{"text": "To investigate the effect of commercial weight loss programmes on macronutrient composition and micronutrient adequacy over a 2 month period.Adults were randomly allocated to follow the Slim Fast Plan, Weight Watchers Pure Points Programme, Dr Atkins' New Diet Revolution, or Rosemary Conley's \"Eat Yourself Slim\" Diet & Fitness Plan.A multi-centre randomised controlled trial.293 adults, mean age 40.3 years and a mean BMI 31.7 (range 27\u201338) were allocated to follow one of the four diets or control group. Subjects completed a 7-day food and activity diary at baseline (prior to randomisation) and after 2 months. Diet records were analysed for nutrient composition using WinDiets (research version).A significant shift in the macronutrient composition of the diet with concurrent alteration of the micronutrient profile was apparent with all diets. There was no evidence to suggest micronutrient deficiency in subjects on any of the dietary regimens. However, those sub-groups with higher needs for specific micronutrients, such as folate, iron or calcium may benefit from tailored dietary advice.The diets tested all resulted in considerable macronutrient change and resulted in an energy deficit indicating dietary compliance. Health professionals and those working in community and public health should be reassured of the nutritional adequacy of the diets tested.NCT00327821 The slimming industry is a thriving and lucrative business in many developed countries throughout the world. In the UK, at any one time, it is estimated that almost two in five women and one in six men are on some kind of reducing diet. The continued rise in obesity supported by the obesogenic environment, combined with a desired society image of a 'slim body' being ideal  suggestsAn excellent example of the impact and resulting confusion that a diet can cause among both the lay public, academics and health professionals is the internationally popular low carbohydrate (CHO) Atkins diet . The potThe scale of the obesity problem and the limitations within health systems to provide weight management advice, mean that the vast majority of people trying to lose weight are likely to do so on their own initiative and use whatever sources of information they have to hand. Thus evaluation of the effectiveness and impact of popular diets is important and has not to date been rigorously investigated. There is very little information on the effect that commercial diets have on the food choices of people undertaking these regimens in an unsupervised free-living population. In particular, there may be implications for the micronutrient sufficiency for adults following energy restricted diets, especially for those following low carbohydrate approaches.'Diet Trials', a large UK multi-site randomised controlled study was designed to compare the relative efficacy of four commercial weight loss programmes on weight and body fat loss and the primary outcomes of this study have recently been published . The dieIn addition to investigating the effectiveness in achieving weight loss, this study provided an evaluation of the degree to which subjects following commercial weight loss programmes with no additional assistance and self-selecting foods were able to make dietary choices that were consistent with the advice provided by the commercial organisations. Another novel aspect of this study was to investigate if any of these programmes compromised micronutrient intakes during a two month period of energy reduction. The nutritional composition of a low CHO diet was compared to the low fat diets (WW and RC). This aspect of the study addresses a shortfall in the literature and will provide health professionals and other bodies with evidence about the nutritional adequacy of popular diets.The aim of this paper is thus to report the dietary macro- and micronutrient changes that occurred in the first two months of dieting in a group of overweight adults taking part in 'Diet Trials'.Ethical approval for the study was obtained from the South East Multi-centre Research Ethics Committee.2, were not actively dieting and lived within a 30 mile radius of their corresponding test centre. Eligible volunteers were required to obtain consent from their General Practitioner to take part in the study and any volunteer was excluded if they had any of the following: prior history of coronary heart disease, known type 1 or 2 diabetes, liver or respiratory failure, gout, taking lipid lowering or anti-hypertensive drugs, history of obesity with known cause , previous gastric or weight-loss surgery, taking any weight loss drug (including Orlistat or Sibutramine), clinical depression, eating disorders, drug or alcohol abuse, any malabsorptive state (including lactose intolerance), treatment for a malignancy, pregnancy or breastfeeding.Three hundred subjects were recruited via media advertising across the UK. Subjects were assigned to attend one of the five regional centres (60 at each centre), situated at the Universities of Surrey, Bristol, Nottingham, Ulster (Coleraine) and Queen Margaret University College, Edinburgh. Standardised assessment instruments and protocols were distributed by the lead centre prior to the study commencing. Participants were considered eligible if they fulfilled the following criteria: aged 18\u201365 years, had a BMI > 27 and < 40 kgmScreening procedures resulted in 293  subjects being screened as eligible for the study. Due to the disproportionate number of female volunteers (70%), subjects were stratified by gender and then randomised across each site to the four diets  and a delayed treatment control group (n = 61). In this way, subjects attending the group based programmes were not located together and furthermore regional food intake differences would be accounted for by a geographically representative spread of participants around the UK. All centres started the study within a 6 week period thus enabling seasonal variation in food availability to be minimised.At baseline (prior to randomisation), and at two months, subjects were asked to complete a 7-day food (with estimated weights) and concurrent with a 7-day activity diary . The actIn addition to the nutrient analysis, the number of fruit (including fruit juice) and vegetable portions were counted assuming a standard portion size of 80 g. Totals per week were then divided by 7 to provide an average daily intake of fruit and vegetables.Activity diaries were coded into minutes per day of time spent in sleeping, light, moderate or vigorous activity. Minutes in each category per day were multiplied by a metabolic equivalent (MET) value to give a total daily MET value using the following values obtained from the Compendium of Physical Activity ,15: sleetmTEE) and 17.7% for measurement errors in predicted TEE (CVwpTEE). The method can be mathematically described as follows:The procedure for identifying mis-reporters of energy intake at baseline followed the method of McCrory et al.  where thwEI = within subject coefficient of variation in energy intakeCVd = number of days of food recordsUsing these equations physiologically implausible energy intake reporters at baseline were identified by excluding rEI +/- 23% (i.e 1 SD). The records not excluded then formed a 'plausible reporters' sub-group which was defined by those who had reported energy accurately at baseline making the recognised assumption that they would continue to record food intake accurately at 2 months . The plaSubjects randomised to the group based programmes (RC and WW) attended their most locally based group due to the wide geographical spread of participants. The cost of attending the group classes were refunded on provision of a receipt. Subjects randomised to the Atkins diet were provided with a copy of the book but no further advice was given . SubjectSummary statistics are presented as mean and standard error. Differences between groups at baseline for continuous outcomes were compared using Student's t-tests, except where the Shapiro-Wilk test indicated non-normal distribution of data, when the non-parametric Mann Whitney U test was used. For categorical variables, the chi-square test was used to investigate between group differences. For normally distributed data where homogeneity of variance was confirmed, ANOVA was used to explore between group differences and where the overall result provided evidence of significant group differences, post hoc comparisons were conducted. For the data that was not normally distributed the non-parametric Kruskal-Wallis test was used. Changes over time for each diet group (baseline to two months) were explored using generalizing estimating equations (GEEs) with an identity link function and an exchangeable correlation structure, thus we were able to adjust for the correlation between repeated measurements on the same participant. Robust variance estimate techniques were used to calculate standard errors and confidence intervals. All p-values were two-sided. To account for multiple comparisons a p-value of less than 0.01 was considered statistically significant. Data were analyzed using Stata version 10.0 .After randomisation to diet group, the average age of subjects participating in the study was 40.3 years , mean BMI was 31.7 kg/m2 , mean waist circumference was 101.4 cm . There were no diet group or centre differences in these baseline characteristics. Smoking was reported in 17% of women and 10% of men.The overall attrition rate after two months was 18% (n = 53), with no significant difference between centres. The main attrition occurred immediately after randomisation primarily in the control group with 23% withdrawing because they did not wish to delay a weight loss attempt  returned 7-day completed diet and activity diaries at baseline. After two months, 172/234 participants returned diaries (74%) with no significant difference in the return of diaries by group or centre.Reported energy intakes are presented in Table At baseline the average % of energy from macronutrients comprised of 42% carbohydrate, 37% fat, 16% protein, 5% alcohol for the whole cohort. In terms of fat content, the average baseline diet contained 30.8 g saturated fat, 14.8 g polyunsaturated and 28.4 g monounsaturated fat. All micronutrient intakes exceeded their respective RNI values with the exception of potassium where the average intake was 95% RNI. There were no significant differences between the baseline diets in terms of macro, micro-nutrient or % energy derived from alcohol. Therefore, all data presented are changes for each diet group from baseline measurement.4,72 = 3.85, p = 0.007), with significant differences between control and all active diet groups. These falls in energy intake are substantiated by the physiological measures of weight loss. Although changes in body weight are fully reported elsewhere [Alteration of energy intake across all diet groups was apparent over the study period with significant falls in total energy intake and energy intake/kg/body weight recorded Table . This walsewhere , in summThe patterns of change in macronutrient composition were in line with the expected changes if subjects were choosing foods consistent with the recommendations of each dietary regimen indicating dietary compliance and was completed using all available data. See Figure In the Atkins dieters, there were highly significant shifts (p < 0.001) in the percentage (%) energy derived from all macronutrients from baseline to 2 months, as follows: 11% increase in protein energy ; 10% increase in energy from fat ; 29% fall in CHO energy  and a 3% fall in energy derived from alcohol . Overall, there was a fall of 30% in mean total energy intake .The RC diet recommends participants chose foods with less than a 4% fat content. Significant dietary shifts did occur with % energy from fat falling on average by 11% . In contrast, protein energy increased by 4% , CHO rose by 8% . There was no significant change in % energy derived from alcohol. Overall there was a fall of 37% in mean daily energy intake .The WW diet recommends a low fat intake, and this was reflected in a significant 7% reduction in dietary fat  between baseline and two months. CHO energy rose significantly by 4%  to provide 47% energy; dietary protein followed a similar pattern with a significant increase from baseline to 2 months . There were no significant changes in % energy derived from alcohol. Overall there was a fall of 38% in mean daily energy intake .The use of meal replacements resulted in the SF group having a significantly lower fat (% energy) diet at 2 months , with corresponding significant increases in CHO energy by 7%  and in protein energy which provided 19% of total energy intake at 2 months . Alcohol consumption also declined from baseline to two months by 2% energy . Overall there was a mean fall of 37% in daily energy intake .In the control group, there was a non-significant (10%) fall in reported energy intake from baseline to two months (p = 0.06), with no significant alterations in % energy derived from fat, protein, CHO or alcohol.Of interest in this analysis, is the effect of the different dietary programmes on the type and amount of fat intake and in particular differences between the low CHO and low fat diets (WW and RC). Change in dietary fat intake was calculated from baseline to 8 weeks in terms of absolute intake (g/day), see Figure Micronutrient intakes reported as a proportion of recommended daily intakes (RNI) are summarised in Table There were significant falls in %RNI for folate, magnesium, calcium, iron and potassium and a significant increase in selenium.There were significant declines in %RNI for riboflavin, niacin, potassium, calcium, magnesium, iron and zinc.There was a significant decline for niacin and a rise in %RNI for zinc after 2 months.There were significant decline in %RNI for magnesium, potassium and zinc.There were no significant alterations in micronutrient %RNI in the control group.Iron is the major micronutrient at risk of deficiency in the UK diet today. An examination of iron intake for those undertaking weight reducing diets is worthwhile as it is often this mineral that is lacking in the diets of women of child-bearing age. In this study, there were significant differences in iron intake between males and females at baseline. At baseline, men had an absolute iron intake  that was significantly higher (p < 0.001) than women (median 11.9 mg range 5.6 \u2013 23 mg/day). Changes in intake of iron (mg/day) from baseline to two months demonstrated a significant effect of diet group for women only .Intake of dietary fibre was on average 17.7 g/day (sem 0.39); with no diet group differences but gender differences were apparent with men consuming on average 3 g/day more dietary fibre than women . Baseline intake of non-starch polysaccharide (NSP) was below the recommended intake of 18 g/day .The median number of fruit and vegetable portions consumed at baseline was 17 portions (IQR 16.25) per week (2.4 portions per day). Only 12% of the entire cohort achieved the UK recommended intake of \u2265 5 fruit and vegetables portions per day at baseline. There were no between diet group differences in portions of fruit and vegetables eaten at baseline.Only the WW diet led to a significant increase  of fruit and vegetables and this amounted to less than one portion per day (0.79 portions per day). There was a trend towards an increase in the RC group ; there were no significant shifts for the other diets tested. Although portions of fruit and vegetables eaten on the Atkins diet did not alter over time, there was a significant reduction in NSP intake in the Atkins dieters. A repeated measures ANOVA of NSP intake from baseline to 2 months of all diet groups showed a significant effect of diet group  and post hoc testing demonstrated a significant reduction in NSP (12.8 g at baseline to 5.1 g at 2 months) for the Atkins group compared to all the other diets.The analyses presented in this paper demonstrate that free living subjects are able to make significant dietary change in line with instructions provided by commercial companies regardless of whether this information is given at group classes or as written instructions. Substantial alteration in participants macro-nutrient composition were recorded and these are supported by the recorded weight losses obtained during the 'Diet Trials' study; indicating compliance with the allocated dietary regimen .In the low CHO Atkins diet, we have shown that subjects were able to reduce their CHO intake substantially without the need for individualised dietary counselling. Furthermore, the energy deficit induced can be attributed to a reduction in overall energy intake with non-replacement of CHO energy and no substantial increase, in absolute terms, of dietary fat. The low fat diets, WW and RC both led to reductions in saturated fat intake, as both proportions of energy and in absolute terms, but it is of interest that the Atkins dieters in this study did not substantially increase their absolute intake of saturated fat which is what might have been anticipated. The effects of these diets on lipid profile are reported elsewhere  but confBaseline intake of fruit and vegetables were lower than the recommended '5 a day' that is encouraged in the UK but was similar to that reported as 'usual' intake for adults in the UK . AlthougAll the other commercial diets encourage an increase in fruit and vegetables partly to increase the satiety of the meals and also to replace high fat, high sugar snacks. A significant increase in fruit and vegetables was only observed in those following the WW diet but this increase was less than one portion per day. These disappointing findings suggest that people remain resistant to the advice to 'eat more fruit and vegetables' even when they are advised to as part of a modified weight loss programme.On the whole, micronutrient intake remained above the RNI for most nutrients on all the commercial diets even with the degree of mis-reporting of energy intake. Bearing in mind the degree of under-reporting established in this study, there is little evidence to suggest that subjects following self-selected weight reducing diets in the long term would be at risk of micronutrient deficiency. Some subjects following the Atkins diet may have been following advice and taking a daily multi-vitamin supplement which is recommended in the book but this was not analysed. Gender differences were apparent, with women tending to reduce their daily iron intake with energy restriction. Those with high iron requirements due to menstrual losses may be at risk of iron deficiency if they were dieting for long periods of time. This could occur on low either of the low fat approach and the low CHO diet. Meal replacement products that ensure adequate micronutrient provision appeared to offer an advantage in this respect.This analysis aimed to compare the nutritional composition of a low CHO diet to low fat diets. We find little evidence of short-term detrimental effects on nutrient intake with a low CHO approach compared to a low fat approach. Folate intake was only just above recommended levels on all the diets tested, although it fell on the Atkins diet at 2 months but still met 93% of the RNI. Women planning a pregnancy would be well advised to take additional folate while following any of these weight reducing regimens. Health professionals should be aware that in the UK, fortified breakfast cereals and bread flour contribute substantially to iron and B complex vitamin intakes and when these foods are restricted, other sources of these nutrients need to be found. The assumption that low CHO diets become very high in protein due to increased consumption of meat is not substantiated by these data.There is little published data on nutrient adequacy in those trying to lose weight. Ashley et al (2007)  reportedThis study provides some information on the usual diet of overweight people in a reasonably large sample of free living subjects from geographically diverse areas of the UK. There are a number of limitations with the methodology used that need to be considered when interpreting the results. First, nutrient intake profiles are the result of self-reported measures of diet which have a number of known limitations, the most significant of which has to be the well documented and long standing issue of mis-reporting of food intake which is common in overweight and obese populations ,27-29. ITo conclude, this is a novel study which provides comprehensive dietary data on a substantial cohort of subjects following four popular diets without supervision. Health professionals generally would consider three of the diets as nutritionally acceptable  and one diet (Atkins) being controversial. Comparisons of pre- and post-intake indicated dietary compliance. Baseline data suggested overall nutritional adequacy and none of the diets resulted in micronutrient insufficiency or an increase in absolute fat intake which has been a common criticism of low CHO diets. An inadequate intake of dietary fibre was noted in this diet. The caveats to this study are the inherent errors of dietary assessment and that findings may not be generalisable to certain subgroups within the population with specific nutritional requirements, particularly women with raised iron requirements and those with increased calcium needs. This analysis provides reassuring and important evidence for the effectiveness and nutritional adequacy of four commercial diets in weight management for the general public which are particularly pertinent for community and public health nutritionists and those working in primary care. It is suggested that commercial companies work in partnership with health professionals to identify and intervene with high risk clients, such as those planning pregnancies, to provide more individualised dietary advice.KRF receives consulting fees for serving on the scientific advisory panel of Slimming World, a company that offers a support service for weight loss. This company was not involved in this trial but as it is similar to Rosemary Conley and Weight Watchers, the conclusions may have implications for the company.All authors were involved with data collection. RH, AH, MS were in particular involved in the diet and nutrient analysis. HT and MBEL drafted the manuscript with RW completing the statistical analysis, all authors have been involved in reviewing the manuscript.4,172 = 5.1, p < 0.001, post hoc differences between control and SF, control and WW, control and RC;ANOVA a: 2 m data: F4,172 = 5.5, p < 0.001, post hoc differences between control and SF and control and WW;ANOVA b. 2 m data: F4,77 = 3.85, p = 0.007, post hoc differences between control and all other groupsANOVA c: 2 m data: F"}
+{"text": "A novel mechanism explains how exercise exerts its beneficial effects on energy balance through an effect at the level of the hypothalamus. Exercise is a mainstay recommendation for fending off obesity and preventing diabetes, cardiovascular problems, or simply to release stress. Although physicians are certain about the many health benefits of exercise, especially for obese patients, a satisfying explanation for why exercise leads to long-term improvements in health profiles isn't nearly as clear. More obviously, exercise can tip the positive imbalance between energy acquisition and energy expenditure  [1]\u2013[5] At a biochemical and physiological level, there is a considerable amount of cross-regulation and integration between the mechanisms controlling food intake, energy expenditure, and fat deposition. Interestingly, increased fat deposition and excessive body weight are associated with allostatic changes aiming to restore energy balance. For instance, increased fat mass is associated with increased production of leptin and other adipose-derived hormones directed to reduce food intake and promote energy expenditure. Conversely, states of reduced food intake, such as fasting, induce an allostatic response directed to save energy stores and increase the drive for food intake. However, despite these allostatic responses to maintain energy balance, under conditions of chronic hypercaloric excess  and/or impedance  the efficiency and accuracy of these regulatory mechanisms are defective. It has been suggested that toxic effects of lipid accumulation in peripheral tissues, such as pancreatic \u03b2 cells, liver, heart, and skeletal muscle may be an underlying cause, though the exact mechanism is unclear. This process is known as lipotoxicity, and it has been linked with the pathophysiology of insulin resistance, type 2 diabetes, liver disease, atherosclerosis, and cardiovascular disease Food intake and energy expenditure are precisely modulated by specific sets of neurons placed in an area of the brain called the hypothalamus, which comprises the major portion of the ventral part of the diencephalon. The hypothalamus is organized in anatomically-defined neuronal clusters, called nuclei, forming interconnected neuronal circuits via axonal projections. The hypothalamus receives multiple inputs of information as diverse as the sensory experience of eating, the process of ingestion, absorption, metabolism and levels of energy storage. Thus, hypothalamic neurons respond to peripheral nutrients, such as glucose and fatty acids, and hormones, such as leptin, ghrelin and insulin, by modifying the synthesis of orexigenic (feeding-promoters) or anorexigenic (feeding-inhibitors) neuropeptides and then adjusting feeding to the body's nutritional energy demands. When energy intake surpasses expenditure, the expression of orexigenic neuropeptides (such as agouti-related protein (AgRP), and neuropeptide Y (NPY)) diminishes and the expression of anorexigenic neuropeptides (such as cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)) increases. Reverse changes occur when energy expenditure exceeds intake. The impairment of this precise homeostatic system elicits hyperphagia, obesity, and type 2 diabetes A typical problem associated with failure of the central mechanism governing energy balance is the development of resistance to peripheral signals, such as insulin and leptin The ER is a sophisticated luminal network in which protein synthesis, maturation, folding, and transport take place PLoS Biology, Jos\u00e9 B.C. Carvalheira and colleagues move our neurobiological understanding significantly forward by suggesting a novel mechanism by which exercise exerts its beneficial effects on energy balance through a hormonal effect at the level of the hypothalamus ob/ob mice, to show that exercise restores food intake, insulin, and leptin sensitivity to the levels of lean (control) animals. These effects are linked to: (1) normalized expression of hypothalamic neuropeptides modulating feeding (such as NPY and POMC) and (2) improvement of hypothalamic leptin and insulin signaling pathways, all of them severely impaired in obese animals. Of note, authors demonstrate that the molecular mechanism under this action is the reduction of hypothalamic IKK\u03b2/NF-\u03baB activation and ER stress  through a novel hypothalamic mechanism involving increased serum and hypothalamic expression of the pro-inflammatory cytokine interleukin-6 (IL-6), which induces the expression of the anti-inflammatory cytokine interleukin-10 (IL-10), also in the hypothalamus , in both normal and genetic models of cytokine deficiency . These data suggest that the IL-6-IL-10-IKK\u03b2/NF-\u03baB axis is a potential therapeutic target for the treatment of obesity The significance of these data is given by several novel findings. First of all, although physical exercise was already a key strategy for the prevention of obesity and related diseases Mens sana in corpore sano,\u201d and that physical exercise is a key factor to maintain us and our brains in a healthy state.In summary, the new study of Carvalheira and colleagues adds further confirmation that hypothalamic ER stress is an important pathogenic mechanism leading to obesity. It provides a novel integrative framework to understand the deleterious metabolic effects of overnutrition and, more importantly, provides a good rationale for the indication of exercise to obese patients. Moreover, it presents a \u201cscientific evidence\u201d to support the famous Latin citation of the Roman poet Juvenal, \u201c"}
+{"text": "We show that transient energy and fat imbalances in the first several days following a diet switch can account for a significant fraction of the total body weight change. We also discovered a time-invariant curve relating body fat and fat-free masses in male C57BL/6 mice, and the shape of this curve determines how diet, fuel selection, and body composition are interrelated.The mouse has become the most popular organism for investigating molecular mechanisms of body weight regulation. But understanding the physiological context by which a molecule exerts its effect on body weight requires knowledge of energy intake, energy expenditure, and fuel selection. Furthermore, measurements of these variables made at an isolated time point cannot explain why body weight has its present value since body weight is determined by the past history of energy and macronutrient imbalance. While food intake and body weight changes can be frequently measured over several weeks , correspondingly frequent measurements of energy expenditure and fuel selection are not currently feasible. To address this issue, we developed a mathematical method based on the law of energy conservation that uses the measured time course of body weight and food intake to estimate the underlying continuous-time dynamics of energy output and net fat oxidation. We applied our methodology to male C57BL/6 mice consuming various  The unrelenting obesity epidemic has resulted in intensive basic scientific investigation into the molecular mechanisms of body weight regulation\u2014with the mouse being the organism of choice for such studies. We know that any mechanism of body weight regulation must exert its effect by influencing food intake, energy output, fuel selection, or some combination of these factors over extended time scales (\u223cweeks for mice). While food intake and body weight can be frequently measured in mice, current methods prohibit corresponding measurements of energy output or fuel selection on such long time scales. We address this deficiency by developing a mathematical method that quantitatively relates measurements of food intake, body weight and body fat to calculate the dynamic changes of energy output and net fat oxidation rates during the development of obesity and weight loss in male C57BL/6 mice. The mathematical model is based on the law of energy conservation, makes very few assumptions, and provides the first continuous-time estimates of energy output and fuel selection over periods lasting many weeks. Application of our methodology to various mouse models of obesity will improve our understanding of body weight regulation by placing molecular mechanisms in their whole-body physiological context. Mouse models of obesity have become critically important research tools for discovering molecular mechanisms of body weight regulation. But understanding these mechanisms in the context of whole-body physiology requires knowledge of food intake, energy output, and fuel selection Expensive indirect calorimetry systems can be used to measure energy expenditure and respiratory exchange over periods of a few days and most systems require removing mice from their normal environment which can alter their behavior Here, we present a mathematical method that quantitatively relates food intake, body weight and body fat to calculate the dynamic changes of energy output and net fat oxidation rates during the development of obesity and weight loss in male C57BL/6 mice. The mathematical model is based on the law of energy conservation, makes very few assumptions, and provides the first continuous-time estimates of energy output and fuel selection over periods lasting many weeks. Our methodology also revealed the relationship between diet, fuel selection, and body composition change in male C57BL/6 mice by identifying a time-invariant curve relating body fat and fat-free masses.ad libitum access to standard chow (C), high fat diet (HF), or high fat diet plus liquid Ensure (EN) for 19 weeks, while some mice were fed the high fat or the high fat plus Ensure for 7 weeks before being switched back to chow for the remaining 12 weeks . As previously described Our model calculated the first continuous-time estimates of the energy output dynamics underlying the observed body weight changes . The 95%Net fat oxidation rates increased sharply at the onset of high fat feeding in the HF and HF-C groups, but did not rise sufficiently to match the increase of fat intake . InteresA useful measure of fuel selection is the respiratory quotient, RQ, where a value of 0.7 reflects a state of pure fat oxidation whereas a value of 1.0 reflects a state of pure carbohydrate oxidation and intermediate values represent a fuel selection mixture see . The estThe mouse has become the most popular organism for investigating molecular mechanisms of body weight regulation. But understanding the physiological context by which a molecule exerts its effect on body weight requires knowledge of energy intake, energy expenditure, and fuel selection. Our simple mathematical method calculates the dynamics of energy output and fuel selection over extended time periods using longitudinal measurements of body weight, food intake, and body composition. We showed that our method can detect both transient changes of energy expenditure and net fat oxidation rates as well as longer timescale changes found with weight gain and loss. Similar methodology has been previously developed by our group to relate human body-composition changes with dynamic adaptations of fuel selection in both adults Our equations extract information about energy output that is already present in the body weight and food intake data. Other than the law of energy conservation, the only assumption was that the relationship between changes of body fat and fat-free mass were described by a well-defined function in accordance with the Forbes theory of body composition change To estimate the net fat oxidation rate and RQ, an additional assumption regarding carbohydrate balance was required see . We foun0.75. This translates to an absolute expenditure rate of less than 10.7 kcal/d for a mouse that was at most 40 grams at the time of measurement In contrast to our method, currently available techniques for estimating energy expenditure are expensive, involve a plethora of assumptions, and can impact the behavior of the mice Our own attempt to use indirect calorimetry to validate the model predictions of energy expenditure and fuel selection highlighted two important issues. First, the mice that were consuming the high energy diets lost significant amounts of weight when moved to the indirect calorimetry cages indicating that their behavior was not representative of the mice not subjected to the procedure. Second, the measured energy expenditure rates were unrealistically high compared to the model predictions for all groups of mice. In fact, the measured energy expenditure rate was higher than the measured energy intake in the chow-fed mice that did not lose weight (an impossibility) and greatly exceeded the expenditure required to explain the weight loss in the mice fed the high energy diets. These discrepancies led us to diagnose a technical problem with the indirect calorimetry equipment. Thus, we were unable to validate the model estimates of energy expenditure and fuel selection.The field of farm animal nutrition has a long and rich history of using mathematical modeling to analyze animal growth and identify nutritional factors that potentially limit growth rate At the next level of complexity, animal growth models prescribe an energy partitioning rule that specifies how body protein will accumulate for a given food intake rate as a function of body weight, age, or body protein. Energy partitioning rules are often complex Once the partitioning rule is specified, the outputs of animal growth models include body fat mass, maintenance energy requirement, as well as body fat and protein deposition efficiencies given the food intake and body weight as model inputs. In contrast, our model outputs are body fat mass, fuel selection, and total energy expenditure which are more relevant for mouse obesity studies and avoids the known problem of arbitrarily distributing total energy expenditure between tissue deposition costs versus maintenance energy requirements A weakness of our methodology is that it does not distinguish the various components of energy output including resting metabolic rate, thermic effect of feeding, adaptive thermogenesis, physical activity, or any changes of energy excreted in urine and feces that are unaccounted for by the estimates of diet metabolizability. Furthermore, the method does not operate on a within-day time scale and therefore cannot address changes between day versus night or transitions between fed and fasted states. Indirect calorimetry is required to address these issues and would provide important information for the interpretation of our calculated longer-term estimates of energy output and fuel selection. We believe that the combination of our continuous-time methodology with indirect calorimetry measurements at judiciously chosen time points can be applied to various mouse models of obesity as a powerful tool for characterizing the metabolic dynamics underlying experimentally observed body weight changes.We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. All procedures were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.TM feeders , and liquid Ensure was provided in a 30-ml bottle with a rodent sip tube  and liquid intake was measured every day. Solid food intake was corrected for any visible spillage and was measured every day for the high fat diet and every other day for the chow diet using a balance with a precision of 0.01 g (Ohaus model SP402). Body composition was measured once per week using 1H NMR spectroscopy  after body weight was determined.Full details of the experiment were previously described F is the body fat mass, FFM is the fat-free mass defined as the measured body weight, W, minus the fat mass, and TI is the total metabolizable energy intake rate corrected for spillage, and E is the energy output rate. We distinguish the energy output rate from the energy expenditure rate since we did not measure any changes of energy excreted in urine or feces. In other words, if the metabolizable energy content of each diet is constant then our calculation of the energy output is equivalent to energy expenditure.We begin with the law of energy conservation, also known as the energy balance equation:\u03b1, that describes the relationship between changes of FFM and F in male C57BL/6 mice:\u03b1 is specified, equation (1) can be solved for the energy output rate as a function of the measured energy intake rate and the rate of body weight change as follows:Analogous to the Forbes theory of human body composition change Note that very few assumptions were made in the development of our equations to estimate energy output. All of the above equations were derived from the law of energy conservation (1) and the only assumption was that there exits a well-defined Forbes relationship, \u03b1, relating changes of body fat and fat-free masses \u2013 an assumption that was directly confirmed by comparison to measured body composition data.P is body protein, G is glycogen, GNG is the gluconeogenic rate, DNL is the de novo lipogenic rate, and FI, PI and CI are the intake rates of dietary fat, protein and carbohydrate, respectively. The oxidation rates of fat, protein, and carbohydrate  sum to the total energy output, E.Since we are also interested in fuel selection, we must consider the fates of dietary macronutrients including their oxidation rates, storage in the body, as well as major inter-conversion fluxes where carbohydrate can be converted to fat  and amino acids can be converted to the carbohydrate glucose . The following macronutrient balance equations represent these changes:FFM is proportional to body protein such thatTo simplify the macronutrient balance equations, we note that glycogen stores are small, especially when compared with daily carbohydrate intake rates. For example, humans have a glycogen pool size of about 500 g which is equivalent to the typical amount of carbohydrate consumed over \u223c2 days and equilibrates on a time scale of \u223c1 day The shape of the Forbes curve has direct implications for how fat oxidation rate is related to changes of body fat. This can be seen by calculating the partial derivative of the net fat oxidation rate with respect to body fat:RQ, is the carbon dioxide production rate divided by the oxygen consumption rate and was approximated by:2 production. Since the carbohydrate oxidation rate is approximately equal to the carbohydrate intake rate on long time scales, the calculated RQ may have slight inaccuracies during rapid transitions immediately after diet switches, but will be reasonably accurate thereafter.The respiratory quotient, To apply our mathematical model to data from our mouse experiment, food intake measurements were averaged over each diet period and we assumed stepwise transitions immediately after each diet switch followed by a smooth approach to the average intake of the final diet period. These curves are depicted as solid black lines in \u03b1, was fit to an exponential function of the body fat mass as shown in \u03b1, is then given by:b, does not influence the Forbes function, we adjusted the FFM data for each group by subtracting the difference between the calculated intercept parameter for each group and its average value across groups. We then simultaneously fit the adjusted FFM data from all groups to arrive at our final Forbes function used for all of the groups.The Forbes body composition function, th and 97.5th percentiles of the posterior distribution of energy output.The parameter values for the Forbes body composition function"}
+{"text": "Aging is a process associated with physiological changes such as in body composition, energy expenditure and physical activity. Data on energy and nutrient intake adequacy among elderly is important for disease prevention, health maintenance and program development.This descriptive cross-sectional study was designed to determine the energy requirements and adequacy of energy and nutrient intakes of older persons living in private households in a rural Filipino community. Study participants were generally-healthy, ambulatory, and community living elderly aged 60\u2013100 y (n = 98), 88 of whom provided dietary information in three nonconsecutive 24-hour food-recall interviews.2 (p = 0.003) for every 1% decrease in total caloric intake as percentage of the total energy expenditure requirements.There was a decrease in both physical activity and food intake with increasing years. Based on total energy expenditure and controlling for age, gender and socio-economic status, the average energy requirement for near-old (\u2265 60 to < 65 y) males was 2074 kcal/d, with lower requirements, 1919 and 1699 kcal/d for the young-old (\u2265 65 to < 75 y) and the old-old (\u2265 75 y), respectively. Among females, the average energy requirements for the 3 age categories were 1712, 1662, and 1398 kcal/d, respectively. Actual energy intakes, however, were only ~65% adequate for all subjects as compared to energy expenditure. Protein, fat, and micronutrients  intakes were only ~24\u201351% of the recommended daily intake. Among this population, there was a weight decrease of 100 g (p = 0.012) and a BMI decrease of 0.04 kg/mThese community living elderly suffer from lack of both macronutrient intake as compared with energy requirements, and micronutrient intake as compared with the standard dietary recommendations. Their energy intakes are ~65% of the amounts required based on their total energy expenditure. Though their intakes decrease with increasing age, so do their energy expenditure, making their relative insufficiency of food intake stable with age. The world population is now experiencing population aging as evidenced by the much more rapid growth of the sector of persons \u2265 60 y compared to that of the general population . Life exThe increasing population of older persons may lead to an increase in the number of people at high risk of disability and morbidity. Older persons have special health needs and nutritional requirements arising from long-standing dietary habits, a lifetime of different disease encounters and changes in body structure and metabolism. In view of this changing trend, the older age group will become a globally significant health, social and economic policy concern of governments. The development of sound, affordable and sustainable policies and effective community-based programs is necessary to respond to their needs.There are limited data on the nutrient requirements of older persons that could be used as a basis for health policy. Partly, the dearth of data may be due to the inherent difficulty of collecting dietary data. Another factor may be the presence of different groups of older persons  in the rural villages of Palsara and Malabanan in the municipality of Balete, Batangas province, Philippines. The poverty prevalence in Balete is 50.1%  . They haNurses and nutritionists conducted house-to-house interviews to obtain socio-economic demographic profiles and medical histories. Information such as the participant's age, educational attainment, family size, household composition, occupation and average monthly income were gathered. A medical history form was used to gather information on the participant's medical history such as past and present illnesses, use of medications and nutritional supplements, and smoking and alcohol drinking habits. Smoking was defined as currently smoking at least one cigarette per week. Drinking was defined as intake of at least one unit per day .2). Medical technologists collected 5 ml of blood samples by venipuncture after an 8-hour fast for blood sugar and cholesterol determinations. These blood indices were reported more fully in an earlier publication [A trained physician conducted a physical examination for clinical signs of illness and validated the information in the medical history form. Weight was measured in light clothing using a platform scale  and height was measured in upright standing position without shoes using a standard portable wooden height measuring board (UNICEF model). Weight was recorded to the nearest 0.1 kg and height to the nearest 0.1 cm. Weights and heights were used to calculate body mass index . Food models and household measuring tools were used by dietitians to aid the subjects in estimating their food intakes. Portion sizes were estimated using measuring cups and spoons and food models. Dietary intakes of fat, protein, carbohydrates, and the micronutrients were assessed by using the Philippine Food Composition Tables . The resThe assessment of physical activity level was done by observation. The field staff followed the subjects during their routine daily activities in the span of 2 months, averaging about 2 to 3 days of observation per subject in the course of data collection and fieldwork . During a single observation period, the field staff stayed with the subjects from 4 to 6 hours at a time. The subjects' activities were variously observed in the morning, afternoon and evening. In this way the field staff were able to collect data on light to moderate intensity activities such as leisure activities, sitting, eating, and commuting which would have been more difficult to recall accurately. The list of physical activities and their level of intensity expressed as multiples of the basal metabolic rate  is shownMean micronutrient intakes were computed as percentages of the recommended intake based on the Philippine Recommended Energy and Nutrient Intakes ; averageSocio-economic status was a score  constructed from a matrix of toilet facilities and availability of drinking water. A socio-economic score (SES) of 1 was given to participants who either had a private water-sealed toilet or had a private source of drinking water . Those with a SES of 0 had neither and those with a SES of 2 had both of these. There was a significant correlation between this socio-economic score construct and the reported total monthly income of the household .Data were encoded using FoxPro version 2.6  and analyzed using EpiInfo version 6.04d  and Stata . Means were computed for quantitative variables while frequency distribution tables were generated for categorical variables. Pearson's chi-square test, or Fisher's Exact test were used to analyze data in Table Multiple linear regression was used to analyze the data shown in Figures Because there were cases in which more than one subject lived in the same household, a multiple linear regression mixed effects model was used with the household number as a random effects parameter and the explanatory variables as fixed effects parameters when controlling for several variables at the same time. For all the tests, a p-value < 0.05 was considered to be statistically significant.Approval to conduct the study was obtained from the Tufts University-New England Medical Center Human Investigation Review Committee and the National Ethics Committee of the Philippine Council for Health Research and Development.A description of the subjects is shown in Table 2) was 22.5%, 37.3% and 47.8% among the near-old, young-old and the old-old respectively.Their mean monthly household incomes were below the poverty threshold  and 42.1The average total energy requirements as calculated based on the basal metabolic rate, specific dynamic action of food, activity levels of the subjects and the analysis using simple linear regression are shown in Table Figure Figure 2  were observed for every 1% decrease in total caloric intake as a percentage of the total energy expenditure requirements. Height was not associated with energy intake percentage.Mean anthropometry measurements are shown in Table The main concern of geriatric nutrition is the conservation of good health and prevention of degenerative diseases . IdeallyThis study provides energy and nutrient intake data from older persons independently living in private households in a rural community in the Philippines. For all subjects, intakes of carbohydrates, protein, and fat provided ~78%, 11%, and 11% of the total energy intake, respectively. While the actual food intake of the old-old were less than those of the near- and young-old, because of the decreasing energy requirements with increasing age, the relative intake of macronutrients as a percentage of energy expenditure was not significantly different for the different age groups. The percentages were not equally distributed among the macronutrients, however, with a much lower relative percentage in fat and protein intakes compared to carbohydrate intake.These results are similar to findings from other recent studies among community-dwelling, community living elderly people in both developing and developed countries ,17,28,29Many age-related changes that might influence dietary and health requirements occur continually throughout the life cycle. Micronutrient deficiencies have been shown to be common in elderly people due to a reduced food intake resulting from dietary restrictions and a lack of variety in the foods they eat. They are particularly vulnerable to dietary restrictions due to social and psychological factors, long standing dietary habits, physiological changes associated with aging  and oralMore than a third of the participants were chronic energy deficient. Low food intake with subsequent low body weight, weakened immune system at old age and poor access to health facilities and services may further increase their risk for many nutrition-related acute or chronic illnesses.Five of the 7 micronutrients with dietary intake data  were approximately 1/3 of the recommended nutrient intake for Filipino elderly. Iron (51%) and niacin (77%) were the exceptions. The dietary intake was particularly low for vitamins A and C. The percentage niacin intake decreased with increasing age. Iron intake as a percentage of the recommendations had significant variability with age, increasing among the young-old and the old-old compared with the near-old. This population also had data on vitamin A status . Total bStudies conducted in neighboring Asian countries show that mean intakes among populations of older persons often fall below the official dietary recommendations. Among 350 elderly Malays  the meanFinancial status may limit one's dietary choices. Though most of the study participants were still economically active, their mean monthly incomes were below the national poverty threshold for rural areas . In thisThe cross-sectional nature of data collection allowed for associations to be identified, but causality cannot be established. Furthermore, because our study participants were community living older persons who were generally healthy, the results of this study may not be directly applicable to institutionalized or home-based elderly even though they are consistent with other study results conducted among this age group ,45-47. TThe study results show that the community living elderly participants in this study had energy intakes that were about 65% of the amounts required based on their total energy expenditure. Their nutritional and health vulnerability, together with a myriad of age-related processes such as body structure changes, and physiological and immune function alterations make this sector of the population at high risk for malnutrition-related diseases. Also their growing population makes them a significant social and economic concern of governments.12 that may be affected by chronic conditions. Thus, there is a need to review current recommended daily nutrient allowances which could be used as basis for the development of guidelines to address the nutritional needs of the growing elderly population.Attempts to provide the elderly with adequate nutrition encounter many practical problems. First, their nutritional requirements are not well defined. Second, the process of aging also affects other nutrient needs. The effect of aging on nutrient requirements cannot be easily quantified. There are changes noted in studies that showed a decrease in energy requirement with advancing age , while oPrograms for this group may be geared towards (a) screening for and assessing nutritional problems, and (b) nutritional counseling, health promotion and disease prevention. Health care and support services should be made accessible and education and awareness on health and nutrition should be promoted not only among the older persons but also among younger adults. Efforts should be made in educating the public on the conditions associated with aging including effective elderly care. The importance of micronutrients in the promotion of health and prevention of later-life disorders should also receive considerable attention in diet-disease national programs.The authors declare that they have no competing interests.MGDR contributed to the acquisition of data, interpretation of data, and preparation of the manuscript. PRS analyzed and interpreted the data, and prepared the manuscript. JDRM and JAAS provided significant advice with respect to the analysis and interpretation of the data and critically reviewed the manuscript. AAB and LWT contributed to the collection of the data and review of the manuscript. FSS contributed to the design of the study and critically reviewed the manuscript.Energy and macronutrient intakes of the study participants, by age and gender. Significant differences in the intake of energy, protein and carbohydrates (but not fat intake) with increasing age was seen using multiple logistic regression analysis, controlling for age, gender and SES.Click here for fileMicronutrient intakes of the study participants, by age, gender and SES score. Intakes of iron, niacin, riboflavin, thiamin, calcium, vitamin A and vitamin C were not significantly different by age, sex or socio-economic status, except for vitamin A intake which was significantly higher for those with the highest SES.Click here for file"}
+{"text": "In 1983, Coates conducted a survey that ranked the side-effects perceived by patients receiving chemotherapy in the order of their severity. Vomiting and nausea were found to be the two most distressing side-effects. They have an impact on quality of life and compliance with treatment. The development of 5HT3 antagonists has been a major step forward in the prevention and treatment of chemotherapy-induced nausea and vomiting. Presently, these antiemetics are routinely used as concomitant therapy in emetogenic chemotherapy regimens. The purpose of this study was to evaluate the impact of 5HT3 antagonists on patient perceptions of the side-effects of chemotherapy. Coates' survey was replicated in patients who received 5HT3 antagonists for acute nausea and vomiting resulting from emetogenic chemotherapy. Patients received the survey to identify those physical and non-physical side-effects that they attributed to chemotherapy and were asked to rank the five most distressing side-effects. Of the 197 patients who consented to take part in the study, 181 were evaluable. Nausea, hair loss and vomiting were described as the three most distressing side-effects of chemotherapy. Eighty per cent of all the patients actually experienced nausea and 57% experienced vomiting. Hair loss appeared to be more distressing to women (P < 0.001) but, in other aspects, gender, age and marital status did not influence the ranking of the three most distressing side-effects. Constipation was ranked as 6th and was not identified as a distressing side-effect in 1983. Nausea and vomiting remain to be the first and third most distressing side-effects of chemotherapy, even though the incidence and severity of acute nausea and vomiting are now significantly reduced."}
+{"text": "Few studies have evaluated the association between incident chronic kidney disease (CKD) and related complications, especially in elderly population. We attempted to verify the association between GFR and concurrent CKD complications and elucidate the temporal relationship between incident CKD and new CKD complications in a community-based prospective elderly cohort.2).We analyzed the available data from 984 participants in the Korean Longitudinal Study on Health and Aging. Participants were categorized into 6 groups according to eGFR at baseline examination  for hypertension was 2.363  in group 4, 5.191 (2.074-12.995) in group 5, and 13.675 (1.611-115.806) in group 6; for anemia, 7.842 (2.265-27.153) in group 5 and 13.019 (2.920-58.047) in group 6; for acidosis, 69.580 (6.770-715.147) in group 6; and for hyperkalemia, 19.177 (1.798-204.474) in group 6. Over a 5-year observational period, CKD developed in 34 (9.6%) among 354 participants with GFR \u2265 60 ml/min/1.73 m2 at basal examination. The estimated mean number of new complications according to analysis of co-variance was 0.52  in subjects with incident CKD and 0.24 (0.19\u20130.29) in subjects without CKD (p = 0.002). Subjects with incident CKD had a 2.792-fold higher risk of developing new CKD complications. A GFR level of 52.4 ml/min/1.73 m2 (p = 0.032) predicted the development of a new CKD complication with a 90% sensitivity.The mean age of study population was 76 \u00b1 9.1 years and mean eGFR was 72.3 \u00b1 17.0 ml/min/1.73 mIn an elderly prospective cohort, CKD diagnosed by current criteria is related to an increase in the number of concurrent CKD complications and the development of new CKD complications. In a prospective cohort study of 1,038 hospital inpatients, the eGFR thresholds used for detecting CKD complications were 50, 44, 40, 39, and 37 ml/min/1.73 m2 for hyperparathyroidism, anemia, acidosis, hyperkalemia, and hyperphosphatemia, respectively [Age is one of the most important risk factors for chronic kidney disease (CKD). In the United States, the prevalence of an estimated glomerular filtration rate (eGFR) of <60 ml/min/1.73 mThis study was conducted as a part of the Korean Longitudinal Study on Health and Aging (KLoSHA), which included a randomly selected, community-based, elderly population . The KLo2; group 2, 75\u201389 ml/min/1.73 m2; group 3, 60\u201374 ml/min/1.73 m2; group 4, 45\u201359 ml/min/1.73 m2; group 5, 30\u201344 ml/min/1.73 m2; and group 6, <30 ml/min/1.73 m2. CKD was defined as a GFR < 60 ml/min/1.73 m2. Anemia was defined as a hemoglobin level < 12 g/dL in women and <13 g/dL in men [2 < 22 mmol/L.The data regarding all medications taken by the participants were collected. Diabetes mellitus (DM) and hypertension were defined as they had been elsewhere . The ser2 at baseline examination to <60 ml/min/1.73 m2 at second examination and persistent CKD in patients who had GFR <60 ml/min/1.73 m2 at both baseline and second examination. Newly developed CKD complications were detected at second examination. The duration of the follow-up period was 59.4 \u00b1 6.9 months. Complications related to CKD were defined as hypertension, hypertriglyceridemia, hypoalbuminemia, hyperkalemia, acidosis, and anemia. Incident CKD was diagnosed in patients who experienced GFR decrease from \u226560 ml/min/1.73 mt-test according to the number of subgroups, and differences in categorical variables were analyzed by Pearson\u2019s chi-square or Fisher\u2019s exact test. The levels of potassium, total CO2, and hemoglobin and the number of complications at baseline examination were estimated by analysis of co-variance (ANCOVA), adjusted by the factors related to each parameter. The risk factors for concurrent CKD complications, incident CKD, persistent CKD, and newly developed CKD complications during follow-up were identified by multiple logistic regression analysis. Formal testing for interactions between GFR groups and aging for each complication was conducted by comparing \u20132 log likelihood in regression models with and without the interaction term (GFR group*age in a 10-year unit). All analyses were performed using SPSS software . Data were presented as the mean \u00b1 SD values for continuous variables and as proportions for categorical variables. Differences in continuous variables were analyzed by one-way analysis of variance or Student 2. The number of participants in each GFR group was as follows: 150 (15.2%) in group 1 (GFR \u2265 90 ml/min/1.73 m2), 348 (35.4%) in group 2 (GFR 75\u201389 ml/min/1.73 m2) 249 (25.3%) in group 3 (GFR 60\u201374 ml/min/1.73 m2), 161 (16.4%) in group 4 (GFR 45\u201359 ml/min/1.73 m2), 59 (6.0%) in group 5 (GFR 30\u201344 ml/min/1.73 m2), and 17 (1.5%) in group 6  included 436 men (44.3%) and 548 women (55.7%) with a mean age of 76.0 \u00b1 9.1 years and a mean estimated GFR of 72.3 \u00b1 17.0 ml/min/1.73 m2 started to decrease from GFR 60 ml/min/1.73 m2 (p<0.001). The estimated number of complications adjusted with related factors was 0.98 (0.88-1.09) in GFR group 1, 1.11 (1.05-1.17) in group 2, 1.09 (1.02-1.17) in group 3, 1.16 (1.06-1.26) in group 4, 1.48 (1.32-1.64) in group 5, and 2.12 (1.83-2.41) in group 6 (p<0.001)  . 2 at basal examination, 34 developed CKD (9.6%) during a 5-year observational period. The risk of incident CKD was found to be 5.896-fold higher in group 3 as compared to group 1 (p = 0.006). Each 10-mmHg increase in SBP was found to increase risk of incident CKD by 1.331-fold (95% CI: 1.087\u20131.629) . Although 46.3% (19/41) of participants with GFR of 45\u201359 ml/min/1.73 m2 at basal examination had a GFR \u226560 ml/min/1.73 m2 at second examination, only 5.6% (1/18) who had a GFR <45 ml/min/1.73 m2 at baseline had the above mentioned GFR at the second examination (p = 0.002). The number of CKD complications at the second examination was higher in participants with persistent CKD than in participants with GFR who experienced a GFR increase to over 60 mL/min/1.73 m2 . Among 354 participants who had a GFR \u2265 60 ml/min/1.73 m7\u20131.629) . Compare2 at basal examination, 90 (25.4%) developed at least one new complication during the follow-up period. These included 40 (11.3%) subjects who developed incident hypertriglyceridemia; 27 (7.6%), incident anemia; 17 (4.8%), incident hypertension; 5 (1.4%), incident hyperkalemia; 3 (0.8%), incident acidosis; and 3 (0.8%), incident hypoalbuminemia. Simple logistic regression analysis indicated that incident CKD increases the risk of developing anemia  and hyperkalemia , and multiple regression analysis indicated that incident CKD increases the risk of developing new complications by 2.792-fold (95% CI: 1.308\u20135.964). ANCOVA adjusted by related factors for development of new CKD complications indicated that subjects with incident CKD developed 0.52 (95% CI: 0.35\u20130.68) new complications, while subjects without incident CKD developed 0.24  new complications. Among participants with a GFR \u2265 60 ml/min/1.73 m2 at baseline examination, a threshold GFR of 52.4 ml/min/1.73 m2 at second examination was found to predict development of any CKD complication with 90% sensitivity .Among the 354 subjects who had a GFR \u2265 60 ml/min/1.73 m2, anemia at GFR < 45 ml/min/1.73 m2, and both acidosis and hyperkalemia at GFR < 30 ml/min/1.73 m2. The rate of incident CKD, as defined by the criterion value of GFR of 60 ml/min/1.73 m2, was 9.6% over the 5-year study period. The rate of the incident CKD was found to increase the incidence of anemia and hyperkalemia and the total number of newly developed complications, while improvement in GFR was found to result in improvement of the severity of complications.In the cohort examined in this study, the concurrent prevalence of hypertension increased at a threshold GFR of <60 ml/min/1.73 mSeveral reports have suggested that a decrease in GFR in older subjects increases the risk for metabolic complications associated with CKD. However, these reports were limited in their analysis of the temporal associations between complications and decrease in GFR because of cross-sectional or retrospective study design ,10. The 2), the higher prevalence of anemia, acidosis, hyperkalemia, hypertension, and CKD complication as a whole were consistently present at GFR levels <45 ml/min/1.73 m2 as the results of elderly subjects from NHANES data [2 which was comparable to the Gubbio study in Italy [The results of this study confirm the existence of a relationship between the concurrent CKD complications and a decrease in GFR without any interactions between GFR and the aging process, indicating that a decrease in GFR attributable to the aging process may not be responsible for these complications. When we adopt the criteria for GFR group , but higher than that in subjects with a GFR of 30\u201344 ml/min/1.73 m2 (**p = 0.001) and those with a GFR < 30 ml/min/1.73 m2 (***p = 0.015).(TIF)Click here for additional data file."}
+{"text": "Alicyclobacillus acidoterrestris is a spore-forming Gram-positive, thermo-acidophilic, nonpathogenic bacterium which contaminates commercial pasteurized fruit juices. The draft genome sequence for A.\u00a0acidoterrestris strain ATCC 49025 is reported here, providing genetic data relevant to the successful adaptation and survival of this strain in its ecological niche. Alicyclobacillus acidoterrestris is capable of surviving extremely harsh conditions, for instance during industrial food processing  and pH conditions (pH 2 to 6); it can also survive pasteurization and is able to grow during food storage  and was kindly provided by Ronit Ben Avraham from Milouda Laboratories (Israel). Genomic DNA was isolated from liquid culture using a genomic DNA purification kit (Sigma-Aldrich) and prepared for shotgun sequencing using the PrepX ILM DNA library kit . DNA was initially sheared using a Covaris S2 acoustic shearing device, and subsequent to sequencing, adapter-ligated fragments were size selected (400\u2013800\u00a0bp) using the Pippin prep automated electrophoresis instrument . Sequencing was performed on an Illumina HiSeq2000 instrument, employing paired-end 100-base reads. Approximately 13\u00a0M reads were generated in pairs and assembled by the de novo assembler within the software package CLC Genomics Workbench v 6.0 . A total of 207 contigs of length \u2265500 bases were generated, with a sum of 4,063,548\u00a0bp, an N50 of 44,524 bases, and an average coverage of >100\u00d7. More than 96% of the reads mapped to the draft genome contigs. The contigs were successfully used for annotation and gene prediction by Rapid Annotations using Subsystems Technology (RAST) , Bacillus licheniformis ATCC 14580 (60% similarity), and B.\u00a0halodurans (57% similarity). The sporulation kinase A (KinA), which activates Spo0A by phosphorylation, was also found to be conserved in A.\u00a0acidoterrestris. Thus, KinA protein shows 32% similarity to the PAS/PAC sensor signal transduction histidine kinase of the A.\u00a0acidocaldarius strains as well as to KinA of B.\u00a0subtilis 168.In response to stressful conditions, bacteria can initiate a developmental pathway leading to the formation of dormant endospores . SporulaAURB00000000.This whole-genome shotgun project has been deposited at GenBank under the accession no."}
+{"text": "Journal of Experimental Botany now tries to provide a state-of-the-art insight into the numerous roles of some major plant peptide classes including CRPs as well as various nonCRPs such as CEPs, CLEs, IDAs, PEPs, PSKs, and others.Due to the presence of cell-wall material separating the surface of neighbouring cells, plants were expected to require many mobile ligands to exchange information among cells, tissues, organs, and with their environment. In particular, the classical plant hormones auxin, cytokinins, gibberellins, abscisic acid, and ethylene have long been considered to regulate growth and developmental processes as extracellular signalling molecules . Until tArabidopsis. For example, 53% of the total number of female gametophyte-specific genes encode members of various CRP subclasses  belong to the post-translationally modified small-peptides and consist of only five amino acid residues. The disulphated peptide was initially described as a growth factor. Arabidopsis. Several IDA-like (IDL) peptides have now been analysed by Arabidopsis also reduced root size when applied to Brachypodium, indicating that CLE signalling pathways are evolutionary highly conserved. Legumes can form nodules as lateral root outgrowths that provide a nurturing environment for rhizobia, the symbiotic bacteria that deliver fixed nitrogen in return for carbohydrates. The carbon costs are high and nodule formation is tightly regulated to occur only under N-limiting conditions. Not surprisingly, plants regulate this expenditure via short and long range (between root and shoot) apoplastic signalling. Arabidopsis. However, redundancies exist on both the peptide and the receptor side, setting the hurdles even higher for the identification of \u2018linear\u2019 signalling pathways and the assignment of a given peptide to a specific receptor and signalling outcome. Furthermore, loss-of-function alleles are either not available for many of the peptides described so far, due to the small size of their genes, or the lack of apparent phenotype(s) in a single mutant situation. Techniques for a more comprehensive functional analysis of peptides are therefore urgently needed, since novel peptides with signalling roles are being described at a rapid pace (see below). The generation of \u2018antagonistic\u2019 or dominant negative versions of a peptide that might block receptor function could provide one solution to this dilemma. et al., this issue), it could greatly benefit from detailed structural information on the peptide\u2013receptor interface. This is urgently required as the number of peptides is steadily increasing. Arabidopsis CLE genes for comparison and for separation into subgroups. CLE peptides from legumes have attracted (and deserve) considerable attention for their important role in the regulation of nodule formation . This work will provide a very useful reference source for future studies.The IDA peptide is another nonCRP and was first described to promote organ abscission in 2+-release, cGMP production, and MAPK activation via the LRR-RLKs PEPR1 and 2. In addition to immune responses, the PEP systems now emerge as controlling root biomass and darkness-induced senescence. Arabidopsis, to other plant species and showed that PEPs are not recognized outside their family of origin. However, signalling events downstream of the PEP receptors, for example, ethylene production, are conserved, and PEPRs are interspecies compatible. Obviously, this finding opens the doors for approaches to re-engineer or exchange PEPRs between species to increase the immune system of crop plants. PEP expression is strongly induced by herbivores, and that PEPR mutant Arabidopsis plants are, consequently, more susceptible to the feeding attacks of Spodoptera littoralis (cotton leafworm). Lateral roots arise from the asymmetric divisions of xylem-pole pericycle cells, and this first cell division is affected by a surplus of GLV6, a peptide that is normally expressed in sites of lateral root initiation and the primary root. Fernandez and coworkers previously found that silencing of GLV6 reduces both primary root length and lateral root density. GLV6 is a member of the GLV/RGF/CLEL family and several peptides of this group were found to promote root meristem growth by controlling PLT transcription factors through RLK dependent signalling pathways. Medicago truncatula CEP1 gene gives rise to five or more CEP peptides consisting of different domains with distinct hydroxylation and arabinosylation patterns. This work emphasizes again the importance of biochemical approaches: peptides must be isolated from the plant itself to define their different mature forms which can vastly affect their biological activities.Other nonCRPs include PIPs, GLV/RGF/CLEL, and CEP peptides. In their review, Arabidopsis, AtCAPE1, which is related to the cysteine-rich tomato immune regulator CAPE1. Surprisingly, they show that AtCAPE1 negatively regulates salt-tolerance and propose a role for this novel peptide in balancing immune and salinity responses. Plant lateral organ primordia are separated from the shoot meristem, their site of origin, by a specialized group of cells, which establish the boundary domain. Arabidopsis can affect organ separation. Both peptides are expressed in multiple tissues and it is likely that additional functions will be discovered upon further investigations. TAX peptides were first discovered in Taxus baccata and shown to modulate the biosynthesis of taxane, a desired anti-cancer drug. In Arabidopsis, TAX1 and 2 might indirectly affect organ separation by controlling the biosynthesis of a (unknown) hormone.The number of new candidate signalling peptides is continuously increasing. Arabidopsis root meristem. This paper points towards important future aspects of peptide signalling research to understand the cellular outputs of peptide\u2013receptor interactions. In conclusion, this special issue of the Journal of Experimental Botany aims to provide an overview of some aspects of this exciting and innovative plant research field and gives some insights into the emerging technologies currently used to stimulate researchers to identify novel signalling peptides, their functions, receptors, and downstream signalling pathways. We hope you enjoy reading the various articles as much as we do.The majority of the peptides discussed here are secreted into the apoplast to control gene expression or function in adjacent cells, but the signalling cascades that they instigate are just single branches within larger gene regulatory networks. In the final paper of this special issue on peptide signalling in plants,"}
+{"text": "In vitro gain-of-function and loss-of-function studies in human renal carcinoma cell lines, demonstrated that miR-28-5p suppressed cell proliferation and migration by directly inhibiting RAP1B, and this effect was reversed by co-transfection with RAP1B. In addition, the stable overexpression of miR-28-5p inhibited tumor cell proliferation in vivo. This newly identified miR-28-5p/RAP1B axis provides a novel mechanism for the pathogenesis of RCC, and molecules in this axis may serve as potential biomarkers and therapeutic targets for RCC.The incidence and mortality rate of renal cell carcinoma (RCC) have been significantly increasing; however, the mechanisms involved in RCC development and progression are unclear. In this study, we found that miR-28-5p was decreased in RCC tumor specimens and several renal carcinoma cell lines. By using a combination of luciferase reporter assays and western blotting, we identified RAP1B, a Ras-related small GTP-binding oncoprotein implicated in a variety of tumors, as a direct target of miR-28-5p in RCC. The RAP1B protein level was increased in RCC tumor specimens and renal carcinoma cell lines, and this was inversely correlated with miR-28-5p expression.  Renal cell carcinoma (RCC) represents 85% of all primary renal neoplasms and is among the top 10 most common malignancies in both men and women. In 2012, it is estimated that approximately 64,000 individuals will be diagnosed with RCC and 13,570 will die from this disease in the United States [in vitro [MicroRNAs (miRNAs), small noncoding RNAs that regulate the translation of many genes by binding to the untranslated region (3\u2032UTR) of target mRNAs, are involved in a variety of physiological and pathological processes, in particular, cancer development. Accumulating evidence shows that miRNAs are aberrantly expressed in many types of cancers, including RCC, and some of these miRNAs function as tumor suppressor genes or oncogenes during tumor development and progression \u20139. Thesein vitro . HoweverRAP1B, a Ras-related small GTP-binding protein that behaves as a GTPase, has been implicated as an oncogene in a variety of tumors, including esophageal squamous cell carcinoma (ESCC), CRC, T-acute lymphoblastic leukemia, non-small cell lung carcinoma, glioma and thyroid carcinoma \u201327. RAP1In this study, we explored the relationship between miR-28-5p and RCC and the potential mechanisms of miR-28-5p in RCC. We found that miR-28-5p was decreased in RCC tissues. Gain-of-function and loss-of-function studies demonstrated that miR-28-5p acted as a tumor suppressor in RCC for multiple antitumor effects by directly inhibiting RAP1B. Taken together, these results indicate that the miR-28-5p/RAP1B axis may provide further insight into the pathogenesis of RCC and may represent a potential novel therapeutic target for RCC.P < 0.05, Figure To examine whether miR-28-5p was dysregulated in RCC, we first determined the expression patterns of miR-28-5p in 33 RCC tissue samples and 33 corresponding adjacent non-tumorous tissues from the same patients using real-time quantitative reverse transcriptase PCR (qRT-PCR). Most of the cancer specimens (87.9%) had the tumor histotype of clear cell RCC . miR-28-We then determined the miR-28-5p expression level in three human renal carcinoma cell lines, A498, ACHN and Caki1, and found that miR-28-5p was markedly deceased in all three cell lines compared with the immortalized primary human proximal tubular cell line HK-2 Figure . Taken tin silico search using three computational algorithms in combination, TargetScan [http://www.targetscan.org/vert_61/), miRanda [http://www.microrna.org/microrna/home.do) and PicTar [http://pictar.mdc-berlin.de/cgi-bin/new_PicTar vertebrate.cgi), to predict the potential target genes of miR-28-5p. Computational analyses identified seven candidate genes containing potential miR-28-5p binding sites    analysis. The clinical information and the relative expression of miR-28-5p for these 20 paired tissue specimens were displayed in ) Figure . More im) Figure .Next, using western blotting, we found that RAP1B protein was highly expressed in another 16 paired RCC tissues and adjacent non-tumorous tissues (4 pairs were remained from the 33 paired samples and the other 12 pairs were newly collected) as well as three tested human renal carcinoma cell lines, including A498, ACHN and Caki-1, compared with HK-2 cells , while nin vitro, we also stably overexpressed miR-28-5p by transfecting a lentiviral expressing vector of miR-28-5p into the above two cell lines. Stable ectopic expression of miR-28-5p in these cells was validated by qRT-PCR  assay. As shown in Figure in vitro results further confirmed that miR-28-5p had a biological effect on proliferation  against RAP1B mRNA or a RAP1B\u2013expression vector. The efficiency of the expression of RAP1B siRNAs and exogenous RAP1B was examined by western blotting  stimulation . Thus, win vitro experiments revealed that miR-28-5p or RAP1B expression was associated with proliferative traits, we next examined the effect of miR-28-5p or RAP1B expression on tumor formation in vivo. We performed a tumor formation assay in a nude mouse model using A498 cells stably expressing miR-28-5p after lentiviral infection or transfected with a RAP1B plasmid to overexpress RAP1B, and then cells were implanted subcutaneously into mice -293T were purchased from Shanghai Cell Bank, Chinese Academy of Sciences  and cultured in the following media: McCoy's 5A , MEM-NEAA , DMEM/F12 (GBICO) and DMEM medium respectively. The A498 cell line was purchased from Cell Resource Center, IBMS, CAMSI/PUMC and cultured in MEM-NEAA medium.The tissue collection and analyses were approved by the Ethics Committee of Jinling Hospital of Nanjing University, and written informed consent was obtained from all participants. Thirty-three pairs of RCC tissues and their matched non-tumorous adjacent tissues were obtained from patients undergoing surgery for RCC at the Jinling Hospital. All patients underwent a tumorectomy prior to the receipt of any adjunctive therapy. Pathology specimens from all patients were centrally reviewed and the definitive tumor stage was established on the basis of operative findings according to the WHO's TNM classification system for RCC . All tisTotal RNA was extracted from tissues or cells with TRIzol reagent  following the manufacturer's protocol. miR-28-5p levels were determined by qRT\u2013PCR using TaqMan assay kits  with the U6 sRNA as an internal reference. RAP1B mRNA was detected by qRT\u2013PCR using a SYBR Premix E \u00d7 Taq Reverse Transcription\u2013PCR kit , and values were normalized to GAPDH. The primers specific for RAP1B and GAPDH were as follows: RAP1B (sense) 5\u2032-GTGAATCCCTTGCT TGCTCAT-3\u2032 and RAP1B (anti-sense) 5\u2032-AATACTGTGGCTCCCTGTTGG-3\u2032; and GAPDH (sense): 5\u2032-GATATTGTTGCCATCAATGAC-3\u2032 and GAPDH (anti-sense): 5\u2032-TTGATTTTGGAGGGAT CTCG-3\u2032. The reactions were performed using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems).miR-28-5p mimics and its negative control (NC) mimics-scramble, miR-28-5p inhibitor and its NC inhibitor-scramble, and the miR-28-5p mutant were obtained from GenePharma , and lentivirus to overexpress miR-28-5p was purchased from Invitrogen. RAP1B siRNAs were obtained from RiboBio Co., Ltd. . Their sequences were as follows: miR-28-5p mimics (sense) 5\u2032-AAGGAGCUCACAGUCUAUUGAG-3\u2032 and miR-28-5p mimics (anti-sense) 3\u2032-C AAUAAGACUGUGAG CUCCUUU-5\u2032; mimics-scramble (sense) 5\u2032-UUCUCCG AACGUGUCACGUTT-3\u2032 and mimic-scramble (anti-sense) 3\u2032-ACGUGACACGUU CGGAGAATT-5\u2032; miR-28-5p inhibitor (sense) 5\u2032-CUCAAUAGACUGUAGCUCCU U-3\u2032; inhibitor-scramble (sense) 5\u2032-CAGUACUUU UGUGUAGUACAA-3\u2032; RAP1B- siRNA1 (sense) 5\u2032-GUGAGUAUAAGCUAGUCGUdTdT-3\u2032; siRNA2 (sense) 5\u2032-AC CUAGUGCGGCAAAUUAAdTdT-3\u2032; and siRNA3 (sense) 5\u2032-UGUUGGUAAUAU UGACdTdT-3\u2032. The miR-28-5p mutant was constructed by replacing the RAP1B binding site with its complimentary sequence. A mammalian expression plasmid encoding the human RAP1B open reading frame (pReceiver-M02-RAP1B) was purchased from GeneCopoeia . An empty plasmid served as the negative control. All constructs were verified by sequencing.The miRNA targets were predicted using the miRanda , TargetSFormalin-fixed, paraffin-embedded RCC tissues were subjected to standard IHC. A rabbit monoclonal anti-RAP1B antibody  was used in this study. The results of immunostaining were scored blindly with no information of the clinical data by Dr. Caiyun Wu and who were guided by a pathologist . The scoring was based on the intensity and extent of staining and was evaluated according to the following histological scoring methods as previously described , 38. BriThe antibodies used for western blotting were as following: rabbit monoclonal anti-RAP1B antibody, anti-p-p38 antibody, anti-Erk1/2 antibody and anti-p-Erk1/2 antibody, and rabbit polyclonal anti-p38 antibody ; mouse monoclonal anti-GAPDH antibody .4 cells/well (A498) or 2 \u00d7 104 cells/well (ACHN), transfected with the indicated miR-28-5p mimics, miR-28-5p inhibitor, plenti-miR-28-5p, RAP1B vector, RAP1B siRNA or negative controls, and incubated for 12, 24, 48 and 72 h. Cell cycle analysis was conducted using flow cytometry after overexpressing or inhibiting miR-28-5p expression through transient transfection following 48 h culture. For colony formation assay, RCC cells were seeded in 2 ml medium per well in the 6-well culture plates (100 cells per well for A498 and ACHN). After cultured for 14 days, cells were fixed with paraformaldehyde and stained with crystal violet.Cell proliferation was evaluated using a WST-8 Cell Counting Kit-8 . A498 and ACHN cells were seeded in 96-well culture plates at 1 \u00d7 10A wound-healing assay was performed to assess cell migration. An artificial wound was created 24 h after transfection using a 10 \u03bcl pipette tip. The A498 and ACHN cells were then cultured with 2% fetal bovine serum for 20 h. To visualize the migrated cells and wound healing, images were taken at 0 and 20 h.5 cells/well on the upper chamber with serum-free MEM-NEAA medium. Cells were allowed to invade the lower compartment for 17 h. Each experiment was performed in triplicate and repeated twice.The transwell migration was tested in a Transwell Boyden Chamber . Cells were harvested 48 h after transfection, and A498 and ACHN cells were seeded at a density of 5 \u00d7 104) were transfected with miR-28-5p mimics, miR-28-5p inhibitor or negative controls, and cell invasion was performed by matrigel invasion assay. Cells in the upper compartment of the chamber were suspended in serum-free medium, and the lower chamber contained medium supplemented with 10% fetal bovine serum. After incubated for 48 h, cells passing through the matrigel membrane were fixed and stained with crystal violet, followed by counting from three random microscopic fields.Cell invasion was evaluated using a Boyden chamber system with a polycarbonate membrane . For cell invasion assay, RCC cells . The mice were sacrificed 4-week after seeding the tumor cells. The tumor weights were then determined and their ellipsoid volumes were calculated using the following formula: Volume = \u03c0/6 \u00d7 (length) \u00d7 (width) \u00d7 (height). All animal care and handling procedures were performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board of Jinling hospital .Four-week-old male nude mice were purchased from the Model Animal Research Center of Nanjing University . After 4 days of acclimatization, a total of 7 \u00d7 10t-test. Comparisons between more than two groups were conducted using one-way analysis of variance (ANOVA), and the differences between groups were subsequently determined by the Fisher LSD test, when appropriate. Spearman's rank correlation analysis was used to examine the correlation between the relative expressions of miR-28-5p and RAP1B. A P value < 0.05 was considered statistically significant. SPSS version 13.0  software was used for all data analyses.Each experiment was conducted at least three times. The results are presented as means \u00b1 s.d. The statistical significance of the differences was evaluated using Student's"}
+{"text": "Symbiont-bearing Larger Benthic Foraminifera (LBF) are ubiquitous components of shallow tropical and subtropical environments and contribute substantially to carbonaceous reef and shelf sediments. Climate change is dramatically affecting carbonate producing organisms and threatens the diversity and structural integrity of coral reef ecosystems. Recent invertebrate and vertebrate surveys have identified the Coral Triangle as the planet\u2019s richest center of marine life delineating the region as a top priority for conservation. We compiled and analyzed extensive occurrence records for 68 validly recognized species of LBF from the Indian and Pacific Ocean, established individual range maps and applied Minimum Convex Polygon (MCP) and Species Distribution Model (SDM) methodologies to create the first ocean-wide species richness maps. SDM output was further used for visualizing latitudinal and longitudinal diversity gradients. Our findings provide strong support for assigning the tropical Central Indo-Pacific as the world\u2019s species-richest marine region with the Central Philippines emerging as the bullseye of LBF diversity. Sea surface temperature and nutrient content were identified as the most influential environmental constraints exerting control over the distribution of LBF. Our findings contribute to the completion of worldwide research on tropical marine biodiversity patterns and the identification of targeting centers for conservation efforts. Reefs of Southeast Asia have been identified as the most extensive  and diverse of the world2 but are also among the most vulnerable3, since coral cover is rapidly declining4. About 75 percent of the world\u2019s reefs are currently threatened3 by rising temperatures, climate change, and direct human perturbations1. This applies particularly to the Philippines that are facing high population pressures coupled with severe exploitation of marine resources5.Warm-water coral reefs are exceptionally diverse ecosystems that are home to more than three million species6. The best explored and resolved biogeographic patterns in the Indo-Pacific are those of corals and shore fish9. To date, a total of 627 species of scleractinian corals, representing 74 percent of all coral species worldwide, have been identified in the Coral Triangle7. The Coral Triangle is defined as a roughly triangular area that includes seascapes of the Philippines, Malaysia, Indonesia, Papua New Guinea, the Solomon Islands and Timor-Leste  are most diverse in the western part of the Coral Triangle (Philippines and large parts of eastern and southern Indonesia)13. Shallow-water benthic foraminifera from the tropical, subtropical, and temperate Indo-Pacific have been extensively studied since the 1800s and were later complemented by comprehensive surveys from previously largely unexplored areas , constitute a polyphyletic functional group that is highly adapted to oligotrophic conditions and restricted to the photic zone in warm waters between latitudes 40\u00b0N and 31\u00b0S21. Large-scale studies on latitudinal diversity gradients have been performed on Atlantic deep-sea foraminifera22 but not on LBF. Biogeographic studies on generic-level show that LBF diversity is highly correlated to that of scleractinian corals19 and assumed to peak within the Indo-West Pacific where it was established during the Miocene26. The center of diversity in Indo-Pacific LBF, however, remains ambiguous.Previous biogeographic analyses on benthic foraminifera have been conducted on morphospecies as well as on genetic phylotypes28. Here, we apply an overlay of single SDMs using MaxEnt and, for comparison, an overlay of minimum convex polygon range maps to identify the center of species richness in Indo-Pacific LBF. The species distribution data is based on occurrence records of 68 LBF species which we identified and synonymized for the Indian and Pacific Ocean. This first evaluation on species-level aims to contribute to the completion of the overall picture of global tropical marine biodiversity patterns and helps to refine the delineation of high-priority areas for conservation.Species distribution modeling (SDM) has become a useful tool to predict and quantify the distribution of taxa in geographic space with applications in such diverse fields as setting up conservation priorities, testing biogeographic hypotheses or assessing the impact of human-induced perturbations29 but not all of them have been examined from their type localities. Several LBF species are particularly rare or endemic and have not yet been subjected to molecular analysis. There is also still disagreement about the number of valid subfamilies, genera, and species among researchers31. In general, benthic foraminiferal diversity is presumed to be rather under- than overestimated32. Several studies have reported high species richness (>30 taxa of LBF) from localities in the Central Indo-Pacific  and Pacific Ocean. These include 3 species within the Alveolinidae, 21 species within the Peneroplidae, 9 species within the Soritidae, 6 species within the Amphisteginidae, 14 species within the Calcarinidae, and 15 species within the Nummulitidae Table\u00a0. To dateic Table\u00a0 and a to34 and records from the scientific literature . Two different methods have been applied in order to improve the informative value: (1) an overlay of individual Minimum Convex Polygon (MCP) range maps with no further consideration given to habitat suitability and (2) an overlay of species ranges as predicted by Species Distribution Models (SDMs). Both richness maps are hybrids as species occurrences that allowed no SDM performance or creation of MCPs were included subsequently by buffering and merging each point data with a 500\u2009km radius. The maps were created using ArcMap 10.3.1 for Desktop and projected onto the WGS 1984 PDC Mercator coordinate system centered at 180\u00b0 longitude. The maps have a 2.5-arcminute (of a longitude/latitude degree) spatial resolution, equivalent to about 4.5\u2009km at the equator. In order to quantify potentially colonizable areas, Coral Reef regions layers from The IUCN Red List of Threatened Species\u2122 were buffered with a 100\u2009km radius and used as a mask, restricting the projection and prediction of the species ranges on areas LBF are generally distributed. The georeferenced landscape is a 1:10\u2009m scale and was obtained from Natural Earth free vector and raster map data.Individual grid-based range maps of the Indo-Pacific symbiont-bearing larger foraminifera species were established and subsequently combined to richness maps. All data for creating the maps and richness gradients were processed in R , pH (ph), dissolved oxygen , phosphate , nitrate , salinity (PSS), silicate , as well as chlorophyll a concentration , cloud fraction , diffuse attenuation coefficient at 490\u2009nm ; photosynthetically available radiation , and sea surface temperature .The original set of environmental variables comprised calcite concentration  over 0.5 (no better than random) to 1 (perfect discrimination). AUC values greater than 0.9 show very good, greater than 0.8 show good and greater than 0.7 show useful discrimination ability of the model41. For the evaluation of the model performance, it is referred to the AUC test values that are given for each of the species involved. For more details on the operating mode of MaxEnt and the interpretation of its output see Elith et al.42.The interpretation and predictive performance of the model can be evaluated through the logistic output that MaxEnt provides per default. The Area Under the Receiver Operating Characteristic (ROC) Curve (AUC) is a common measure of model accuracyAs SDM predicts the occurrence of species solely based on selected abiotic environmental variables, occurrences have been provided with a buffer of 2,500\u2009km for selected species with limited distribution ranges Table\u00a0.The SDM output was subsequently used for creating boxplots in a 3\u00b0 resolution over latitudinal and longitudinal grid space. These boxplots were then merged into area charts to visualize richness/suitability gradients for LBF in the Indo-Pacific realm.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The maps derived from the Minimum Convex Polygon (MCP) overlay and the Species Distribution Model (SDM) overlay show largely congruent main richness patterns by revealing the Central Indo-Pacific, and particularly the central Philippines as the center of species richness in symbiont-bearing larger benthic foraminifera  and the inversely related importance of diffuse attenuation and chlorophyll a concentration , indicated by their highly negative contribution  and maximum cloud cover (cloudmax), and strongly negatively correlated with dissolved oxygen (dissox) and maximum photosynthetic available radiation (parmax). PC3 is slightly positively correlated with sea surface temperature variables  and strongly positively correlated with mean photosynthetic available radiation (parmean). PC4 and PC5 are strongly positively correlated with nutrient variables , and PC5 is mostly driven by a slightly positive correlation with the range of chlorophyll a concentration (chlorange). The evaluation of the variable contribution implies that for most of the species PC4 had the highest explanatory power  transformed the set of original environmental variables obtained from Bio-ORACLE into different sets for modeling application in MaxEnt. PC1 is slightly positively correlated with sea surface temperature variables . At around \u2212140\u00b0 a final peak occurs around the Tuamotus (max. 20 species) in the tropical central Southern Pacific. Further to the eastern Pacific margin, species richness/suitability declines sharply and remains low (<6). It drops significantly within the longitudinal range of the Eastern Pacific Barrier, an area that stretches diagonal south-eastwards from Hawaii through the Pacific. Longitudinal maximum values (<20) of richness at the eastern Pacific margin refer to very restricted and isolated areas of a few kilometers.The latitudinal gradient of Indo-Pacific LBF richness shows a broad unimodal and asymmetric pattern with the highest peak around 10\u00b0 North corresponding to the center of richness in the Philippines Fig.\u00a0. Almost 43. LBF species richness decreases from its center in latitudinal and longitudinal directions. The decrease towards higher latitudes and towards the eastern margins of the oceans is a general pattern in tropical marine biodiversity44. The asymmetric pattern of the LBF latitudinal richness gradient is in agreement with previous analyses on single and overall latitudinal marine richness gradients45. It has been identified to be mainly driven by temperature and is most sharply delineated by the extent of the 20\u2009\u00b0C winter isotherm46. Our findings of a sharp decline between 15 to 20\u00b0 North and South support the notion that the 20\u00b0 winter isotherm has a strong effect on the richness pattern in LBF. The longitudinal gradient, in turn, very well reflects the dependence on available shallow water habitat. Peaks in richness correlate directly with the position of islands, shallow seas, and continental shorelines. The extreme low at around \u2212120\u00b0 reveals the impact of deep water expanses (i.e. Eastern Pacific Barrier). Both land-barriers and deep water expanses have been previously identified as the most effective boundaries for tropical marine shelf biotas46.The species richness suggested by the Species Distribution Models (SDMs) correlates well with the stacked Minimum Convex Polygon (MCP) pattern, and generally well with empirically observed records of regional LBF species richness. This first LBF species-level modeling provides strong support for previous observations assigning the Central Indo-Pacific as the center of tropical marine biodiversity12 and especially with patterns of shore fish and invertebrates12,. The area of maximum richness identified for habitat-forming taxa , in turn, is somewhat extended and includes large parts of southern and eastern Indonesia12. So far, the highest number of coral species was recorded in the Sulu Sea ecoregion and comprises Palawan, parts of Borneo, and parts of Mindanao47. However, as research is ongoing and several ecoregions are under revision, these scores are subject to change7.In both of the models, richness is highest in tropical and subtropical waters in the Indian and Pacific Ocean. Additionally, this evaluation is the first to identify a defined geographic region of maximum species-richness in LBF, namely the central Philippine archipelago. Our analyses show the highest species richness scores and the largest extent of most suitable area within the Philippine Visayas region (56 species max.). The area where most distribution ranges overlap appears to also offer the most suitable environmental conditions for LBF. The main pattern agrees with findings reported on overall tropical marine biodiversityAlthough the Philippines might seem well-sampled Fig.\u00a0, most ofContrary to the MCP, the SDM projection allows a distinction of habitat suitability over the full geographic space including unsampled areas, and thus identifies coldspots and hotspots of potential LBF species richness within the center of biodiversity. According to the SDM, larger areas with high scores outside the Philippines are also indicated for central and eastern Indonesia, the northernmost coast of Australia, southern Papua, the Great Barrier Reef and the Torres Strait.43. Areas with a deep photic zone and moderate hydrodynamic energy are most likely to exhibit high numbers of LBF species, as this promotes the occurrence of species that are specialized to narrow ranges of light intensities along the depth gradient24. Species diversity is further known to be linked to available area/habitats25. Larger areas usually offer higher spatial diversity (i.e. habitat heterogeneity), and also allow species to have larger spatial ranges and a larger population size. This, in turn, reduces the risk of extinction and promotes vicariance. Available nearshore habitat (i.e. coastline length), was recently identified as the best predictor for species richness and overall biodiversity in the Central Indo-Pacific, followed by habitat heterogeneity and sea surface temperature12. Most available nearshore habitat is found in the central Philippines and in eastern Indonesia, as both areas harbor several hundreds of smaller islands and islets that offer a great variety of habitats and resources12. Additionally, the geological history of the Philippines is highly complicated48 and may have contributed significantly to the diversification of the region8. It is assumed that island integration events during the Miocene and Pliocene promoted bioconcentration and an amalgamation of separately evolved faunas8. Vicariance events49 like the Pleistocene isolation of sea basins50 might have further stimulated speciation, especially within the central Philippines8. Today, the Indo-West Pacific, the Great Barrier Reef, and the tropical western Indian Ocean are characterized by a high level of connectivity and were identified acting as a source for larval dispersal51.Unevenly distributed species diversity across taxa within the center of biodiversity is related to strong dependency on habitat heterogeneity, e.g. cross-shelf gradients in salinity, turbidity, water energy levels and substrate types44. Only a few\u201dtranspacific\u201d species of reef organisms are found both in the central Indo-Pacific and in the Eastern Tropical Pacific (ETP)52. Our analysis reveals the same general pattern. Among all 68 LBF species analyzed in this study, at least eight have a transpacific distribution with occurrences in the ETP (Table\u00a0Dendritina? culebraensis (McCulloch)) is probably endemic to the ETP (Table S2).The decrease of tropical marine biodiversity towards the coasts of the Eastern Pacific is steeper than towards the western margins of the Indian Ocean, especially beyond the Central PacificTP Table\u00a0. They re44. Prevalent environmental constraints are the limitation of available shallow water habitats, the eastward decrease of sea surface temperatures, the impact of the cold-water Peru and California currents, the lowering of the thermocline, the presence of upwelling zones at the eastern margins of the Pacific, and the isolation by the Eastern Pacific Barrier (EPB)44.The reef fauna of the ETP went largely extinct after the built-up of the Panama Isthmus during the Pliocene and, to date, remains depauperate53. To cross this most efficient marine biogeographic barrier, organisms are required to possess long-lived pelagic larvae or propagules that tolerate temporary exposure to low sea surface temperatures54. Based on individual survival capabilities, foraminiferan propagules settle and survive in a cryptic state at least for weeks to months in environments that are potentially unfavorable for adult specimens55. Adult forms, in turn, are dispersed passively by ocean currents, by attaching to floating objects or migrating organisms, by anthropogenic vectors, or within the fecal pellets of herbivorous fish57. Oceanic currents function as vectors for dispersal, or as physical barriers for passive dispersing of marine biotas where the directionality of the prevailing current impedes faunal connectivity and gene flow56. Although El Nino events may potentially facilitate eastward directed range extensions of tropical species53, eastward dispersal across the EPB and a replenishment by western population sources towards the ETP are considered highly unlikely58. This suggests that the ETP LBF biotas have been largely isolated from central Pacific propagule supplies.The EPB is a broad and deep stretch of open ocean lacking stepping stone islands or atolls facilitating the dispersal of warm water biotas across biogeographic boundaries. It has a long geological record and has persisted throughout the Cenozoic59. Our SDMs provides strong support for this observation , that represent transitional zones between the central Indo-Pacific and the western Indian Ocean biotas60, may facilitate dispersal and range expansion over large distances and potentially act as stepping stones60. However, in contrast to the Maldives that have a generally rich benthic foraminiferal fauna61 and are characterized by the presence of several central Indo-Pacific LBF taxa13, the LBF fauna of the Chagos Archipelago is relatively depauperate62. The low LBF diversity is reflected by the islands isolated position62 and the restricted richness shown in our SDM projection. In the western Indian Ocean province that includes the Chagos Archipelago60, characteristic central Indo-Pacific LBF taxa  are absent and partially substituted by morphologically similar species (e.g. Cycloclypeus carpenteri by Heterocyclina tuberculosa)13.The overall richness pattern in the Indian Ocean reveals a fundamental difference from that in the Pacific Ocean as tropical marine biodiversity increases towards the western margin of the Indian Ocean ion Fig.\u00a0. The coa35 but possibly identify priority sites for future colonization. Because previous analyses indicate that the observed LBF diversity matches on average 35% of the true diversity19 additional sampling activities may be required. For the Red Sea, the second most diverse region of the Indian Ocean59, the number of LBF species predicted to occur was consistent with the total number of species recorded so far (20 species).Within the northern Mozambique Channel, the SDM projection suggests suitable environmental conditions for up to 32 species. Actual species records reveal a range overlap of 21 species in this area with maximum records of about 14 species for individual sites .The geographic coverage on the distribution of recent Indo-Pacific LBF has steadily improved since the mid to late 1990s. The delineation of biogeographic patterns, however, still constitutes a challenge as LBF distribution is often patchy and species-level taxonomy requires further efforts. Most of the studies included herein focused on shallow water depths within the limit of recreational diving (<50\u2009m). Fewer studies included grab-sampled stations at greater depth and deeper dwelling species of LBF generally occur more sporadically in the literature. Future developments in LBF biogeography, including continuous efforts to resolve molecular species identifications, are likely to improve the resolution of the observed distribution ranges and taxonomic relationships. However, the species richness pattern identified herein is strongly supported by its remarkable similarity to those of other tropical marine taxa in revealing the Philippines as the bullseye of tropical marine biodiversity.71. This study contributes to the completion of the overall picture of tropical marine biodiversity and the knowledge of richness patterns may aid our understanding to target conservation actions72.In view of today\u2019s rapid global warming, the outlook for tropical reef calcifiers is alarmingSupplementary InformationSupplementary Table S2"}
+{"text": "Periodontitis is a chronic disease that begins with a period of inflammation of the supporting tissues of the teeth table and then progresses, destroying the tissues until loss of the teeth occurs. The restoration of the damaged dental support apparatus is an extremely complex process due to the regeneration of the cementum, the periodontal ligament, and the alveolar bone. Conventional treatment relies on synthetic materials that fill defects and replace lost dental tissue, but these approaches are not substitutes for a real regeneration of tissue. To address this, there are several approaches to tissue engineering for regenerative dentistry, among them, the use of stem cells. Mesenchymal stem cells (MSC) can be obtained from various sources of adult tissues, such as bone marrow, adipose tissue, skin, and tissues of the orofacial area. MSC of dental origin, such as those found in the bone marrow, have immunosuppressive and immunotolerant properties, multipotency, high proliferation rates, and the capacity for tissue repair. However, they are poorly used as sources of tissue for therapeutic purposes. Their accessibility makes them an attractive source of mesenchymal stem cells, so this review describes the field of dental stem cell research and proposes a potential mechanism involved in periodontal tissue regeneration induced by dental MSC. Periodontitis is a chronic inflammatory disease of the supportive tissues of the teeth. This disease is caused by specific microorganisms or groups of specific microorganisms, which result in a pathological disinsertion of the collagen fibres of the cementum; progressive destruction of the periodontal ligament and alveolar bone with increased probing depth formation, recession, or both; and apical migration of the union epithelium .When these conditions last over time, they cause the tissue to continue to be destroyed until the tooth is lost due to lack of support. This not only has repercussions at the local level that affect the chewing, phonation, and aesthetics of the patient but it is also related to other pathologies that affect quality of life .Although the disease can be treated successfully in its early stages, unfortunately, it is diagnosed when it affects the periodontal ligament, which causes most patients to seek dental care when the disease is very advanced, and the chances of keeping the tooth in the mouth are minimal. Consequently, different therapeutic options focus on recovering the lost health of the tissues . The conventional treatment consists of emphasizing hygiene, performing scaling and root planing, providing antibiotics, and, occasionally, performing flap surgery to access the root surfaces to debride them properly .These actions stop the acute phase of the disease, and sometimes a significant amount of new connective tissue insertion is recovered; however, the regeneration of the complex structure of the periodontium is not achieved. Conventional treatment relies on natural and synthetic materials that fill defects and replace lost dental tissue, but these approaches are not substitutes for a real regeneration of tissue with a physiological architecture and function. To address this, there are successful engineering initiatives. The purpose of tissue engineering is the regeneration of tissues through the combined use of biomaterials and biologic mediators in order to create living tissues that can replace structures or functions that have been lost. In this sense, there are several approaches to tissue engineering for regenerative dentistry, among them, the use of stem cells. In particular, stem cells have great versatility at the level of tissue regeneration for many different characteristics and can modulate chronic inflammation, a central feature in periodontitis. Given the characteristics of these cells, they are considered a potentially useful tool for efficient regeneration of periodontal tissues .Therefore, this mini-review presents the basic concepts of periodontitis, the mesenchymal stem cells of dental origin that are used in the treatment, and the possible molecular mechanism involved in the regeneration of periodontal tissues.Periodontitis is a condition that destroys the tissues around the tooth, which evolves to tooth loss and triggers various complications at the local and systemic level ,6.Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and, specifically, those of the red complex: Porphyromonas gingivalis , Tannerella forsythia (T. forsythia), and Treponema denticola [Periodontitis is one of the diseases in which the importance of an imbalance of species of pathogenic and beneficial microorganisms is well demonstrated. Dysbiosis in the initial stage is generally induced by poor hygiene that causes the accumulation of different types of bacteria such as enticola .Although periodontitis is initiated by an imbalance that causes the accumulation of these bacteria and their lipopolysaccharides (LPS), the destruction of the supporting tissues of the tooth is mainly due to an exacerbated immune response of the host in susceptible individuals, which prevents the acute inflammation from being effectively resolved and initiates chronic periodontitis . Figure. In thesThis allows the macrophages that have arrived at the area of the lesion to produce prostaglandin 2 (PGE2). High levels of this molecule and IL-1\u03b2 increase the binding of PMNs and monocytes to endothelial cells, exacerbating inflammation, which, together with IL-6 and TNF-\u03b1, induce osteoclasts to activate and reabsorb the alveolar bone ,11.Meanwhile, local capillaries release a large amount of serum as a result of the release of histamine and complement molecules, which leads to increased vascular permeability. This serum is converted into a tissue fluid that contains inflammatory peptides  that are carried into the gingival sulcus. Increased gingival fluid causes the tissues and the amount of gingival crevicular fluid to increase in volume . MacrophUnder normal conditions, antioxidant mechanisms protect the tissues from damage mediated by ROS. However, if the body\u2019s antioxidant capacity is insufficient against ROS, oxidative stress (OxS) occurs that damages the hard and soft tissues of the periodontium ,15.OxS causes oxidation of important enzymes, stimulation of release of more proinflammatory cytokines, lipid peroxidation, and damage to DNA and proteins. These mechanisms affect the gingival tissues, periodontal ligament, and alveolar bone that support the teeth ,17. In aIf this situation is sustained, the epithelial adhesion is destroyed, and the alveolar crest loses its height, which translates clinically into dental mobility and formation of periodontal pockets, causing the accumulation of more bacteria that increase the problem, thereby completely destroying the periodontal ligament; the alveolar bone becomes atrophied, and the tooth is lost ,20.To avoid this outcome, conventional treatment for periodontitis patients is divided into three different phases, which often overlap. The initial phase is focused on stopping the progression of destruction of periodontal tissues by eliminating local factors through oral hygiene instructions combined with scaling and root planing. The second phase is corrective and is aimed at restoring the function and aesthetics of tissues, while the last phase is considered periodontal maintenance that is intended to prevent the recurrence of periodontitis .Even when this treatment is carried out with rigor, the results are mostly aimed at the stabilization of the disease and not the regeneration of the lost periodontal tissues . TherefoDespite the use of these treatments, the original anatomy and physiology has not been restored, and in some cases, periodontal aberrations such as ankylosis, gingival recession, and formation of compact bone have developed ,24. TherMesenchymal stem cells (MSCs) are adult connective tissue cells that originate from the mesoderm. A stem cell is an undifferentiated cell that is distinguished by its capacity for autoregeneration (which perpetuates the population of stem cells) and by its capacity for asymmetric division, which indicates a daughter stem cell and another with greater commitment to proliferation and cell differentiation ,28 Figu.Stem cells can be isolated in the early stages of embryogenesis (embryonic stem cells) or in various postnatal tissues (adult stem cells). Although embryonic stem cells have a long lifespan and potential for high differentiation, the bioethical aspects involved in their production have focused the research on adult stem cells, which are considered pluripotent and found in most tissues, with the potential to repair damaged tissues or renew cell populations that are constantly being replaced ,30,31,32MSCs are involved in growth, wound healing, and replacement of cells that are lost daily by exfoliation or in pathological conditions. Different studies have shown that they induce repair in neuronal, hepatic, and skeletal muscle after infusion in both preclinical and clinical models ,34,35. TAnother advantage of MSCs is that they can be obtained from various sources of adult tissues such as bone marrow, adipose tissue, skin, and tissues of the orofacial area ,42,43,44Within the orofacial area, there are several sources of MSCs, including periodontal ligament stem cells (PL-MSC), apical papilla derived stem cells (SCAP), dental follicle cells (DFC), and dental pulp mesenchymal stem cells (DP-MSC) from both deciduous (SHED) and permanent teeth (DPSC). They all have attracted scientific interest, given their similarity to MSCs derived from bone marrow, their immunoregulatory capacity, and their minimally invasive obtention procedure ,49.The human periodontal ligament (PDL), a fibrous connective tissue that surrounds and supports the tooth, contains a small subpopulation of MSC that is responsible for maintaining and regenerating periodontal tissue structure and function. These cells form osteoblasts, fibroblasts, and tooth cementoblasts that form cementum- and periodontal ligament-like tissues .SCAP and DFC are stem cells that are located only in the developing tooth germ before they erupt into the oral cavity; SCAP is at the tips of growing tooth, and DFC is in a connective tissue sac surrounding the enamel organ and dental papilla. These cells form adherent clonogenic clusters and similar to other MSC populations; they differentiate into adipocytes and odontoblasts/osteoblasts , progeniDP-MSC are cells in the pulp chamber of the tooth that originate from the cranial neural crest. They have clonogenic capacity and rapid proliferative rates, can differentiate into various cell types such as chondrocytes, adipocytes, and odontoblasts, and can even be reprogrammed into myocytes or neurons. Similar to bone marrow, the type of surface markers they express are Stro-1, CD29, CD73, CD90, CD105, and CD166, and they are negative for haematopoietic markers such as CD14, CD45, CD34, CD25, and CD28 .The differentiation of DP-MSCs and PL-MSC into the different cell strains is determined by the microenvironment surrounding the cells, such as growth and transcription factors, molecular receptors, and extracellular matrix proteins. In addition, both in vitro and in vivo studies have shown that both have the ability to form mineralized tissues ,57,58,59One of the advantages of MSCs of dental origin is that they can be safely used in allogeneic transplants, because they have immunosuppressive properties such as those found in bone marrow-derived cells . Good imIn addition to their immunomodulatory function, they also have an anti-inflammatory function, which suggests that they can stop the development of lesions and allow the intrinsic regenerative processes to have a greater possibility of success. In this sense, MSCs of dental origin may have a direct or indirect effect on the control of OxS processes and the inflammation characteristic of periodontitis, since it has been suggested that these cells have an anti-inflammatory effect that could help to reduce the high levels of ROS and cytokines that are present in periodontitis .These cells have demonstrated a strong ability to induce bone formation in vivo. According to investigations, DP-MSCs of exfoliated teeth cannot differentiate directly into osteoblasts but may induce the formation of new bone by forming an osteoinductive template to recruit osteogenic cells from the host . As DP-MOn the one hand, histological findings in animal models suggest that the application of MSCs of dental origin can promote the regeneration of infra-bone defects. Rats with bone defects that were treated with PL-MSCs presented a higher percentage of bone-type filler after surgery, as well as the generation of structures similar to cementum and periodontal ligament after 21 days of treatment . SimilarOn the other hand, a study that aimed to understand the effect of DP-MSCs in a preclinical miniature pig model demonstrated that allogeneic transplantation of DP-MSCs from deciduous teeth can effectively repair the loss of hard and soft tissue caused by periodontitis without adverse effects .To accelerate the repair of artificially created dehiscence to emulate the tissue destruction of patients with advanced periodontitis, a study was conducted in sheep in which PL-MSCs were placed in a collagen scaffold. The results showed extensive coverage of new alveolar bone with a cell line similar to osteoblasts at 4 weeks of treatment .On the other hand, it has been observed that a functional connection between the cementum, ligament, and alveolar bone requires cells, a series of chemical signals and a physical support called scaffolding. In this regard, it has been shown that DP-MSCs promoted regeneration in periodontal defects when placed in combination with biomaterials .Likewise, it has been found that DP-MSCs placed in a collagen scaffold can be used to repair bone defects in humans, since this allograft produces rapid regeneration of alveolar bone in both quality and quantity of tissue compared with techniques that use bone from various sources for guided regeneration .In this sense, Aimetti et al. described a clinical case of a periodontal defect in a 56-year-old male treated with DP-MSCs obtained from one of his third molars. At one year after surgery, it was observed clinically and radiologically that the defect had been completely filled by bone-like tissue .For the transplantation of dental origin MSCs to regenerate injured periodontium, it is essential to have at least three factors: blood supply, adequate molecular signals and proliferation, and differentiation towards the cellular precursors that are capable of regenerating the lost tissues. .During the formation of new periodontal tissues, it is necessary to have sufficient blood supply, because the molecules that are necessary for regeneration will arrive through vessels. DP-MSCs can exert a neovascularization effect that induces early granulation tissue, which, in the case of periodontitis, is followed by the formation of a vascular network in the periodontal niche ,87. There is even evidence that after transplantation of DP-MSCs from deciduous teeth, they can differentiate into functional blood vessels that connect to the recipient vessels .To have a suitable means for DP-MSC differentiation, it is necessary to have signals that arrive through neoformed blood vessels such as insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor \u03b21 (TGF-\u03b21), and hepatocyte growth factor (HGF) , which hTNF-\u03b1 has no direct effect on the proliferation or cell cycle of DP-MSCs; however, it increases the mineralization of the extracellular matrix and the expression of mineralization-related genes such as bone morphogenic protein 2 (BMP2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and collagen I (COL-I) via the NF-\u03baB pathway during osteogenic differentiation ,92.On the other hand, sufficient amounts of IGF-1 promote collagen synthesis in fibroblasts and differentiation into osteoblasts via the mTOR pathway and stimulate Runx2 and the production of osteocalcin . OsteocaThus, while stimulating the synthesis of an extracellular matrix that is later to be mineralized is one factor, there are also others that play a crucial role in periodontal regeneration such as extracellular matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) . MSC delOn the other hand, both monocytes and Th2 lymphocytes that travel in the bloodstream produce IL-10, which in turn favours the expression of osteoprotegerin (OPG) and negatively regulates RANKL, NFATc1, and M-CSF, thereby decreasing differentiation and activation of osteoclasts by reducing bone resorption. At the same time, the inflammatory environment caused by Th1 is reduced, and the expression of Th2 and the anti-inflammatory microenvironment are favoured .The third element to be taken into account is the DP-MSCs that are placed in the damaged periodontal tissue, which can proliferate and differentiate into the precursors of cementoblasts, osteoblasts, and fibroblasts that are responsible for the generation of alveolar bone, root cementum, and periodontal ligament, respectively.Oncostatin M (OSM), which is a cytokine of the IL-6 family, as well as IL-6 itself, induces differentiation into preosteoblasts, since IL-6 is related to the production of ALP, which is required for the formation of osteogenic nodules, promotes the mineralization of the extracellular matrix, and increases the expression of genes associated with osteogenesis using the JAK3/STAT3 signalling pathway ,98,99.In an in vivo analysis of tissue formation, it was shown that the DP-MSCs from permanent and deciduous teeth can differentiate and form bone . Finally, it has been observed that DP-MSCs differentiate into cementoblasts and fibroblasts, which, respectively, secrete the CEMP-1 protein and COL-1 for the synthesis of radicular cementum and collagen fibres of the periodontal ligament, respectively. In this way, the three elements contribute to functional periodontal regeneration . Figure.Research on mesenchymal bone marrow stem cells is a widely explored field of knowledge; however, those of dental origin are in an incipient stage. In this sense, it could be argued that we still lack the requisite biological understanding of these cells to apply them to a human patient. Even something as elementary as the choice of differentiation markers that would allow a unified international understanding of the use of MSC of dental origin is a recurrent but elusive theme in research. However, this does not seem to impede the race to position MSCs as an alternative therapy.Indeed, along with cutting-edge clinical trials, we continue to address issues that are related to the supply and procurement of enough cells for therapeutic purposes, their senescence prevention, and their guaranteed proliferation and expansion without detriment to their ability to differentiate. Beyond the questions of basic science, which will undoubtedly have to be resolved in the future, there are still issues that are related to in vivo functionality. Studies of xenotrasplant have shown that it is possible to form complex dentin, root cementum, periodontal ligament, tissues such as bone, etc. By transplanting MSCs of dental origin in various animal models, we have clear evidence that these stem cells have the potential to induce the formation of complex structures such as dental tissues. . HoweverThe available human studies suggest that there are no adverse effects or ethical implications, but there are still very few of them that are needed to reach definitive conclusions. The history of medicine has taught us that an emerging procedure can rise to triumph only to eclipse later due to the development of unacceptable side effects . This isBiological, molecular, and clinical evidence supports the potential use of MSCs as an alternative therapy in diverse pathologies. In this sense, the use of MSCs of dental origin to repair and regenerate periodontal tissues can offer advantages over other more exploited cellular sources. Because of its similarity to target tissue and remarkable accessibility, it seems reasonable to conclude that using MSCs of dental origin to treat periodontitis is the most logical option. In addition, it would entail the least risk, since the therapeutic cells come from the same tissue that is intended to be cured, which would not be the case of MSCs that originated from bone marrow or adipose tissue. While this assumption needs to be verified in future studies, it does not, however, diminish the prospects for therapeutic use of MSC."}
+{"text": "When it is coupled with magnesium polysulfide, which has high reactivity and is homogeneously distributed on carbon matrix, the Mg/S cells deliver a good cycling stability with a high discharge capacity over 1000 mAh g\u22121 for more than 50 cycles.Rechargeable magnesium/sulfur (Mg/S) batteries are widely regarded as one of the alternatives to lithium\u2010ion batteries. However, a key factor restricting their application is the lack of suitable electrolyte. Herein, an electrolyte additive that can reduce the polarization voltage is developed and 98.7% coulombic efficiency is realized. The as\u2010prepared Mg\u2010ion electrolyte exhibits excellent Mg plating/stripping performance with a low overpotential of 0.11 V for plating process, and high anodic stability up to 3.0 V (vs Mg/Mg Unlike bis(trifluoromethane)sulfonimide magnesium (Mg(TFSI)2), hexafluorophosphate magnesium (Mg(PF6)2), borofluoride magnesium (Mg(BF4)2), etc., MgCl2\u2010AlCl3 inorganic salt will not decompose on the surface of Mg anode and affect the Mg ion plating/stripping behavior. However, with a higher standard reduction potential (Al (vs SHE) = \u22121.66 V) compared with Mg, Al will codeposit with Mg, making a low columbic efficiency of magnesium anode. After a carefully screening, yttrium chloride (YCl3) is selected here as a potential candidate of electrolyte additive. This is because that Y ion has a low standard electrode potential (Y (vs SHE) = \u22122.372 V), preventing yttrium from precipitation during the magnesium plating process. Moreover, YCl3 is very reactive with water,2\u2010YCl3 as an inorganic electrolyte can avoid the decomposition of magnesium salt.Among all the energy storage devices, rechargeable lithium\u2010ion batteries (LIBs) represent the most advanced technology and dominate the commercial market. However, there exists concern on the limited availability of resources such as Co, Ni, and Li to meet the tremendous demand in the electric vehicles and grid energy storage. Therefore, exploring battery chemistries with abundant resources and low cost is necessary. With regard to these demands, one ideal cathode material is abundant, lightweight element sulfur which has been extensively investigated in lithium/sulfur battery with a theoretical energy density of 2500 Wh Kg3 is drastically different from AlCl3 used as a common additive in previous studies. In most electrolyte systems of Mg/S battery with AlCl3, the cycling life is poor and the capacity decay is fast from the second cycle.3 compared with Mg, necessitate an electrolytic conditioning process for efficient Mg electrodeposition.3 an unsatisfactory additive.YCl\u22121 and a long cycle\u2010life for more than 50 cycles is demonstrated for the first time.Another factor that results in a high polarization voltage and rapid capacity decay in Mg/S batteries is the solid sulfur cathode, which always suffers from uneven distribution of S and high polarization.Scheme2 (1 molar equiv.) with YCl3 (2 molar equiv.), was reacted at 120 \u00b0C in an ionic liquid, n\u2010methyl\u2010(n\u2010butyl) pyrrolidinium bis(trifluoromethanesulfonyl)imide (PYR14TFSI) . In the following step, equivalent volume of diglyme (DG) was added into the solution, and a transparent Y\u2010based electrolyte is obtained.The synthetic route for the Y\u2010based electrolyte is shown in Figure2+ on Pt. In the gavalnostatic test, a symmetric Mg/Mg cell is cycled at 2 h charging and discharging intervals under a current density of 0.5 mA cm\u22122 ) and polyether carbon (286.0 eV (CH2O)) . The surface species were also confirmed by O 1s spectra where the carbonyl (531.0 eV (C=O)), ether oxygen (533.0 eV (C\u2014O\u2014C)) was observed in O 1s spectra , which indicate that organo\u2010Mg compound and magnesium carbonate (MgCO3) are the main components at the fluctuation stage. While after conditioning, the SEI layer contains magnesium oxide (MgO) (530 eV) only . Oxide layer (specific resistance: 1 m\u03a9 cm to 1 G\u03a9 cm) usually has a better conductivity than carbonate and organo layers (specific resistance: 0.1\u20130.1 \u03c4\u03a9 cm), so carbonate and organo magnesium components in the SEI layer result in the high polarization voltage and fluctuation in the initial stage.To verify that yttrium is electrochemically stable during the Mg deposition process in the Y\u2010based electrolyte, the composition of deposited Mg is investigated by energy\u2010dispersive spectroscopy (EDS). A strong peak of Mg is shown in 2 Figure c. It kee2 Figure c inset a\u22121 || Mg (100 mAh cm\u22122) cell was measured under 0.5 mA cm\u22122 , where the polarization voltage did not show a dramatic increase within 146 cycles, illustrating an average CE higher than \u224897.3%. In contrast, in the Al\u2010based electrolyte, Al was codeposited with Mg during the process of Mg deposition . The oxidative stability of Al\u2010based electrolyte is narrow (2.8 V vs Mg/Mg2+) on Pt , and the polarization voltage is as high as 1.5 V at a current density of 0.5 mA cm\u22122 , which is not competitive compared with the Y\u2010based electrolyte.To study the electrochemical Mg plating/stripping behavior in the as\u2010prepared electrolyte, cyclic voltammetry (CV) measurements were performed at a scan rate of 25 mV sScheme8 catholyte. Mg powder (1 equiv.) and S powder (8 equiv.) are reacted in a basic solvent of N\u2010methylimidazole (N\u2010MeIm) at 95 \u00b0C overnight.8 is resulted. The MgS8 is then attached onto a graphene/carbon nanotube (G\u2010CNT) matrix to form the magnesium polysulfide (MgPS) composite cathode. The details are described in the Experimental Section in the Supporting Information.Figure3/2 peak at 164 eV  , the peak at 495 nm can be assigned to S82\u2212, however, the peaks of S62\u2212 (358 nm) and S3\u00b7\u2212 (616 nm) show up, due to partly disproportioned reaction occurs in Mg polysulfide solution.x, and S\u2014S bond in MgSx, respectively, and indicate the existence of polysulfide anions. The XPS results validate MgSx as the major component in the polysulfide electrolyte and are consistent with the XAS data and UV\u2013vis data. The presence of MgS results from the disproportionation reactions of MgPS. Figure 3 additive and AlCl3 additive, we compare the electrochemical performances of two different Mg/S cells: MgPS cathode in Al\u2010based electrolyte, and MgPS cathode in Y\u2010based electrolyte. Figure\u22121. When the MgPS cathode is used with the conventional Al\u2010based electrolyte, the performance in the first 10 cycles is maintained at 530 mAh g\u22121; however, its capacity starts to decay very quickly in the following cycles. Remarkably, when the Y\u2010based electrolyte is introduced, the capacity is greatly improved to more than 1000 mAh g\u22121 and the cell can be cycled for 50 cycles, in a sharp contrast with the previous case. It should be noted that the slightly lower capacity in the first few cycles can be ascribed to the activation process of MgPS in the Y\u2010based electrolyte. The typical voltage profiles of the cells with and without Y\u2010based electrolyte are compared in Figure 3\u2010based electrolyte and the homogeneously distribution of MgPS on the G\u2010CNT matrix. Electrochemical impedance spectroscopy (EIS) is performed for the two different Mg/S cells in order to compare their electrochemical kinetics. As shown in Figure In order to examine the different effects of YClFigure3 additive and MgPS cathode can promote the decomposition of aggregated MgS particles, which results in a reduced polarization voltage and a significantly increased sulfur utilization. To investigate the different effects between the YCl3 and AlCl3 additives on the decomposition process of MgS, density functional theory (DFT) calculations were performed to derive the energy profiles of reactions. In the charge process, two Cl will transfer from YCl3 (or AlCl3) additives to Mg on the surface of MgS, resulting in the decomposition of MgS and formation of MgCl2. The transferring processes of Cl can be divided into two steps and expressed in the following reactionsTo understand the different performances of these two cells, we performed post\u2010analyses in SEM. The SEM images of the different cathodes loaded on carbon nanofiber (CNF) at the discharged and charged states are shown in 3 (or AlCl3) to the surface Mg. Figure S8 (Supporting Information) shows the adsorption conformations of YCl3\u2212x + MgClx and AlCl3\u2212x + MgClx  on MgS (001) surface. It shows that the adsorption conformation of YCl3\u2212x is similar to that of AlCl3\u2212x. Figure S9 (Supporting Information) schematically illustrates the energy profiles of transferring processes of Cl from YCl3 and AlCl3 to the surface Mg on MgS (001) surface. The first Cl transferring from YCl3 (or AlCl3) to the surface Mg with energy barrier of 1.80 eV (1.63 eV), followed by the second Cl from YCl2 (or AlCl2) to MgCl with barrier of 2.57 eV (2.94 eV). The higher energy barrier of the two steps determines the overall energy barrier of the decomposition of MgS and the formation of MgCl2. The overall energy barrier of YCl3 with 2.57 eV is less than that of AlCl3 by 0.37 eV, which indicates that YCl3 additive can improve the decomposition of MgS in comparison with AlCl3 additive.We took the (001) surface of MgS in NaCl\u2010type structure as the model to investigate the transferring processes of Cl from the YCl2+), an ultrahigh Mg plating/striping Coulombic efficiency (\u224898.7%), and a long Mg plating/striping cycle life. When the Y\u2010based electrolyte is used together with the MgPS/G\u2010CNT cathode, the Mg/S cell demonstrates an ultrahigh capacity of 1000 mA h g\u22121 with an excellent cycling performance over 50 cycles. This new electrolyte additive offers new avenue for further development of better Mg electrolytes and practical Mg/S cells.In conclusion, a new type of inorganic Y\u2010based electrolyte with high plating/stripping coulombic efficiency is developed for Mg/S batteries. The newly designed electrolyte exhibits superior properties, including a good anodic stability , aluminum chloride , and anhydrous DG were received from Sigma\u2010Aldrich, PYR14TFSI was purchased from MTI Corporation, and YCl3 was prepared by Prof. Y. M. Yao in Soochow University. All reactants and solvents, unless otherwise stated, were used as received. All the samples were handled in an argon\u2010filled glovebox with water below 0.5 ppm and oxygen below 15 ppm. MgCl2 (19 mg) and anhydrous YCl3 (78 mg) (or MgCl2 (19 mg) and anhydrous AlCl3 (53.3 mg)) were added to a 10 mL glass vial, which was then vigorously stirred at 120 \u00b0C in 1.5 mL PYR14TFSI overnight. Then 1.5 mL DG was added with stirring overnight at room temperature to form the Y\u2010based electrolyte. Treatment of 16.7 mg of Mg (0.688 mmol), 180 mg of sulfur (0.702 mmol), and 3 mL of N\u2010MeIm at 95 \u00b0C for 12 h to obtain a red solution. Then 360 mg G\u2010CNT was added and stirred overnight. 60 mg polyvinylidene fluoride (PVDF) dissolved in NMP was added to the above slurry. The resulting slurry was uniformly spread via a doctor blade on pyrolytic graphite. G\u2010CNT was pasted on pyrolytic graphite first and then MgPS solution was dropped onto G\u2010CNT. Then the electrode was dried at 60 \u00b0C in the glovebox. Typically, each electrode contains about 0.7\u20131.0 mg cm\u22122 of the active material.Materials Characterization: The morphologies of the samples were investigated using an FEI XL30 Sirion SEM operated at an accelerating voltage of 5 kV. XPS analysis was performed on an SSI SProbe XPS spectrometer with monochromatic Al K\u03b1 (1486.6 eV) radiation.Electrochemical Measurements: Electrochemical experiments were performed using CR2032 coin cells assembled in an argon\u2010filled glovebox with magnesium metal as the counter and reference electrodes and MgS8@G\u2010CNT as cathode. Galvanostatic cycling measurements were evaluated with a LAND battery test system. Cyclic voltammetry measurements were performed on a VMP3 potentiostat (Bio\u2010logic). EIS data were obtained on the same potentiostat from 200 kHz to 100 mHz with an AC voltage amplitude of 10 mV at the open\u2010circuit potential.Computational Details: Spin\u2010polarization DFT calculations were performed by using Vienna Ab initio Simulation Package (VASP).\u22121. MgS(001) surface was modeled by a five\u2010layer slab with 4 \u00d7 4 supercell and only \u0393 point was used. The bottom two atomic layers of the slab model were fixed during structural optimizaiton. To eliminate the interaction between periodic slabs, the vacuum layer was set to 20 \u00c5. DFT\u2010D3 method was used to describe van der Waals force.The authors declare no conflict of interest.SupplementaryClick here for additional data file."}
+{"text": "Alternatively, the thermodynamically favorable conversion reaction may circumvent the sluggish Mg2+ diffusion kinetics. In this review, the focus will be laid on promising cathodes beyond the typical intercalation-type materials. We will give an overview of the recent emerging Mg systems with conversion-type and organic cathodes.Rechargeable magnesium (Mg) batteries are an attractive candidate for next-generation battery technology because of their potential to offer high energy density, low cost, and safe use. Despite recent substantial progress achieved in the development of efficient electrolytes, identifying high-performance cathode materials remains a bottleneck for the realization of practical Mg batteries. Due to the strong interaction between the doubly charged Mg Efficient and cost-effective electrical energy storage system is regarded as the feasible solution for the implement of the renewable energy and carbon-free transportation. Lithium ion batteries (LIBs) have emerged as the state-of-the-art technology for powering today's portable electronics and have also been introduced for e-mobility and stationary storage applications. However, with the constantly increasing demands for long-lasting customer electronics and extended driving range for electric vehicles (EVs), the current advanced LIBs are not able to meet the energy requirements and the safety issue remains unresolved , high abundance in the earth crust and dendrite-free metal deposition , high theoretical volumetric capacity . The strong electrostatic forces between the Mg ions and the surrounding anions in crystal matrix induce the intrinsically slow kinetics of Mg2+ ion insertion and diffusion within the host cathodes. Consequently, when Mg2+ ions intercalate, the conventional oxide based cathode materials suffer from low capacity, high voltage hysteresis and undergo irreversible conversion reactions at the cathode surface. Use of \u201csoft\u201d chalcogenides would improve the performance, but it compromises the high voltage of the intercalation cathode yielding a low energy density for the full cell . In fact, the major factors hindering Mg2+ insertion/diffusion kinetics and can be a potential alternative to the intercalation chemistry. Conversion electrodes undergo a redox reaction that involves a chemical transformation by breaking and creating chemical bonds. Analogously to the concepts in Li-ion chemistry, the conversion reactions for Mg batteries can be generally expressed asConversion-type electrodes have been proposed as alternative candidates for Mg batteries because of the high theoretical capacities with multi-electron transfer and low cost . Among them, S and Se have been considered as particularly promising cathode materials for various battery systems including Li and multivalent metal-based systems  and halogens , low toxicity and abundance of sulfur as a cathode candidate. Lithium-sulfur (Li-S) battery, possessing a high theoretical energy density of 2,800 Wh l\u22121 and, has been suggested as low-cost post Li-ion system. However, use of the Li-metal anode induces severe safety issues associated with growth of Li dendrite during the cycling of the batteries. In contrast, dendrite-free deposition of Mg enables Mg metal an ideal anode material. Coupling a sulfur cathode with a Mg metal anode, based on a two-electron conversion reaction of Mg2+ + S + 2e\u2212 \u21c4 MgS, Mg-S battery offers a theoretical cell voltage of 1.77 V and an energy density of 1,722 Wh kg\u22121 and 3,200 Wh l\u22121, respectively, which renders Mg-S battery an attractive option for future battery generations  battery chemistries are receiving increasing attentions because of the high theoretical capacity  for Mg plating/stripping , followed by liquid\u2013solid reduction forming insoluble MgS2 and MgS  was first reported by Liebenow et al. . With th1 Figure   and improved reversible capacity upon cycling of the Mg-S batteries  filled with sulfur powder and a CNF-coated separator  cathode, respectively  electrolytes are generally comprised of binuclear [Mg2(\u03bc-Cl)3(THF)6]+ cation and aluminate anions in equilibria and their initial electrochemical properties could be severely deteriorated by the dissolved cathode or intermediates species  and selenium-sulfur 2 in dimethoxyethane (DME), in which the cells were operated at a low current rate imide  with Mg and MgCl2, where the S-CNT cathode could provide a relatively stable reversible capacity of about 400 mAh g\u22121 at a current rate of 500 mA g\u22121 for more than 100 cycles  compared to other reports. It needs to mention that the use of Cu current collector was shown to be beneficial for the Mg-S cell performance although the electrolyte was measured to be less stable on Cu. The corrosion issue caused by Cl\u2212 ions in these electrolytes would be a drawback for their practical use.Zhang et al. prepared boron-centered anion-based magnesium (BCM) electrolyte and probed its suitability for Mg-S and Mg-Se batteries 2 (MMC) is supposed to be non-nucleophilic but have not been tested for Mg-S battery so far. The ionic compounds of Mg fluorinated alkoxyborates and Mg alkoxyaluminates fulfill multiple requirements as electrolyte salts and can be universally incorporated with any type of cathode and anode materials (CF3)2) has been employed as the highly efficient and non-corrosive electrolyte 4]2 electrolyte. The discharge/charge profiles illustrate that discharge voltage remains at about 1.5 V for the extensive cycles at a current rate of 0.1 C. A discharge capacity of ~400 mAh g\u22121 was retained after over 30 cycles 2 battery has been demonstrated with a non-aqueous dual-electrolyte i.e., the catholyte comprised of Mg(TFSI)2 in glymes and the anolyte composed of Mg(TFSI)2 in an ionic liquid with additional MgBr2  can be used as a conversion-type cathode material, where I2 can be reduced into a soluble intermediate tri-iodide followed by a rapid reversible two-electron reaction of the \u2212 redox pair that can provide a theoretical capacity of 211 mA h g\u22121. With the bis-amide electrolyte, the couple of Mg-I2 has been demonstrated as a promising high power and high energy battery system 2 in \u03b3-butyrolactone as electrolyte, the redox reaction was confirmed with a 3-electrode setup 2 in EC/PC, Mg(ClO4)2 in PC and Mg(TFSI)2 in diglyme with poor electrochemical properties results in inferior battery performance, e.g., low discharge voltage, large charge over-potential and low reversibility  (PAQS) and various non-nucleophilic electrolyte based on Mg(HMDS)2-AlCl3, MgCl2-AlCl3, and Mg(TFSI)2-MgCl2 2-MgCl2 electrolyte with Mg powder anode. However, the capacity gradually dropped upon extended cycling and only <50 mAh g\u22121 was remained after 100 cycles 2-MgCl2 electrolyte in a mixed solvent of tetraglyme and 1,3-dioxolane. The characteristic IR band of the C = O functional group allows the convenient analysis of the reversible electrochemical mechanism in the quinone-type cathodes  and a mixture of TEGDME and 1.3-dioxlane (DOL)   for NP. The cells using the electrolyte in TEGDME/DOL showed superior electrochemical behavior with slow capacity fading and cycling good Coulombic efficiency (99%).A naphthalene-hydrazine diimide polymer (NP) has been tested as a cathode material for Mg organic batteries in two different electrolytes i.e., Mg(TFSI)\u22121 (based on the weight of the composite) at 1C for over 200 cycles in the Mg batteries using THF solution of phenylmagnesiumchloride-AlCl3 as electrolyte  based cathode material for both Mg and Al battery systems has been demonstrated In addition, organic materials are of great potential to be used as high capacity cathode for Mg batteries. The main drawbacks of organic compounds such as high solubility and low electric conductivity could be circumvented by molecular design and synthetic approaches. One of the particular advantages of organic electrodes for Mg batteries is that they are structurally diverse and their properties can be tuned by modifying the chemical structure, which provide an excellent opportunity to overcome some fundamentally limitations of Mg-ion transport achieving high-performance Mg batteries.ZZ-K conceived the concept of this review and wrote the manuscript. All authors discussed and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Cancer cells are highly reliant on certain molecular pathways, which support their survival and proliferation. The fundamental concept of molecularly targeted therapy is to target a protein that is specifically deregulated or overexpressed in cancer cells. However, drug resistance and tumor heterogeneity are major obstacles in the development of specific inhibitors. Additionally, many driver oncogenes exert their oncogenic property via abnormal expression without having genetic mutations. Interestingly, recent accumulating evidence has demonstrated that many critical cancer genes are driven by a unique class of enhancers termed super-enhancers. Genes associated with super-enhancers are relatively more susceptible to the inhibition of general transcriptional machinery compared with genes that are regulated by typical enhancers. Cancer cells are more sensitive to treatment with small-molecule inhibitors of CDK7 or BRD4 than non-transformed cells. These findings proposed a novel strategy to identify functionally important genes as well as novel therapeutic modalities in cancer. This approach would be particularly useful for genetically complicated cancers, such as adult T-cell leukemia (ATL), whereby a large mutational burden is present, but the functional consequences of each mutation have not been well-studied. In this review, we discuss recent findings on super-enhancers, underlying mechanisms, and the efficacy of small-molecule transcriptional inhibitors in ATL. Recent advancements in molecularly targeted therapy have yielded remarkable improvements in outcome for several cancers. Since the success of tyrosine kinase inhibitors such as imatinib and gefitinib in the early 2000s ,2, a numThe most fundamental concept of molecularly targeted therapy is to target a protein that is specifically deregulated or overexpressed in cancer cells to avoid non-specific cytotoxicity in non-transformed cells. Thus, many of the small-molecule inhibitors that have been developed to date were designed to directly target an oncoprotein (disease gene). On the other hand, several new drugs that affect general cellular machinery have been recently developed and shown to possess potent anti-tumor cytotoxicity. These new drugs include small-molecule inhibitors of CDK7, CDK9, and BRD4, which block RNA polymerase II or histone activation, leading to transcriptional inhibition ,17,18. AEnhancers are regulatory elements that are bound by transcription factors and activate transcription through interactions with promoter elements ,22,23,24T-cell receptor (TCR) or immunoglobulin (Ig) gene locus in lymphoid malignancies, which replaces the regulatory element of a translocation partner gene and drives its expression [MYC, BCL2, and TAL1, were identified from the breakpoints of chromosomal translocation in leukemias and lymphomas [TAL1 oncogene in a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases [TAL1 expression. Similarly, mutations or single nucleotide polymorphisms (SNPs) in regulatory elements at the LMO1 or LMO2 gene locus have been reported in neuroblastoma and T-ALL cases [TERT gene promoter that enhance its expression have been found in several cancers [EZH2, DNMT3A, MLL, and ARID1A are often found in hematological malignancies and solid tumors, leading to global alterations of the gene expression program [The abnormal activation or repression of enhancers is often found in cancer cells, and is closely associated with the misexpression of cancer genes ,24. A clpression . Many prymphomas . In thesL) cases . This inLL cases ,28,29. M cancers ,31,32. T cancers ,34. Mise program . TherefoGiven the importance of regulatory elements in normal development and pathogenesis, a current area of major research interest is the identification of regulatory elements using genome-wide technologies, such as ChIP-seq . This leOct4, Sox2 and Nanog\u2014were associated with super-enhancers in mESCs [MyoD were regulated by super-enhancers [TAL1 enhancer locus that was generated by a mutation [GATA3, RUNX1, and MYB genes, which are involved in the TAL1-induced core regulatory circuit [CDK6 oncogene. The association of super-enhancers with oncogenes is also evident in many other cancers. For example, super-enhancer-associated genes include IRF4 and MYC in multiple myeloma [PAX5, MYC, and IRF4 in diffuse large B-cell lymphoma [MYCN and ALK in neuroblastoma [CDK6, MYC, and EGFR in glioblastoma [In early studies by the Young laboratory, super-enhancers were analyzed in mouse embryonic stem cells (mESCs) and various differentiated cells. They were shown to be associated with critical genes involved in regulating cell fate and identification during normal development . For exain mESCs . In diffnhancers . Likewisnhancers . Similarnhancers . In T-ALmutation ,26 , were not associated with super-enhancers  of RNA polymerase II at the serine 5 residue, which is required for the initiation of transcription  Figure , middle.r 100 nM . T-ALL cith THZ1 . In contnhancers A, left, The aforementioned studies on BRD4 and CDK7 inhibitors highlighted the feasibility of inhibiting the general transcriptional machinery as a therapeutic approach against cancer. However, this result has also raised additional questions. Why are super-enhancer-associated genes more sensitive to these inhibitors than other genes? Is the super-enhancer functionally different from typical enhancers?Fundamentally, super-enhancers are defined by bioinformatics analysis B. Super-Another possible explanation is that super-enhancer-associated genes may have different kinetics in transcription. These transcripts could be relatively short-lived, and thus, transcription needs to be continuously activated to maintain their expression. It has been shown that the inhibition of CDK9, which facilitates transcription elongation, primarily affects the accumulation of transcripts with short half-lives, including genes encoding for anti-apoptosis family members and cell cycle regulators ,59. SimiThe development of transcriptional inhibitors provided a new option for cancer therapeutics. Additionally, determination of the susceptibility to transcriptional inhibition can be a novel strategy to identify the critical genes that are required for the maintenance of cancer cells. Early studies on super-enhancers and transcriptional inhibitors were done in T-ALL and neuroblastoma ,42, whicATL is a hematological malignancy derived from T-cells ,62,63. THBZ, which is continuously expressed throughout the course of the disease and is required for the maintenance of ATL cells and the immunophenotype of HTLV-1-infected cells [Importantly, the transformation process of ATL is highly complicated, and involves multiple mechanisms. The development of ATL is closely associated with the infection of human T-lymphotropic virus type-I (HTLV-1), which is endemic in certain parts of the world, including the southern part of Japan, the Caribbean, and South American and African countries . The vired cells . Notablyed cells ,62,63. Eed cells .PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%), and CCR7 (11%) in their cohort. They found that many of the mutated genes, such as PLCG1, PRKCB, CARD11, and STAT3, are involved in the T-cell receptor (TCR)\u2013NF-\u03baB signaling, T-cell trafficking, and immunosurveillance pathways. The accumulation of additional mutations in genes involved in the TCR-NF-\u03baB pathways, along with the inactivation of TP53 and CDKN2A and other mutations, is likely required for the transformation of T-cells into fully malignant cells.The significance of genetic abnormalities was further elucidated by a recent study using whole genome and exome sequencing technology . KataokaEZH2 gene, which encodes a component of the Polycomb repressive complex 2 (PRC2), was found to be significantly highly expressed and accompanied with enhanced H3K27me3 repressive chromatin marks in ATL cells compared to its normal CD4+ counterparts [In addition to genetic abnormalities, several groups have reported the involvement of epigenetic abnormalities in ATL pathogenesis . The EZHterparts . Integraterparts . All of these findings indicate that multiple oncogenic mechanisms are involved in ATL. Given the high mutational prevalence of ATL cells, it is difficult to determine the functional property of each mutated gene. Since ATL also exhibits high heterogeneity among patients, it is more reasonable to identify and target molecular pathways that are commonly deregulated across different ATL cases and are functionally important for pathogenesis. Therefore, it would be feasible to apply super-enhancer profiling to identify critical cancer genes and test transcriptional inhibitors in ATL.IL2RA/CD25, CD28, TNFRSF8/CD30, FYN, and NFATC1/2 were identified in our study. Interestingly, many of these genes were enriched in the TCR pathway, substantiating the finding by Kataoka et al., which reported that the TCR pathway was often affected by genetic mutations in ATL cells [In our latest study by Wong and Ngoc et al. , we perfTL cells .CD2, which has been recognized as a T-cell marker. The second consisted of genes that were associated with super-enhancers in ATL and normal mature T-cells  but not in normal thymus or T-ALL cells, including the CD25/IL2RA gene. This result supported previous findings that ATL arises from CD4+CD25+ mature T-cells [TP73, which is a member of the p53 tumor suppressor family. This gene is normally induced upon intracellular stress, such as DNA damage [TP73 super-enhancer may reflect the intracellular status of ATL cells. Taken together, our results supported previous findings in other cell types that super-enhancers are enriched in genes that characterize cell identity and fate.We then compared this result with profiles in normal T-cells  and T-ALL cells. Importantly, three distinct sets of genes surfaced from this analysis. The first set consisted of genes that were commonly associated with super-enhancers in ATL cells, normal T-cells, and T-ALL cells. This set included  T-cells ,74. LastA damage ,76,77, a50 values below 50 nM. THZ1 treatment efficiently blocked the phosphorylation of RNA polymerase II in sensitive ATL cell lines with a concomitant induction of apoptosis. This study highlighted the feasibility of CDK7 inhibition as a therapeutic option in ATL. Hence, we also performed the gene expression profiling after CDK7 inhibition to find genes that were susceptible to the transcriptional inhibition in ATL cells. We combined this result with super-enhancer profiling to identify potential cancer genes.In the same study, we also tested the efficacy of targeting CDK7 kinase in ATL cells . SeveralCCR4 gene to be regulated under super-enhancers in all of the examined ATL samples, but not in normal T-cells or T-ALL cells. The CCR4 protein has been known to be highly expressed in the majority of ATL cases [CCR4 was significantly downregulated upon THZ1 treatment, further indicating the involvement of this gene in the pathogenesis of ATL. Additionally, from this analysis, we identified a previously uncharacterized gene, TIAM2. This gene was also associated with super-enhancers in all of the examined ATL cases, but not in normal T-cells. The genetic knockdown of TIAM2 inhibited cell growth and induced apoptosis in ATL cells, whereas the overexpression of TIAM2 in T-ALL cells promoted cell growth. This result implicated TIAM2 as a novel candidate cancer gene in ATL. Thus, our approach has successfully demonstrated the potential application of super-enhancer profiling to identify critical cancer genes in genetically complicated cancers.Importantly, we have identified the TL cases ,79,80,81The use of small-molecule inhibitors of CDK9 has also been explored in ATL and HTLV-1-infected T-cells. The kinase CDK9 is the catalytic subunit of the P-TEFb complex, which activates RNA polymerase II by phosphorylating the serine 2 residue in CTD to facilitate transcription elongation  Figure , right. MYC and MCL1 expressions. The administration of BAY 1143572 in immunocompromized NOD/Shi-scid/IL-2R\u03b3null (NOG) mice xenografted with patient-derived ATL cells greatly reduced the infiltration of ATL cells into organs, such as liver and bone marrow. Decreased human soluble IL2R levels in serum were also observed, which indicated a reduction of ATL tumor burden. This study revealed the importance of CDK9-mediated transcription in ATL pathogenesis and implicated CDK9 inhibitors as a novel therapeutic agent for ATL.Recently, Narita and Ishida et al. investigated the therapeutic efficacy of this CDK9 inhibitor on ATL . BAY 114The small-molecule inhibitor of BRD4 has also been tested in HTLV-1-infected cells. It is noteworthy that the inhibition of BRD4 can exert an additional anti-cancer mechanism besides the inhibition of general transcriptional machinery in ATL or HTLV-1-infected cells. It has been reported that BRD4 mediates the activation of NF-\u03baB. Acetylation of the p65 subunit of NF-\u03baB at lysine 310 and the subsequent recruitment of BRD4 have been shown to be essential for the activation of NF-\u03baB in response to various stimuli . Upon thIL2RA and inhibited the growth of HTLV-1 infected cells. Of note, JQ1 also inhibits the proliferation of some Tax-negative HTLV-1 infected cells [Interestingly, treatment with the BRD4 inhibitor JQ1 has been shown to inhibit proliferation and induce apoptosis in Tax-expressing rat fibroblasts and Tax-positive HTLV-1 infected cells . JQ1 treed cells . This fiRecent mutational profiling by whole genome and exome sequencing has provided a large catalog of mutations in protein-coding genes. However, drug resistance and tumor heterogeneity are major obstacles in the development of specific inhibitors. Additionally, many driver oncogenes exert their oncogenic property via abnormal expression due to enhancer abnormalities, and do not have mutations within protein-coding elements. Therefore, it is critical to determine the driver abnormality that directly contributes to tumorigenesis. This is particularly evident for genetically complicated cancers, such as ATL, whereby a large mutational burden is present, but the functional importance of each mutation has not been well-studied.MYCN-driven neuroblastoma cells in which prolonged treatment with THZ1 resulted in an upregulation of the drug transporters responsible for extruding a variety of drugs [In this review, we discussed the therapeutic efficacy of transcriptional inhibitors as well as potential applications of super-enhancer profiling to identify cancer genes. Recent studies suggested that super-enhancers would be required to maintain the expression of critical cancer genes. This would be a common and fundamental mechanism to support cancer cell survival and proliferation, although the acting oncogenes could be different among various cancer types. Given the biological importance of super-enhancers in cancers, super-enhancers represent feasible therapeutic targets. The blocking of super-enhancer-driven transcription resulted in the acute and concurrent disruption of multiple oncogenic machineries to which cancer cells are addicted, regardless of their mutational profiles. Thus, the use of small-molecule inhibitors targeting transcriptional regulators, such as CDK7, CDK9, and BRD4, is an ideal approach. However, we are not optimistic, as no single inhibitor is likely to achieve a maximal anti-cancer effect. A combination treatment with existing therapies is required. Moreover, drug resistance towards THZ1 has already been reported in some cases of of drugs . Resistaof drugs . Hence,"}
+{"text": "Information on the association between earlobe crease (ELC) and peripheral artery disease is limited. We assessed this association in community-dwelling older adults. A total of 294 Atahualpa residents aged \u226560 years were enrolled. ELC were visually identified by two raters. The ankle-brachial index (ABI), used as a surrogate of peripheral artery disease, was categorized using American Heart Association criteria. Using logistic regression and probability models, adjusted for demographics and cardiovascular risk factors, we assessed the relationship between ELC and abnormal ABI determinations, as well as the influence of age on this association. ELC was identified in 141 (48%) individuals, and abnormal ABI determination was carried out in 56 (19%). The association between ELC and abnormal ABI was nonsignificant in logistic regression and probability models with individuals stratified according to their median age. The association between ELC and abnormal ABI determinations is probably attenuated by the high prevalence of both conditions in older persons. ELC might not be useful for identifying candidates for ABI determination. Earlobe crease (ELC), also known as Frank's sign, is a wrinkle extending from the tragus to the outer border of the earlobe . StudiesA recent study showing that ELC may detect asymptomatic individuals with peripheral artery disease (PAD) is promising , since uAtahualpa represents an optimal setting for the practice of epidemiological studies. As previously described, villagers are homogeneous regarding race/ethnicity, lifestyle, socioeconomic status, and diet . The IRBBoth earlobes were examined with the subject in the sitting position. According to Rodriguez-Lopez et al. , an ELC ABI determinations followed the recommendations of the American Heart Association . A manuaDemographics and cardiovascular risk factors, selected as confounding variables, were assessed through interviews and procedures previously described in the Atahualpa Project. These included age, sex, smoking status, physical activity, diet, the body mass index, BP, fasting glucose, and total cholesterol blood levels .x2 or Fisher exact test as appropriate. Using logistic regression models, we evaluated whether ELC was associated with an abnormal ABI (dependent variable), after adjusting for demographics and cardiovascular risk factors. We anticipated a potential effect modification of age in the association between ELC and abnormal ABI determinations and fitted multivariate probability models with participants stratified according to the median age of the population.Data analyses are carried out by using STATA version 14 . In univariate analyses, continuous variables were compared by linear models and categorical variables by Of 437 Atahualpa residents aged \u226560 years identified during door-to-door surveys, 294 (67%) underwent ABI determination and earlobe examinations. Of the 143 nonincluded individuals, earlobe deformities did not allow characterization of ELC in 14, and the ABI could not be obtained in 72 cases because of refusal to consent (n=57), leg amputations (n=10), and uncontrolled tremor in the legs (n=5). In addition, 57 subjects had died or emigrated between enrollment and the invitation.A logistic regression model, adjusted for demographics and cardiovascular risk factors, revealed no significant association between ELC and abnormal ABI determinations . In this model, the only significant covariate was age (p<0.001). Probability margins of abnormal ABI determinations, with individuals stratified according to their median age (69 years), showed that probabilities of abnormal ABI determinations nonsignificantly increased among older individuals with ELC .Results of this study suggest that the association between ELC and abnormal ABI determinations could be attributed to age. These findings might be explained by the high prevalence of both variables in older adults, confirming previous hypotheses that ELC and atherosclerosis could be related to the effects of aging , 11.In a study aimed at correlating ELC with ABI determination , 136 of  \u201cone of the underlying pathogenetic mechanisms involved in the progression of atherosclerosis, possible related to collagen metabolism, may also occur in the skin\u201d [There is limited information about pathogenetic mechanisms explaining how ELC might correlate with atherosclerosis. In an autopsy-based report, it was suggested thathe skin\u201d . It has he skin\u201d .The present study has limitations. Results may not be generalized to other races/ethnic groups but are of value for Amerindians, a population where the association between ELC and atherosclerosis has not been previously investigated. In addition, we cannot rule out the presence of some hidden confounders (such as asymptomatic coronary artery disease), which can modify the final results obtained in the multivariate model. Also, the possibility of a statistical type 2 error cannot be completely ruled out, because the statistical power of our sample was 57.5%. Major strengths include the population-based design with unbiased enrollment of participants and the models used to evaluate the influence of age on the association between ELC and abnormal ABI determinations.This study suggests that the association between ELC and abnormal ABI determinations is attenuated by the high prevalence of both conditions in older persons. ELC might not be useful for identifying candidates for ABI determination."}
+{"text": "Lipids are key molecules in various biological processes, thus their quantification is a crucial point in a lot of studies and should be taken into account in lipidomics development. This family is complex and presents a very large diversity of structures, so analyzing and quantifying all this diversity is a real challenge. In this review, the different techniques to analyze lipids will be presented: from nuclear magnetic resonance (NMR) to mass spectrometry (with and without chromatography) including universal detectors. First of all, the state of the art of quantification, with the definitions of terms and protocol standardization, will be presented with quantitative lipidomics in mind, and then technical considerations and limitations of analytical chemistry\u2019s tools, such as NMR, mass spectrometry and universal detectors, will be discussed, particularly in terms of absolute quantification. The concentration ratio [A]/[B] between two compounds A and B can be easily calculated by employing the following expression:The most important fundamental relationship of quantitative NMR is that the signal integration n in fatty acids (FAs)) will be overlapped and the detailed characterization of lipid species is unfeasible. However, it is possible to identify different types of lipids such as cholesterol, esterified cholesterol, triacylglyceride, phospholipids, and unsaturated fatty acids. Very detailed assignments of the chemical shifts were summarized in the review by Vosgaard and Guo [In proton NMR spectroscopy, each magnetically non-equivalent hydrogen nucleus in each lipid species, will exhibit an NMR signal at a characteristic resonance frequency, which is measured as a chemical shift relative to a standard compound. We know that the chemical shift depends on the chemical environment of the proton, and protons of similar molecules will give signals with close chemical shifts. Lipid species contain many long-chain fatty acids, so many signals of protons  can be easily quantified using the signal integration and an internal or external standard. Srivastava et al., determined the absolute concentration of triacylglycerides (TG), total phospholipids (PL) and total cholesterol in serum samples using quantitative proton NMR spectroscopy and an external standard . The sigrofiling . This to1H diffusion NMR experiment was recorded to detect large molecules  and lipoprotein profile characterization was performed from NMR data using Liposcale test [1H NMR-based lipidomic analysis of HDL lipoprotein of plasma samples, isolated from non-HDL lipoproteins by precipitation, helps in the characterization of the atheroprotective function of HDL and the identification of novel biomarkers of cardiovascular risk [Lipoprotein profiling of plasma samples without extraction, can be useful for the study of diseases . A 1D 1Hale test . In thislar risk . Jimenezlar risk .31P NMR spectroscopy is the most selective analytical tool. The 31P nucleus has a natural abundance of 100% which results in a high sensitivity and each PL has only one phosphorus with a characteristic chemical shift. Assignment of PL species can be confirmed using two dimensional 31P-1H NMR spectroscopy or by addition of each individual PL species in the sample. Using an internal standard, like triphenylphosphate with known concentration, it is possible to quantify the different classes of phospholipids. As discussed by Li et al [3, MeOD, D2O-EDTA) to obtain a well resolved spectrum. Bettjeman et al. determined the absolute concentration of phospholipids in five marine tissues using quantitative 31P NMR spectroscopy [13C, 13C NMR is not as sensitive as 1H and 31P, and no study reported absolute quantification of lipid species using 13C NMR spectroscopy.For quantification of phospholipids, Li et al , it is ntroscopy . They qu1H NMR is an appealing solution in order to obtain a better resolution of the lipid signals, as it allows to split the signal in two dimensions. These experiments are difficult to apply to lipidomics, as they involve long experiments , and absolute quantification requires calibration procedures. Marchand et al used 2D UF COSY (Ultra-Fast COrrelation SpectroscopY) NMR experiment of serum samples of pigs exposed to ractopamine [Alternatively, two dimensional (2D) topamine . Relativ1H NMR has some drawbacks such as low sensitivity, signal overlapping and discrimination of resonances. However, 1H NMR spectra of the lipids in biological matrices provide a fast overview of the major lipid classes , and its spectral linearity avoids the use of multiple internal standards for quantitative estimation. 31P NMR is a powerful technique for the quantification of phospholipid classes. NMR is therefore a very promising tool for lipidomics, as illustrated by a recent perspective on the subject [Contrary to MS, the detailed characterization of lipid species by proton NMR spectroscopy is unfeasible as many protons in FA with similar chemical environment give largely overlapped resonances and the quantity of material to acquire an accurate spectrum is 100 times higher than what is needed for mass spectrometry analysis.  subject .Numerous chromatographic approaches can be applied to analyze lipids in biological matrices. Normal phase high-performance liquid chromatography (NP-HPLC) and hydrophilic interaction liquid chromatography (HILIC) are usually used for the separation of lipid classes, whereas reversed-phase high-performance liquid chromatography (RP-HPLC) is used for the separation of lipid molecular species. Supercritical fluid chromatography (SFC) can also be used in the field of lipid analysis . When ansvD) can be measured based on Nukivama and Tanasawa\u2019s formula [\u03c3, is the surface tension of liquid (dyne/cm); \u00b5, liquid viscosity (poise); l\u03c1, liquid density (g/cm3); gQ, gas flow rate (cm3/s); gu, gas velocity (cm/s); lQ, mobile phase flow rate (cm3/sec); and lu, liquid velocity (cm/sec). This equation has two terms containing some factors that can influence the nebulization and therefore the particle size. In both terms, when parameters concerning gQ or gu are increased, a decrease in particle sizes is observed. In addition, the use of solvents with a high density can also decrease the size of these particles. Thereafter, the evaporation can decrease the particle size, which can be calculated using this formula:d is the particle size after evaporation, D0 is the size of initial droplet, c is the concentration, and s\u03c1 is the density of the analyte. Concerning Corona-CAD, the mechanism depends on charge transfer to the particles after collision with nitrogen that is already positively charged due to the Corona needle. Finally, the charged particles are detected by an electrometer. Generally, the attributed charge depends on the particle size. For ELSD, the response is distinguished by the diffusion of light by several phenomena . When arriving at the detection cell, the particles are crossed by a light beam. The amount of scattered light is measured by a photomultiplier. The light scattering depends on the size and the morphology of the particles, the angle of collection of the light, the surface, and the wavelength of the incident light. Both universal detectors have a nonlinear response, which can be described by the following equation:Y is the detector response and m is the mass of the analyte; a and b are parameters that depend on the analyte and chromatographic conditions. The response is independent of the chemical structure of the analyte like the degree of unsaturation and chain length. However, the detector signal depends on the mass of analytes [Charlesworth et al. described the principle of ELSD , whereas formula :(4)Dsv=5analytes . Thus, t\u00ae), which represents a real benefit for lipid analysis using NP-HPLC coupled to universal detectors with complex gradients. Retention properties of PVA were evaluated by Deschamps et al. for lipid class analysis sphingolipids, glyceroglycolipids and phospholipids using ELSD with a binary elution gradient [v/v) TEA and an equimolar amount of formic or acetic acids to the mobile phase improved the ELSD response and peak resolution [Universal detectors are compatible with all solvents that are more volatile than the analyte , as wellgradient . Godoy Rgradient . A ternagradient . Mobile solution . Other psolution , brain [solution , egg yolsolution , plasma solution , etc., wFew applications have addressed the use of a HILIC system coupled to ELSD for lipid separation. Donato et al. showed quantitative analysis of five PL classes, after solid phase extraction (SPE) extraction, from milk samples in 55 min using HILIC-ELSD. The LOD and LOQ were between 1.6\u20134 \u00b5g/mL according to the analyzed PL classes . ELSD waRegarding the use of the most recent detector Corona-CAD, a method in NP-HPLC was firstly developed for quantitative analysis of non-polar lipid classes  in vegetable oils with a LOD of 1 ng . Other mCoupling CAD to the LC system under HILIC conditions was mentioned by Le Bon et al. to separate and quantify PL classes in olfactory tissues of rats and mice . OtherwiAs shown in the various applications above, the universal detectors are largely used for quantitative analysis of lipid classes and species. These examples showed the possibility of coupling these detectors with different methods of liquid chromatographic separation with more applications in NP-HPLC to quantify lipid classes and some limitations in SFC. Moreover, experiments conducted in NP-HPLC led to less noisy baseline, especially when using Corona detector. As the response of universals detectors is independent of chemical structure, coupling these detectors to RP-HPLC could be used to quantify lipid species without impacting the response by ion suppression effects . Comparam/z) and relative abundances. An MS instrument consists of three components: the ion source for producing gaseous ions from the studied substance, the analyzer for resolving the ions into their characteristic mass components according to their mass-to-charge ratio, and the detector system for detecting the ions and recording the relative abundance of each resolved ion specie. MS suppliers propose different ion sources, such as ESI [Mass spectrometry is a powerful analytical technique used to quantify known materials, to identify unknown compounds within a sample, and to elucidate the structure and chemical properties of different molecules. The complete process involves the conversion of the sample into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios  followed by their extraction from the TLC and their quantification. It can be carried out by total phosphorous colorimetric quantification which is very efficient but not really sensitive , or thro13C standard per natural lipid molecule should be used, but these standards are not available for each natural lipid molecule. Thus, one lipid molecule per class of lipids, having two saturated FA, usually not present in the natural extract, is sometimes used as internal standard [Liquid chromatography coupled to mass spectrometry (LC-MS/MS) is an attractive alternative because of its flow, sensitivity and specificity. As was discussed, the use of internal standards of lipid species is necessary to perform the quantification. Ideally, one standard . HoweverArabidopsis thaliana, Nannochloropsis sp., Phaeodactylum tricornutum, Aurantiochytrium limacinum and Saccharomyces cerevisiae. This strategy is based on the glycerolipidome knowledge of the studied samples. For each new studied organism, a large batch of the type of cells to be analyzed is cultivated and its lipid is extracted by an adapted Folch method [n). With these technics, lipid classes are respectively quantified with their FA distribution and lipid molecules are identified with their fatty acid localization on the glycerol backbone as described in [n. The TLC plus GC-FID method allowed a direct access of the FA profile in a given class, whereas the FA profile must be reconstructed with MS technique, knowing the proportion of each molecule constituting this class and the nature of their FA previously established with the MS trap. Profiles obtained with both techniques were found to be comparable, i.e., the major FAs with one method are also the major FAs with the other one [To circumvent the discrepancy of ionization efficiency and the absence of available labeled standard for all lipid molecules, we can use a quality control (QC) sample to normalize and correct the quantification. This QC corresponds to a known lipid extract mimicking the studied samples, quantified once by TLC plus GC-FID, and then systematically run with the samples to be analyzed by LC-MS/MS . This meh method . The lipribed in . The glyther one . This fi31P NMR, or of simple mixture of lipids by 1H NMR, it can permit the absolute quantification of PL, TG, CE, FC or \u03c9-FA for instance. NMR is non-destructive method, then the lipids extract can be analyzed with other techniques afterwards. After a chromatographic separation, the detector used is decisive and can influence the quantification of lipids. With the use of universal detectors such as ELSD or CAD, the detector response is not influenced by the chemical structure (chain length and number of unsaturations) of molecular species in the same lipid class. Therefore, quantification of each lipid class is possible using one standard per concerned class. Concerning mass spectrometry detection method, despite its sensitivity and its practicality, the use of this tool for the quantification of lipids is not without disadvantages. The difference of ionization efficiency between lipid species, depending on their structure, is indeed a real limitation of this technique. The situation is quite convenient for simple lipids because pure standards are available. Thus, calibration curves can be performed to correct detector sensitivity. This tool is then really convenient to propose absolute quantification paying attention to the validation of the method. For complex lipids, the situation is more complicated because pure molecular species are not available for all molecules. Consequently, the lack of calibration curves prevents the absolute quantification of lipids either in shotgun or in flow injection analysis. Thus, absolute quantification for complex lipids should not be established with mass spectrometry detection, we should only propose data in relative quantification (or close/accurate quantification with caution) and respect guidelines to obtain and validate the data (https://lipidomics-standards-initiative.org/guidelines/lipid-species-quantification).Due to the structural complexity of lipids, their quantification is not easy to perform. Different strategies and techniques should be addressed depending on the targeted molecules. Despite the low sensitivity of NMR, it is a quantitative tool and allows the absolute quantification of lipids containing phosphorus by"}
+{"text": "The Richmond Agitation-sedation Scale (RASS) is a reliable and valid scale for assessing sedation in critically ill pediatric patients. This investigation evaluates the inter-rater reliability of the RASS in mechanically ventilated pediatric patients before and after an educational intervention.This prospective, interventional quality improvement study was completed in a 20-bed pediatric intensive care unit from July 2013 to July 2014. Children 0\u201318 years of age requiring mechanical ventilation and receiving sedative or analgesic medications were eligible. Staff completed simultaneous paired RASS assessments in 3 phases: baseline, after educational intervention, and maintenance.P < 0.001). The improvement was maintained months later with weighted \u03ba 0.78 . In subgroup analysis, there was an increase in weighted \u03ba in patients less than 1 year of age (0.41\u20130.87) and those with developmental delay (0.49\u20130.84).Staff completed 347 paired assessments on 45 pediatric intensive care unit patients: 49 in the baseline phase, 228 in the postintervention phase, and 70 in the maintenance phase. There was a significant increase in the weighted \u03ba after the intervention, from 0.56  to 0.86 (95% CI, 0.77\u20130.95; The RASS is a reliable tool for sedation assessment in mechanically ventilated, sedated pediatric patients after implementation of an educational intervention. It is also reliable in patients less than 12 months of age and patients with developmental delay. The ability to easily educate providers to utilize a valid, reliable sedation tool is an important step toward using it to provide consistent care to optimize sedation. When delivered optimally, sedation and analgesia medications can alleviate pain and anxiety, enhance patient safety, decrease hospital length of stay, and facilitate performance of invasive procedures.8 The Richmond Agitation-sedation Scale (RASS) is a valid and reliable tool used for sedation assessments in adult patients.18 The RASS is becoming a common tool to evaluate sedation level in pediatric patients as supported by its inclusion as a component in 2 pediatric delirium assessment tools and a recent study by Kerson et al.,19 which demonstrated that it is valid and reliable in assessing sedation in critically ill pediatric patients.21 However, to date, no studies exist that explore the use of an educational intervention to improve and maintain the reliability of the scale over time in pediatric patients.A multidisciplinary panel from the United Kingdom recommended the regular assessment and documentation of sedation level using a formal sedation assessment scale.25 the reliability in these 2 groups was also specifically evaluated.This investigation evaluates the inter-rater reliability (IRR) of the RASS in mechanically ventilated pediatric patients before and after an educational intervention in a PICU in which staff used the RASS as a sedation assessment for over 5 years. The research team chose the RASS, given its quick and easy use at the bedside compared with other scales; the staff\u2019s prior familiarity with the scale; and its existing integration into the medical record for documentation. We hypothesized that the reliability would improve significantly with an educational intervention. Because the pediatric population is unique in that assessments of sedation and pain can be affected by young age and developmental delay,13 found to be valid and reliable in adult ICU patients . The physician\u2013physician weighted \u03ba decreased slightly but was still near goal at 0.67. Analysis of subgroups revealed that in patients less than 1 year of age, the IRR improved from a weighted \u03ba of 0.41 before intervention to 0.87 after the intervention. Similarly, in patients with developmental delay, the IRR improved from a weighted \u03ba of 0.49 before intervention to 0.84 after the intervention.To our knowledge, this is the first investigation of an educational intervention to improve the IRR of the RASS in a pediatric population. The high weighted \u03ba in the postintervention phase demonstrates the RASS to have the potential to be an extremely reliable tool for assessment of sedation status in mechanically ventilated, sedated PICU patients when a multi-modal educational curriculum is employed. Sustained IRR months after education and implementation ensure the feasibility of continued reliable use with time. Additionally, the IRR remained above goal despite an institutional transition to a new electronic medical record system between the postintervention and maintenance phases.Previous studies demonstrate the utility of the RASS as an objective tool to assess sedation. However our experience did not reflect this as evidenced by poor reliability in the baseline phase despite the use of the RASS in daily sedation assessment by bedside nurses in the PICU for over 5 years. Several factors likely contributed. During the standard orientation, nurses received minimal training on how to correctly perform the RASS. Investigators noted the inconsistent use of the RASS by the medical team when describing sedation goals, varied nursing practice in the frequency of RASS assessment and documentation for mechanically ventilated patients, and difficulty in finding RASS documentation within the electronic medical record system. The implementation of a focused, multi-modal educational intervention by a multidisciplinary team was fundamental in improving the knowledge and utilization of the RASS with a subsequent significant improvement in IRR. Staff from multiple disciplines worked congruently to develop the various educational tools and to ensure each tool was appropriate for their colleagues. Multiple types of learning modules also accounted for different adult learning styles. One factor that was likely responsible for broad acceptance and utilization of the scale was the incorporation of after-evaluation feedback and debriefing sessions. These sessions identified opportunities for further education and discussion.It is important that a sedation scale be reliable among differing intra- and inter-professional providers. Among nurse-to-nurse and nurse-to-physician pairs, there was a significant sustained improvement in RASS IRR after the educational intervention. However, the physician\u2013physician pairs did not show similar improvement. Only the 2 physicians on the research team participated in the preintervention assessments, likely skewing the results and contributing to the high initial IRR compared with the postintervention results. Assessments in the postintervention and maintenance phases included both research and nonresearch physicians, thus contributing to slightly lower IRR scores. There were also only 2 physician\u2013physician pairs in the maintenance phase assessments, potentially not demonstrating an accurate assessment of the IRR for this phase.Importantly, the RASS can be a reliable tool in 2 unique patient populations frequently encountered in the PICU\u2014children less than 1 year of age  and developmentally delayed children . The use of a slightly adapted version of the original RASS scale to include eye-opening, not just eye contact, for certain levels of sedation allowed the scale to be inclusive for patient populations in which the level of arousal is difficult to determine due to their developmental abilities.There were several limitations to the study. First, the study evaluates use in a single institution. Also, several children required mechanical ventilation for a prolonged course during the postintervention phase. Thus, despite having over 200 RASS assessments, they were performed on a small number of patients. The timing of assessments on weekday shifts may have played a factor in the IRR. Many of the staff in the unit alternate between days and nights, but those working mainly nights represented a lower percentage of the RASS assessments obtained. Lastly, there was a paucity of assessments in the agitated range of the RASS. This result is largely due to the patient population included in the study as described previously. However, there were rare situations when research members were unable to complete RASS assessments on applicable patients due to too much activity in the room .The RASS is a reliable scale for assessing sedation in pediatric patients after introduction of a multi-modal educational curriculum. Without appropriate training and education, the RASS should not be presumed to be a reliable scale among users. Ongoing education at least annually is likely important to ensure maintenance of knowledge, especially in units with high staff turnover. In the future, the educational framework used can be applied to improve the education of other tools utilized in critically ill pediatric patients.The authors have no financial interest to declare in relation to the content of this article."}
+{"text": "In vivo, glioma growth was inhibited by depletion of LINC00662 in nude mice. Mechanistically, LINC00662 directly interacts with miR-107. The High-mobility group box 1 protein (HMGB1) is a known target of miR-107. Moreover, rescue assays reveal that HMGB1 overexpression (or miR-107 inhibition) reverses the glioma growth inhibition caused by LINC00662 knockdown. In conclusion, our results indicate that LINC00662 acts as an oncogene in glioma by modulating the miR-107/HMGB1 axis, suggesting that LINC00662 could be a novel therapeutic target for glioma treatment.Increasing evidence revealed that the aberrant expression of long non-coding RNAs (lncRNAs) has been implicated in tumorigenesis. However, the role and mechanisms of LINC00662 in glioma have not been elucidated. Here, we show that upregulation of LINC00662 expression in glioma is associated with advanced clinical features and poor prognosis. Our results from loss-of-function assays suggest that LINC00662 silencing suppresses the proliferative and invasive abilities of glioma cells.  Glioma is an intracranial malignancy of the glial cells that originates in the neuroectoderm and has a high mortality rate Long non-coding RNAs (lncRNAs) are transcripts consisting of >200 nucleotides Mechanistically, lncRNAs function as competitive endogenous RNAs (ceRNAs). ceRNAs are lncRNAs that modulate the expression of specific genes by sponging miRNA Here, we examined the role of LINC00662 in glioma and found that it promotes glioma deterioration by regulating the LINC00662/miR-107/HMGB1 axis, suggesting its potential as a novel therapeutic target against glioma.In summary, 41 glioma specimens and 11 normal brain specimens were collected from patients admitted to The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology. No patients received chemotherapy or radiotherapy before surgery. Informed written consent was obtained from all the patients before the study commenced. Isolated tissues were immediately frozen in liquid nitrogen immediately after sampling. Ethical clearance for this study was provided by the Medical Ethics Committee of our hospital . Glioma patients' information was shown in Table 2.NHA  and six glioma cell lines  were procured from the Chinese academy of sciences . These cells were grown in RPMI-1640  supplemented with 10% FBS  in a humidified incubator at 37\u00baC, 5% COMiR-107 mimics, and miR-NC were designed by GenePharma , while siRNA against LINC00662  and negative control siRNA (si-NC) were bought from Ribobio. The above-mentioned oligonucleotides and plasmids were transfected to the cells using lipofectamine 3000 reagents .-\u0394\u0394Ct method. The PCR cycle parameters were as following: denaturation: 95\u00b0C, 30 s, 40 cycles; annealing: 60\u00b0C, 30 s; extension: 72\u00b0C, 30 s. Primer were as following: LINC00662, 5\u2032-TGGACATCTGTCTGGAGG-3\u2032 (forward) and 5\u2032- GGCTGAGGCATAAGAATCG-3\u2032 (reverse); miR-107, 5\u2032-AGCAGCATTGTACAGGGCTATCA-3\u2032 (forward) and 5\u2032- GCGAGCACAGAATTAATACGAC-3\u2032 (reverse); HMGB1, 5\u2032-CCAGCGATAGTCCCACTGAT-3\u2032 (forward) and 5\u2032- CCTTCTCCTTGGCAGACATC-3\u2032.Cells were homogenized with TRIzol reagent (Invitrogen) to isolated total RNA and reverse transcribed using a commercial reverse transcription kit . RT-qPCR was performed on Bio-Rad CFX96 system  using SYBER-Green Real-Time PCR Kit . Fold expression in mRNA levels were estimated with the 2After transfection, cells were titrated onto 96-well plates, and 10 \u03bcL of CCK-8 solution  was added into each well. The optical density of each plate was read by a microplate reader .Transfected glioma cells were cultured in 6-well plates for 14 days then mixed with 4% formaldehyde (Sigma). Next, cells were stained with 0.1% crystal violet (Sigma), after which the number of colonies formed was assessed manually.Migration was tested by wound healing assay. Transfected cells were plated in 12-well dishes reaching a confluence of 80%. Then cells were scratched across the surface of well by a pipette. After 24 h, the scratches were observed.Transfected glioma cells were plated in the upper chamber of Matrigel-coated membranes , and the lower chamber was filled with 10% FBS culture medium. After 24 h, transfected cells in the lower chamber were fixed (by methanol) and stained with crystal violet (0.1%).The part of nuclear and cytoplasm were extracted by Nuclear and Cytoplasmic RNA Purification Kit. Then, the relative expressions of LINC00662, GAPDH and U6 in cytoplasm or nuclear of the cells were explored by RT-qPCR assay. U6 and GAPDH were served as endogenous controls.Plasmids harboring the predicted binding sites or mutant sites were purchased from Promega , while plasmids containing the sequences for miR-107 were purchased from Sangon . MiR-107 mimics were co-transfected with LINC00662-Wt, LINC00662-Mut, HMGB1-Wt, or HMGB1-Mut using lipofectamine 3000. Dual-Luciferase Reporter Assay System (Promega) was used to explore the luciferase activity.The RIP assay was done by a Magna RIP kit (Millipore) according to the manufacturer's instruction. The lysates were incubated with magnetic beads with anti-IgG as control and anti-Ago2 in RIP buffer. The levels of LINC00662 and miR-107 in the precipitates were quantified by RT-qPCR.Cell lysates of U87 and N251 cells were incubated with LINC00662 no-biotin probe or LINC00662 biotin probe and streptavidin beads (Invitrogen) were later added. RT-qPCR was applied to analyze expression levels of various miRNAs, separately.Western blot analysis was performed according to a previously described method 6) were stably transfected with sh-LINC00662 or control vector (sh-NC) were subcutaneously injected to the back of female BALB/c nude mice . The size of tumors was monitored weekly by measuring the length (L) and width (W) using a caliper. Tumor volume (V) was calculated using the formula: V = 1/2\u00d7L\u00d7W2. After 6 weeks following injection, the mice were euthanized, and tumors were harvested and weighed. The protocol used for animal experiments was authorized by our hospital.U87 cells  of 3 independent experiments. Data analysis was determined by using SPSS17.0. One-way ANOVA or student's t-test was used to assess for statistical significance between the treatment means of different treatments. Sequence alignment by the NCBI BLAST revealed that LINC00662 is located on chromosome 19q12.3 and spans between nucleotides 27,678,814 to 27,796,263  relative to normal human astrocytes (NHA)  have been implicated in glioma tumorigenesis. For instance, the upregulation of DANCR promotes glioma progression via Wnt signaling Recently, multiple studies have implicated LINC00662 was dysregulated and had potential clinical significance in tumors. For example, Xu et al. showed that LINC00662 promoted oral squamous cell carcinoma cells proliferation and migration abilities As the lncRNA-miRNA-mRNA axis is the most common mechanism involved in the regulation of lncRNAs, we sought to identify the possible miR-107 target by bioinformatics analysis. Recently, Takahashi et al. showed that miR-107 induced cell cycle arrest in lung cancer cell lines The high mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein that translocates to the nucleus to interact with histone nucleosomes and transcriptional factors In conclusion, our study uncovered an oncogenic role and mechanism of LINC00662 in the development and progression of glioma. LINC00662 might exert its functions by regulating the miR-107/HMGB1 axis in glioma. Therefore, our data highlights the potential of this lncRNA as an anti-glioma therapeutic target.LINC00662 was upregulated in glioma;LINC00662 promoted glioma cell proliferation and invasion;LINC00662 enhanced glioma progression by regulating the miR-107/HMGB1 axis.JSW, HRZ designed the research; JSW, XLG, DXX performed the experiments and analyzed the data; JSW, HRZ wrote the manuscript. All authors read and approved the final manuscript."}
+{"text": "Among men who have sex with men (MSM) and transgender women (TW), stimulant use is high and has been associated with an increased risk of HIV infection, suicide and cardiovascular disease (CVD) mortality. We used epidemic modelling to investigate these intersecting health harms among MSM/TW in Lima, Peru and assess whether they could be mitigated by prioritizing HIV pre\u2010exposure prophylaxis (PrEP) and harm reduction interventions among MSM/TW who use stimulants.We adapted a dynamic model of HIV transmission among MSM/TW in Lima to incorporate stimulant use and increased HIV risk, suicide and CVD mortality. Among 6% to 24% of MSM/TW using stimulants (mostly cocaine), we modelled an increased risk of unprotected anal sex (RR\u00a0=\u00a01.35 [95%CI: 1.17 to 1.57]) obtained from local data, and increased risk of suicide (SMR\u00a0=\u00a06.26 [95%CI: 2.84 to 13.80]) and CVD (SMR\u00a0=\u00a01.83 [95%CI: 0.39 to 8.57]) mortality associated with cocaine use based on a global systematic review. We estimated the proportion of health harms occurring among MSM/TW who use stimulants in the next year (01\u20102020/01\u20102021). We also investigated the 10\u2010year impact (01\u20102020/01\u20102030) of: (1) PrEP prioritization for stimulant\u2010using MSM/TW compared to random allocation, and (2) integrating PrEP with a theoretical intervention halving stimulant\u2010associated risk.MSM/TW in Lima will experience high HIV incidence, suicide mortality and CVD mortality  in 2020. Despite stimulant using MSM/TW comprising an estimated 9.5% (95%CI: 7.8 to 11.5) of all MSM/TW, in the next year, 11% 95%CI (i.e. 2.5% to 97.5% percentile) 10% to 13%) of new HIV infections, 39% (95%CI: 18% to 60%) of suicides and 15% (95%CI: 3% to 44%) of CVD deaths could occur among this group. Scaling up PrEP among all stimulant using MSM/TW could prevent 19% (95%CI: 11% to 31%) more HIV infections over 10\u00a0years compared to random allocation. Integrating PrEP and an intervention to halve stimulant\u2010associated risks could reduce new HIV infections by 20% (95%CI: 10% to 37%), suicide deaths by 14% (95%CI: 5% to 27%) and CVD deaths by 3% (95%CI: 0% to 16%) over a decade.MSM/TW who use stimulants experience a disproportionate burden of health harms. Prioritizing PrEP based on stimulant use, in addition to sexual behaviour/gender identity criteria, could increase its impact. Integrated substance use, harm reduction, mental health and HIV care among MSM/TW is needed. Higher While there are no approved medications to treat either cocaine or meth/amphetamine dependence , harm reEpidemic modelling can be used to quantify the contribution of groups to intersecting epidemics, and the potential population\u2010level impact of interventions, thereby guiding evidence\u2010based policymaking . As in m22.1We modified a previously published epidemic model of HIV transmission among MSM and TW in Lima, Peru , to inco2.2The model was mainly parameterized using data from multiple\u00a0local MSM/TW surveillance rounds, local epidemiological studies and global reviews and meta\u2010analyses. Regarding stimulant use, the 2011 surveillance round among MSM/TW is the most recent source of comprehensive\u00a0drug use\u00a0and sexual behaviour data among MSM/TW in Peru. Using these data, we calculated the prevalence of stimulant use in the past 3\u00a0months in each group in Lima, corresponding to 6.2% (95%CI: 4.6 to 7.8) among homosexual identified MSM, 13.3% (95%CI: 10.6 to 16.1) among heterosexual/bisexual identified MSM, 23.61% (95%CI: 20.7 to 26.5) among MSW and 17.8% (95%CI: 14.3 to 21.4) among TW. Virtually all MSM/TW (98%) who reported stimulant use were using cocaine and/or cocaine paste   associated with suicide among people who use cocaine obtained from a recent global systematic review   was simulated with 10,000 different parameter sets randomly sampled through Latin hypercube sampling. The log likelihood of each simulated epidemic trajectory was calculated based on time series HIV prevalence data from 1985 to 2011 for the total MSM/TW population and for each group , time series HIV incidence data from 1999 to 2014 for the total MSM/TW population (5 data points), the proportion using stimulants per group in 2011 and the total ART coverage for 2011 : \u03c2k,ft=Xk,21/\u2211fXk,f1 corresponding to the proportion using stimulants in each group.Scenario 2 : \u03c2k,ft is the PrEP coverage at time point (t) among those in sexual behaviour group k and stimulant use group f  and Xk,f1 corresponds to the number of susceptible individuals in each sexual behaviour/stimulant use group.Where 2.6Due to a lack of effective treatment for stimulant use, we simulated the potential benefits of a hypothetical harm reduction intervention package assumed to reduce the excess risks of HIV, suicide and CVD mortality associated with stimulant use by half. This could be as a result of decreases in the intensity/frequency of stimulant use or through combined harm reduction interventions including condom use distribution and promotion, psychological/psychiatric treatment to prevent suicide and CVD treatment to prevent CVD mortality.2.72.7.1While evidence is mixed , some st2.7.2We performed a sensitivity analysis assuming turnover between groups using and not using stimulants, with an average duration of stimulant use of 5\u00a0years.2.7.3We assumed a two to sevenfold higher rate of suicide among MSM  and a one to threefold higher rate among TW compared to MSM, to acknowledge evidence of higher suicide rates among these populations.2.7.4We implemented a scenario in which 100% of TW would be covered, with the remainder given to MSW to compare the effectiveness of this established strategy to that of PrEP prioritization by stimulant use.33.1Our model estimates MSM and TW in Lima experience a high burden of HIV incidence, suicide mortality and CVD mortality, with estimates of 1.63 per 100 person\u2010years (/100\u00a0py) : 0.83 to 2.51), 0.018/100\u00a0py (95%CI: 0.008 to 0.040) and 0.13/100\u00a0PY (95%CI: 0.03 to 0.5), respectively, in 2020.The burden of each of these health harms differs by group, with 7.1% (95%CI: 4.7 to 10.0), 5.6% (95%CI: 3.6 to 8.7) and 4.7% (95%CI: 3.3 to 7.1) of new HIV infections, suicide deaths and CVD deaths, respectively, occurring among TW when they comprise 4.5% (95%CI: 2.9% to 6.7%) of the total MSM/TW population and 12.4% (95%CI: 8.7 to 16.9), 12.2% (95%CI: 4.2 to 22.9) and 9.0% (95%CI: 3.3 to 17.2) of new HIV infections, suicide deaths and CVD deaths, respectively, occurring among MSW when they comprise 8.3% (95%CI: 3.4% to 15.0%) of the total MSM/TW population. The ratios of the proportion of HIV infections, suicides and CVD deaths among each group in one year divided by the proportion of individuals in each group are shown in Figure\u00a03.2Our modelling indicates that despite MSM/TW who use stimulants comprising an estimated 9.5% (95%CI: 7.8 to 11.5) of the overall MSM/TW population in Lima, between 2020 and 2021, 11% (95%CI: 10% to 13%) of new HIV infections, 39% (95%CI: 18% to 60%) of suicides and 15% (95%CI: 3% to 44%) of CVD deaths would occur among this group. The ratios of the proportion of HIV infections, suicides and CVD deaths among MSM/TW who use stimulants by the proportion of MSM/TW in this group are shown in Figure\u00a03.3Scaling up PrEP among all (100%) MSM/TW who use stimulants in each group between 2020 and 2030 would prevent 17.9% (95%CI: 9.1 to 34.9) of new HIV infections  of new HIV infections, 14% (95%CI: 5% to 27%) of suicide deaths and 3% (95%CI: 0% to 16%) of CVD deaths between 2020 and 2030  more effective, compared to 19% (95%CI: 11% to 31% with no adherence differences).Assuming a five\u2010year duration of stimulant use, the proportion of new HIV infections occurring among MSM/TW who use stimulants was virtually unchanged at 11.1% (95%CI: 9.7% to 13.3%) and the relative increased impact of the PrEP prioritization scenario was 17% (95%CI: 9% to 31%) under this shorter duration of stimulant use (vs. 19% (95%CI: 11% to 31%) at baseline), indicating a strategy of prioritization by stimulant use may be slightly less effective under shorter durations of stimulant use.Assuming higher suicide rates among MSM (two to sevenfold) and TW (one to threefold higher than among MSM), translated to a suicide incidence of 0.08/100\u00a0py (95%CI: 0.02 to 0.19) versus 0.018/100\u00a0py  of suicide deaths (vs. 5.6% (95%CI: 3.6% to 8.7%) at baseline) occurring among TW  reduction in new HIV infections between 2020 and 2030, which is 23% greater than obtained through the prioritization strategy based on stimulant use.4Our study quantified the burden of HIV, suicide mortality and CVD mortality in association with stimulant use among MSM and TW in Lima, Peru. We chose Peru as a useful case study due to the high prevalence of HIV and stimulant use among MSM and TW, comparable to other global settings. We found that MSM/TW who use stimulants are disproportionately affected by incident HIV infection, suicide mortality and CVD mortality. In addition, male sex workers and TW had a higher burden of these health harms compared to homosexual and heterosexual/bisexual MSM (who do not engage in sex work) due to increased sexual risk behaviours and stimulant use. Indeed, substance use among TW in Lima has been described , 56 as pBox 1Our modelling indicates that PrEP prioritization for MSM/TW who use stimulants could increase its impact in Lima, even with potentially lower PrEP adherence among this group. In practice, this would entail integrating substance use and sexual risk assessment within one clinical visit when prescribing PrEP, which currently does not happen systematically. More importantly, even when questions on substance use are asked, this information is rarely acted upon, meaning that referral to substance use counselling, treatment, or harm reduction strategies are not delivered. In this study, we also showed that providing harm reduction interventions in combination with PrEP among MSM/TW who use stimulants would result in substantial overall reductions in HIV, suicide and CVD mortality. Our sensitivity analysis showed that given higher HIV incidence among TW and MSW, a PrEP prioritization strategy based on gender identity/sexual behaviour would have a larger impact compared to a prioritization strategy by stimulant use, but overall our study\u00a0suggests that stimulant use should be included as an additional criterion in current guidelines  see Box. ImportaTo our knowledge this is the first modelling study examining the impact of stimulant use among MSM/TW on HIV, suicide and CVD, but it supports recent modelling highlighting the excess risks of stimulant use on HIV and related infections among PWID . The estBox 2Our study has limitations. First, like all models ours was limited by data uncertainties. For example we lacked MSM/TW and setting\u2010specific data on suicide and CVD mortality overall and by stimulant use. As a result, our model might underestimate overall infection/mortality burden and dilute differences between groups. While we implemented a sensitivity analysis to address this in relation to suicide rates, the study should be updated when MSM/TW\u2010specific data become available see Box . ImportaSecond, we simulated a theoretical intervention package as there are no effective pharmacotherapies for stimulant use, and psychosocial therapies have weak or nonspecific effects. Our study therefore presents the potential impact if a stimulant use treatment were developed, or if a package of interventions were provided to address HIV, suicide and CVD among MSM/TW who use stimulants. Studies have shown low to moderate effectiveness of psychosocial interventions including motivational interviewing, contingency management and cognitive behavioural therapy, on reducing both methamphetamine use and risky sexual behaviours among MSM . Among TThird, while our model incorporates much complexity in terms of sexual behaviours and stimulant use by sexual orientation, gender identity and engagement in sex work; a full range of intersecting health risks exists that we did not explore. Associations between HIV infection and suicidality have been consistently reported. Similarly, there is evidence for higher risk of CVD among HIV\u2010positive patients , 67, 68.5Our modelling indicates that prioritization of HIV PrEP among MSM/TW in Lima who use stimulants could enhance PrEP prioritization strategies based on sexual behaviours, or sexual orientation/gender identify. Stronger integration of interventions that address stimulant use among MSM/TW in HIV programmes is key to reducing multiple associated harms among this population. Importantly, these interventions should be complementary to broader interventions addressing the vulnerabilities and structural gaps that affect the wellbeing of sexual minorities in Peru. Interventions to guarantee their fundamental rights, including the right to an official identification reflecting their gender, the right to protection from harassment and violence, and the right to employment and health, are key to reducing persistent health disparities.NM has received unrestricted research grants from Gilead and Merck unrelated to this work. All other authors declare no competing interests.AB and NM conceptualized the study analyses and wrote the first draft of the manuscript. AB developed the model and generated model results and figures. LT, LD, RM, SK and MF conducted the systematic reviews and meta\u2010analyses to inform model parameterization. RA provided data to inform the model and provided guidance in its analyses and interpretation of results. KR and KK conducted statistical analyses to inform the model parameterization. CC, AS and JC aided in manuscript drafting. All authors critically reviewed and approved the manuscript for submission.Integrating services for HIV and related comorbidities: modelling to inform policy and practice Supplement was supported by the US National Institutes of Health, Fogarty International Center. Additional support was provided by the National Institute on Drug Abuse , the National Institute of Allergy and Infectious Diseases (R01AI147490 [NM]), the Australian National Drug and Alcohol Research Center [MF], the National Health and Medical Research Fellowship [LD], the Curtin Senior Research Fellowship [RM], UNITAID through the ImPrEP study [CC] and [KK].This article as part of the Appendix S1. Model specifications, equations, parameterization and analyses.Click here for additional data file."}
+{"text": "Tumor necrosis factor (TNF)-alpha polymorphisms have been repeatedly indicated to play a pathogenetic role in various mental disorders, but none of these studies focused on the susceptibility to suicidal behavior in SCZ. We recruited 1087 chronic inpatients with SCZ and 576 controls to assess the psychopathological symptoms of SCZ using the Positive and Negative Syndrome Scale scales. We selected 2 polymorphisms (-308G>A and -1031C>T) in the TNF-alpha gene and analyzed their associations with SCZ and suicide. Our results showed that TNF-alpha -308G>A and -1031C>T were not related to SCZ and suicide. However, we found that suicide attempters with the C allele carriers exhibited suicidal behaviors significantly later than those with TT genotype in the SCZ patients. The haplotype containing the T allele of the -1031 was significantly associated with the age of suicide initiation. Further logistic regression analysis showed that -1031C>T interacted with psychopathological symptoms and drinking, age of smoking, and related to the initiation age of suicide attempts. Our study demonstrated that the TNF-alpha variants may affect the age at which suicide attempts started among SCZ suicide attempters.Patients with schizophrenia (SCZ) exhibit higher suicide rates than the general population. However, the molecular mechanism remains poorly understood.  Patients with schizophrenia (SCZ) have a high risk of attempting and completing suicide. Suicide is considered as one of the most common causes of premature death in SCZ patients . The aveRecent findings have recognized the possible role of the abnormal immune system in the primary pathophysiological mechanism of suicidal behavior \u20134. It isTNF-alpha polymorphism and suicide in SCZ. At this time, only the -308G>A polymorphism in the TNF-alpha gene has been studied in relation to suicide attempts in individuals with MDD [Several lines of studies of peripheral biomarkers have shown higher serum TNF-alpha levels in those persons with suicide attempts, suicidal ideation, or suicide \u201313, but with MDD .TNF-alpha gene has multiple frequent polymorphisms [TNF-alpha gene displayed better cognition functions [Inflammation is not only related to suicidal behavior, but also to mental disorders. Many studies by our team and others have suggested that the pathogenesis of schizophrenia involves an alteration of peripheral immune system that leads to altered blood levels of TNF-alpha \u201323. A reorphisms  and seveorphisms \u201327. Our unctions  and decrunctions . Taken tTNF-alpha gene and alteration of TNF-alpha serum levels in SCZ, it would be beneficial to investigate the association between the TNF-alpha gene and suicidality of SCZ patients. Based on previous literature, we selected -308G/A (rs1800629) and -1031T/C (rs1799964) single nucleotide polymorphisms (SNPs) to assay among the patients with SCZ in this study. -308G/A polymorphism has been confirmed by several studies to be a susceptibility factor for suicide in patients with MDD [Considering the high suicide rate in the patients with SCZ and the pathogenic role of with MDD , while -with MDD . Thus, wThe demographic characteristics and clinical data are shown in TNF-alpha -308G>A (rs1800629) and -1031C>T (rs1799964). Also, there was no genotypic or allelic correlation between the two SNPs and attempted suicide  and PANSS total scores  reached significantly different values in the subgroup analysis. However, the association between -308G>A and suicide still was not statistically significant after a logistic regression analysis (p>0.05). Given that most of the samples were male patients, we performed stratified analysis for each sex and still did not find significant differences in the allele and genotypic frequencies of the two SNPs between male suicide attempters and male non-attempters or female attempters and female non-attempters .No deviation from Hardy-Weinberg equilibrium was found in the patients and controls, or in the subgroup analysis based on suicide attempt. No significant differences in the genotype and allele frequencies between patients and controls were found for  suicide . In a stTNF-alpha polymorphism and clinical variables, the quantitative trait test was used to determine the relationship between TNF-alpha -308G>A or -1031C>T and individual phenotype. We found no association between -308G>A or -1031C>T and any clinical phenotypes, including gender, age, education, onset of illness, BMI, antipsychotic treatment , hospitalization, duration of illness as well as the symptoms of schizophrenia that were measured by the PANSS  and -1031C>T genotype  to be related to age at suicide attempt initiation.As shown in TNF-alpha gene and risk for attempted suicide in Han Chinese patients with SCZ, showing no relationship between TNF-alpha gene -308G>A and -1031C>T polymorphism and SCZ and suicide attempt. However, we found a link between the -1031C>T polymorphism and the age at which SCZ patients began to attempt suicide. Moreover, the age of suicide initiation was significantly positively correlated with age of symptoms onset in the patients.This is the first investigation to explore the relationship between TNF-alpha gene and SCZ, but with inconsistent results [TNF-alpha gene 1031T-863A-857C-308G, 1031C-863C-857C-308G, and 1031C-863C-857T-308G and increased risk of SCZ [TNF-alpha polymorphism and SCZ might be due to ethnic differences, as the frequency of -1301C>T and -308G>A genotypes varied significantly between different ethnicities.Several studies have investigated the association of polymorphisms in the  results , 30\u201333.  results \u201336, espe results . MoreoveTNF-alpha gene, our results indicate that there was no association between two tag SNPs in the TNF-alpha gene and suicide attempt. The patients with SCZ were associated with higher risk of suicide than the general population, and previous studies speculated that the pathophysiology of suicide might originate as a disturbance within the immune system [TNF-alpha gene in suicide. Results from these groups showed that -308G>A was related to suicidal behaviors in MDD [TNF-alpha gene polymorphism and suicide in SCZ [TNF-alpha gene polymorphism. Furthermore, neither study showed any association between suicide and TNF-alpha haplotype in patients. We attributed the failure to find a relationship between suicide and TNF-alpha polymorphism to low sample size of suicidal patients with SCZ in both studies, since there were only 120 suicide attempters in this study and 74 attempters in the above study for the genetics analysis. The sample size of suicide attempters was small, resulting in an even smaller number of patients found to have the CC genotype of -1031 polymorphism, which may produce false negative results. Therefore, additional studies with larger samples containing several ethnic populations are needed to confirm these findings. It is worthy of mentioning that in a stepwise logistic regression analysis for relationships between attempted suicide and TNF-alpha polymorphisms, with age, gender, education, BMI, smoking and clinical symptoms shown on PANSS scores as covariates, we found that -1031C>T genotype distributions  and PANSS total score  reached significance. This result suggests that under the influence of clinical variables, combined with clinical psychopathological symptoms, the TNF-alpha -1031C>T polymorphism may play a role in attempted suicide in SCZ patients. Why was there no significant difference in the allele or genotype distribution of TNF-alpha -1031C>T polymorphism between suicide attempters and non-attempters in SCZ patients, and then this difference became significant when combined with clinical variable, especially psychopathological symptoms? However, due to our cross-sectional design in this study, we are currently unable to provide a reasonable explanation for this phenomenon, which deserves further exploration in future research.Regarding the relationship of suicide attempt and the e system \u201339. Howes in MDD \u201321 and te in SCZ . HoweverAnother important finding was that the -1031C/T polymorphism may have an effect on the age at which suicidal behavior initiation occurred in SCZ. Patients attempting suicide with the -1031C allele started committing suicide later than those who carried the TT genotype. Further haplotype analyses found that the age at which suicidal behavior initiated in patients carrying 308(A)-1031(T) was earlier than those with haplotype counterparts, which further supports that the -1031TT genotype may be associated with earlier age of suicide initiation. Suicide is a complex behavior, and recent scientific literatures analyzed the age of suicide initiation of suicidal attempters as a potential candidate marker to characterize different subgroup attempters and reduce the heterogeneity of suicidal behavior \u201342. ThesTNF-alpha -1031 TT genotype and the earlier age at which suicide initiation occurred in the patients with SCZ. Many studies have indicated that the two tag SNPs (-1031C>T and -308G>A polymorphisms) have an effect on the protein production; although these results were contradictory [TNF-alpha mRNA was associated with impulsivity and hopelessness after controlling for demographics and substances used [It would be extremely meaningful to explore the possible mechanism underlying the associations between the adictory . For exaadictory . Anotheradictory . Moreoveadictory . Taken tadictory \u201351. Howeadictory . In partces used . In addices used . Taken tTNF-alpha gene knockout mice showed increased emotional responses when exposed to stress compared with wild-type mice [On the other hand, our previous results demonstrated that -1031C/T polymorphism was related to the onset age of SCZ in long-term hospitalization patients with SCZ . Interesype mice . Hence, ype mice \u201356. Animype mice , while pype mice , 58. Furype mice . Activatype mice . A recenTNF-alpha gene and suicidal tendencies in SCZ was preliminary, and further validation needs to be carried out in a larger, independent sample to increase statistical capacity before a definitive conclusion can be drawn. Second, there are several known polymorphisms in the promoter of TNF-alpha gene, but we had measured only two of those polymorphisms. Whether the other variants affected the results of this study is unclear. A complex analysis of the influences of polymorphisms in the gene should be examined in further researches. Third, the levels of TNF-alpha and other inflammatory cytokines in the cerebrospinal fluid and blood were not measured in this study. Therefore, we were unable to assess whether the T allele of the -1031T/C caused a decrease in TNF-alpha levels in the brain. Fourth, one of the major limitations of the current study is short of data on suicide method and associated lethality. Therefore, suicide attempts were not classified as serious suicide attempts. Therefore, we were able to only explore various factors related to suicide attempts, rather than the severity of the suicidal behaviors. In further investigations, an appropriate tool to evaluate the severity of suicide attempts would be warranted. Fifth, the hidden demographic stratification in our sample may be a confounding factor. We had a small number of female patients that may cause a discrepancy in results. Sixth, all SCZ patients enrolled in this current study were inpatients with long-term duration of illness. Hence, our findings were limited to chronic inpatients with longer duration of symptoms and more severe psychopathological symptoms compared to the typical psychiatric outpatients. Therefore, our findings in this study could not be generalized to other patients. Seventh, it is important to tease out how the genetic variation at the specific locus is getting translated into a phenotypic variation. However, we did not test the functional relevance of -1031C/T by using an in vitro cellular model, which should be remedied in the future study. Finally, although previous study showed that the C allele of TNF alpha-1031C/T was associated with higher expression of TNF-alpha , we did not measure TNF-alpha levels in this study. It would be better if the blood level of TNF-alpha could be provided in the same patient sample to increase the validity of this paper. Thus, we could not provide the mechanistic explanation linking the C allele to later age of suicide attempt in SCZ patients, which deserves further investigation.The study has several limitations that should be noted. First, although the sample was large, only 152 patients with suicidal behaviors were included. Therefore, the relationship between TNF-alpha gene did not appear to modify the propensity to develop schizophrenia and suicidal behaviors, but they did influence the age of suicide initiation in patients with SCZ. In addition, environment factors, such as drinking status, smoking, and psychotic symptoms, interacted with TNF-alpha gene and appeared to be related to the age at suicide initiation in SCZ. This suggests that the interaction between the genetics and environment might influence some pathological aspects of suicide in SCZ. However, our results may have appeared by chance, due to limited sample size of suicide subgroup. Therefore, the current association finding in this study should be confirmed in other independent samples.In conclusion, we found that the -1031C>T and -308G>A in the 1087 inpatient with schizophrenia were randomly recruited from Beijing HuiLongGuan hospital and HeBei Province Veteran Psychiatric Hospital. The current study was conducted from August 2015 to September 2017. All patients were of the chronic type, with duration of illness for at least 5 years. The inclusion criteria in the study were as our previous studies in detailed descriptions: age 20-75 years; and diagnosis of SCZ according to the Structured Clinical Interview for DSM-5 (SCID) by the trained psychiatrists. All subjects had been taking stable doses of oral antipsychotic medication for at least one year prior to recruitment. Antipsychotic medications consisted mainly of drug monotherapy, including clozapine (n = 488), risperidone (n = 239), chlorpromazine (n = 79), sulpiride (n = 55), perphenazine (n = 51), quetiapine (n = 47), haloperidol (n =38), aripirazole (n = 32), and others (n = 58). According to the method provided by Woods , the aveAccording to the definition of the World Health Organization (WHO 2014), the above four psychiatrists evaluated the history of suicide attempts based on chart review and interview data. Attempted suicide refers to any non-fatal suicide, which is intentional self-inflicted poisoning, injury, or self-harm, with or without fatal intentions or consequences (WHO 2014). In this study, there were 152 SCZ patients with a history of suicidal attempt and suicidal ideation and 935 SCZ patients without suicidal behaviors. The mean number of attempted suicide was 1.51 \u00b1 0.94 times (ranging from 1 time to 5 times).In this study, 576 healthy controls were recruited. They were randomly recruited through advertisements in the local community, and they matched patients with fewer years of education. Patients and healthy controls had comparable socioeconomic backgrounds. Psychiatrists used the standardized SCID diagnostic assessment to rule out controls with Axis I disorders. Individuals taking psychoactive drugs  were also excluded.The current study was approved by institutional Review Board of Beijing HuiLongGuan hospital. All subjects provided signed informed consent form to participate in this study.On the same day of blood drawing, the symptoms of schizophrenia were assessed by PANSS. Four psychiatrists participated in a training session on the use of PANSS and conducted repeated assessment tests prior to the study. In this present study, a five-factor model of SCZ symptoms was analyzed, labeled as \u2018positive factor\u2019, \u2018negative factor\u2019, \u2018cognitive factor\u2019, \u2018depression factor\u2019, and \u2018excitement factor\u2019 .TNF-alpha gene based on previous studies, -308G>A (rs1800629) and -1031C>T (rs1799964). These two polymorphisms were genotyped as the protocol described in our previous study [Based on previous published studies, we selected 2 tag SNPs in the >A rs18009 and -10A technician who was blind to clinical conditions genotyped each DNA sample of all subjects twice for the accuracy of genotyping. For quality control, 5% of all samples were randomly selected for repeated genotyping. Quality control tests showed that the error rate of regenotyping data was less than 0.1%.http://analysis.bio-x.cn). Analysis of variance (ANOVA) for continuous variables and the Chi-Square test (\u03c72 test) for categorical variables were used to analyze the differences in patients and controls. ANOVA and 2\u03c7 test were used to find the associations between demographic and clinical variables and the suicidal behaviors. The differences in allele and genotypic frequencies of -308G>A and -1031C>T polymorphisms between patients and healthy controls and between suicide attempters and non-attempters within the patient group were tested by the 2\u03c7 test. SHEsis software was utilized to analyze pairwise LD statistics for two tag SNPs, haplotype frequency, haplotype block, and haplotype association with schizophrenia or suicidal behavior. Quantitative trait tests were conducted to analyze the relationship between gene polymorphisms and characteristics of suicidal behavior. Less than 1% of rare haplotypes were excluded in the association analysis. The effects of the TNF-alpha genotypes on the demographic characteristics and clinical symptoms in schizophrenia attempters were examined by ANOVA and effects of genotypes on the age of suicide initiation were examined by one-way analysis of covariance (ANCOVA) to adjust for clinical confounding factors using the SPSS 18.0 software. Bonferroni corrections were used to each test to adjust for multiple testing.Adherence to Hardy-Weinberg (HW) equilibrium was evaluated by SHEsis (TNF-alpha genotypic association with suicidal behavior within the patient group while considering recessive, dominant and codominant genetic models. Only the significant genetic models in the binary logistic regression and those variables with significant associations in the ANOVA analyses between schizophrenia attempters and non-attempters were included in the stepwise multiple logistic regression analyses. In the model, the genetic models and confounders were used as independent variables, and the diagnosis was used as a dependent variable.Furthermore, we used binary logistic regressions to calculate Statistical power of the sample was computed using Quanto Software with known risk allele frequencies and the suicide attempt prevalence in the patients with schizophrenia."}
+{"text": "Soybean oil is a complex mixture of five fatty acids . Soybean oil with a high oleic acid content is desirable because this monounsaturated fatty acid improves the oxidative stability of the oil. To investigate the genetic architecture of oleic acid in soybean seeds, 260 soybean germplasms from Northeast China were collected as natural populations. A genome-wide association study (GWAS) was conducted on a panel of 260 germplasm resources.Glyma.11G229600.1 on chromosome 11 and Glyma.04G102900.1 on chromosome 4 were detected in a 2-year-long GWAS. The candidate gene Glyma.11G229600.1 showed a positive correlation with the oleic acid content, and the correlation coefficient was 0.980, while Glyma.04G102900.1 showed a negative correlation, with a coefficient of \u2212\u20090.964.Phenotypic identification results showed that the oleic acid content varied from 8.2 to 35.0%. A total of 2,311,337 single-nucleotide polymorphism (SNP) markers were obtained. GWAS analysis showed that there were many genes related to oleic acid content with a contribution rate of 7%. The candidate genes Glyma.04G102900.1 on chromosome 4 and Glyma.11G229600.1 on chromosome 11 were detected in both analyses (2018 and 2019). Glyma.04G102900.1 and Glyma.11G229600.1 are new key candidate genes related to oleic acid in soybean seeds. These results will be useful for high-oleic soybean breeding. Glycine max (L.) Merrill] originated in China and has been cultivated for more than 3000\u2009years [Soybean [00\u2009years . Soybean00\u2009years . In ChinThe fatty acid composition of soybean oil is approximately 5 to 11% linolenic acid, 43 to 56% linoleic acid, 15 to 33% oleic acid, and 11 to 26% saturated acids . The culArabidopsis [Genome-wide association analysis (GWAS) presents a powerful tool to connect this trait with the underlying genetics. With the rapid development of next-generation sequencing technology, GWAS has been successfully applied to plants such as rice and bidopsis , 10. A lbidopsis . In soybbidopsis . A soybebidopsis . These rScientists have successfully used the GWAS method to obtain a large number of candidate genes \u201316. HoweSpecifically, 260 soybean germplasms from Northeast China  were collected as natural populations. The soybean lines were planted in the field of Jilin Agricultural University from 2018 to 2019. The fatty acid content in the soybean seeds as determined by a NIRS DS 2500 instrument after harvest. Specific-locus amplified fragment sequencing (SLAF-seq) technology was used to sequence the genomes of the 260 soybean materials, and GWAS was used to screen candidate genes related to soybean oleic acid content.From 2018 to 2019, the oleic acid content of each soybean line was analysed by SPSS 22.0 software. The oleic acid content of the seeds approached the normal distribution  values decayed to half of the maximum value within 9.5\u2009kb , fastlmmc software, and Emmax software were used for the GWAS. SNP markers significantly correlated with the oleic acid content of soybean seeds were detected, and the linkage disequilibrium (LD) distance was set to 8.9\u2009kb. The Manhattan and QQ (quantile-quantile) diagrams for the oleic acid content in 2018 and 2019 are shown in Fig.\u00a0lectin gene (GenBank: A5547\u2013127) was used as the reference gene. The results showed that the candidate gene Glyma.11G229600.1 in soybean seedlings was expressed in different tissues, but the relative expression level of the gene was differed significantly, ranging from 1.23 to 4.31 in soybean leaves, from 10.21 to 39.56 in stems, and from 16.21 to 43.14 in roots  in the leaves of the soybean line q001, which has the lowest oleic acid content. The relative expression level of Glyma.11G229600.1 in the seeds of the soybean line q001 was also the lowest (25.26). The relative expression level of the candidate gene Glyma.11G229600.1 in the leaves of the soybean line q353 was 4.3 times higher than that in the leaves of the soybean line q001, and the level in seeds was 10 times higher than that in leaves   by using qRT-PCR. The ots Fig.\u00a0a. The caGlyma.04G102900.1 was also analysed in soybean leaves, stems, roots and seeds. The relative expression level of Glyma.04G102900.1 in leaves ranged from 9.62 to 44.41 and that in stems ranged from 3.18 to 28.11 , with a correlation coefficient between Glyma.04G102900.1 and oleic acid content of \u2212\u20090.964\u2009~\u2009\u2212\u20090.998  and stearic acid , stearic acid was significantly positively correlated with oleic and arachidic acid, while stearic acid had an inverse association with both linoleic and linolenic acids [Soybean oil is composed of five fatty acids: palmitic acid (16:0), stearic acid 18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) , and the:0, oleicGlyma.11G229600.1, located on chromosome 11, was simultaneously detected by GWAS during 2018 and 2017. Glyma.11G229600.1 belongs to the plant BAG protein family. The BAG proteins are a broadly conserved gene family with homologs spanning wide evolutionary distances, including yeast, animals, and plants [Arabidopsis thaliana [Glyma.11G229600.1 may increase the drought tolerance of soybean, thus affecting the accumulation of oleic acid in soybean seeds.thaliana . The BAGthaliana . Droughtthaliana . The fatthaliana . Severe thaliana . It can Glyma.04G102900.1 belongs to the plant GRAS protein family. GRAS proteins constitute an important family of plant-specific proteins named after the first three members discovered: gibberellic acid insensitive (GAI), repressor of gai (RGA) and scarecrow (SCR). At least 33 GRAS genes have been identified in A. thaliana and rice [Glyma.04G102900.1 and Glyma.11G229600.1 have been reported to be associated with the oleic acid content. Hence, further studies should be conducted to support this finding. Our results provide a basis for deciphering the mechanism underlying the determination of fatty acid composition in soybean. Moreover, the SNP markers identified here demonstrate that marker-assisted selection is a powerful strategy for identifying genes of interest in soybean and can be used in breeding programmes aimed at optimizing fatty acid profiles in seeds.and rice . Two GRAand rice . In thisGlyma.04G102900.1 on chromosome 4 and Glyma.11G229600.1 on chromosome 11 were detected in both analyses (2018 and 2019). Glyma.04G102900.1 and Glyma.11G229600.1 are new key candidate genes related to oleic acid in soybean seeds.In this study, the genome-wide association study (GWAS) technique was used to find SNP markers correlated with oleic acid content. In 2018, 20 new candidate genes related to oleic acid content were detected, and in 2018, a total of 8 new candidate genes related to oleic acid content were also detected. 2 (26\u2009\u00d7\u200910\u2009m), and each block was subdivided into eight sections. Each section was subdivided into 260 subsections. Natural drying (sunlight) was allowed to occur, and then, the seeds were threshed for oleic acid determination. Fourteen soybean varieties with significantly different oleic acid levels were selected to test candidate gene expression. The names of the soybean lines and the fatty acid content are shown in Table\u00a0The 260 soybean materials provided by the Biotechnology Center of Jilin Agricultural University were planted in the experimental field of Jilin Agricultural University  from 2018 to 2019 . A randomized complete block design was used. Each soybean line was examined using three biological replicates. The field was divided into three blocks of 260 mThe levels of oleic acid and four other fatty acids  in soybean seeds were determined by a NIRSTM DS 2500 instrument  after harvesting. SPSS version 22.0 software  was used to calculate the correlation coefficients of fatty acids in soybean seeds.RsaI-HaeIII. The sequencing service was provided by Beijing Biomarker Biotechnology Company, PR China.Total genomic DNA was extracted from the leaves of each soybean line using the CTAB method according to Murray & Thompson (1980) . The 260Principal component analysis (PCA) was used to assess the population structure using the EIGENSOFT software package. Based on the neighbour-joining method, MEGA5 software was used to construct a phylogenetic tree that included each sample.K matrix and finally obtain a correlation value for each SNP marker. The results of each model of each trait were annotated based on a 0.000001 level of significance. In this experiment, both the Manhattan map and QQ map were constructed using Haploview software. The Manhattan map was used to represent the correlation between genotype data and phenotypic data. The QQ map was used to represent the level of difference between observed and predicted values. In this study, the candidate genes were predicted by using the Swiss-Prot and NR databases.Based on the SNP markers obtained by SLAF-Seq technology, the correlation values between SNP markers and oleic acid content were obtained by using the five models  in TASSEL software. TASSEL software can calculate the Q matrix of the sample population structure according to the Glyma.11G229600.1 gene were as follows: 95\u2009\u00b0C for 10\u2009min; 35\u2009cycles of denaturation at 95\u2009\u00b0C for 30\u2009s, annealing at 67\u2009\u00b0C for 30\u2009s, and extension at 72\u2009\u00b0C for 30\u2009s; and extension at 72\u2009\u00b0C for 10\u2009min. All of the above reactions included 40\u2009cycles. After amplification, the dissolution curve was calculated by the 2-\u0394\u0394Ct method [Glyma.04G102900.1 were as follows: predenaturation at 95\u2009\u00b0C for 10\u2009min; 35\u2009cycles of denaturation at 95\u2009\u00b0C for 30\u2009s, annealing at 59\u2009\u00b0C for 30\u2009s, and extension at 72\u2009\u00b0C for 35\u2009s; and extension at 72\u2009\u00b0C for 10\u2009min. Three biological replicates were used for each gene.qRT-PCR analysis was performed using a Bio-Rad CFX system . Total RNA was extracted using an Eastep\u00ae Super Total RNA Extraction Kit . The amplification reaction conditions were as follows: predenaturation at 95\u2009\u00b0C for 10\u2009min, denaturation at 95\u2009\u00b0C for 10\u2009s, followed by annealing at 53\u2009\u00b0C for 20\u2009s, and extension at 72\u2009\u00b0C for 15\u2009s. The amplification reaction conditions for the t method . The ampThe phenotypic data were measured and recorded using Microsoft Excel 2010 software. Differential saliency analysis, analysis of variance, correlation analysis and descriptiveness analysis were performed by using SPSS 19.0  software . The posAdditional file 1 Table S1. Heritability of fatty acid content in soybean seeds.Additional file 2 Figure S1. Population structure of the soybean germplasm collection. (PPTX 182 kb)Additional file 3 Table S2. SNPs identified as being associated with oleic acid content in 2 years (P\u2009<\u20090.000001).Additional file 4 Figure S2. Genome-wide linkage disequilibrium (LD) decay for all 260 accessions. (PPTX 223 kb)Additional file 5 Figure S3. Expression of Glyma.11G229600.1 in different tissues of soybean lines. (PPTX 354 kb)Additional file 6 Table S3. Correlation between Glyma.11G229600.1 expression and oleic acid content.Additional file 7 Figure S4. Expression of Glyma.04G102900.11 in different tissues of soybean lines. (PPTX 233 kb)Additional file 8 Table S4 Correlation between Glyma.04G102900.1 expression and oleic acid content."}
+{"text": "Medullary thyroid carcinoma (MTC) accounts for 1%\u20132% of thyroid cancer in the United States based on the latest Surveillance, Epidemiology, and End Results (SEER) data, and this study aimed to construct a comprehensive predictive nomogram based on various clinical variables in MTC patients who underwent total thyroidectomy and neck lymph nodes dissection.  Data regarding 1,237 MTC patients who underwent total thyroidectomy and neck lymph nodes dissection from 2004 to 2015 were obtained from the SEER database. Univariate and multivariate Cox regression analyses were used to screen for meaningful independent predictors. These independent factors were used to construct a nomogram model, a survival prognostication tool for 3- and 5-year overall survival, and cancer-specific survival among these MTC patients. n\u2009=\u2009867) and the test group (n\u2009=\u2009370). Univariate and multivariate Cox regression analyses were used to identify meaningful independent prognostic factors (P < 0.05). Tumor size, age, metastasis status, and LNR were selected as independent predictors of overall survival (OS) and cancer-specific survival (CSS). Finally, two nomograms were developed, and the predicted C-index of overall survival (OS) and cancer-specific survival (CSS) rate in the training group was 0.828 and 0.904, respectively. The predicted C-index of overall survival (OS) and cancer-specific survival (CSS) rate in the test group was 0.813 and 0.828.  A total of 1,237 patients enrolled from the SEER database were randomly divided into the training group ( Nomograms constructed by using various clinical variables can make more comprehensive and accurate predictions for MTC patients who underwent total thyroidectomy and neck lymph nodes. These predictive nomograms help identify postoperative high-risk MTC patients and facilitate patient counseling on clinical prognosis and follow-up. Medullary thyroid carcinoma (MTC) is a neuroendocrine malignant tumor derived from parafollicular cells, accounting for 1%\u20132% of thyroid cancer in the United States based on the latest Surveillance, Epidemiology, and End Results (SEER) data . The cur\u2217Stat software . The patients were limited to being diagnosed with MTC and underwent total thyroidectomy and neck lymph nodes dissection between January 1, 2004, and December 31, 2015. We extracted the following clinical information from the database: age at diagnosis, race , sex, year of diagnosis, histologic type, clinical TNM stage, tumor size, tumor extension, number of nodules, surgery of primary site, number of removal lymph nodes, the number of regional lymph nodes by pathological findings, the number of positive lymph nodes (pLNs), survival time, cause of death, and survival status, and based on the regional lymph nodes by pathological findings and the numbers of positive lymph nodes (pLNs) of each patient, we calculated the lymph node ratio (LNR). The exclusion criteria were set as follows after obtaining data: (1) patients with missing or unknown clinical information, (2) patients with survival time less than 1 month, and (3) patients without local lymph node dissection. Finally, data of 1,237 patients were collected from the SEER database based on the inclusion and exclusion criteria. X-tile software was used to find out the optimal cutoff values for age, tumor size, the number of regional lymph nodes by pathological findings, the number of pLNs, and LNR (n\u2009=\u2009867) and a test cohort (n\u2009=\u2009370) in the ratio of 7\u2009:\u20093. The training cohort is used for model construction and the test cohort for external verification. The endpoints of this study are overall survival (OS) and cancer-specific survival (CCS); OS refers to the period from the time of surgery to the death of any cause or the date of the last follow-up, while CSS was calculated from the time of operation to the date of cancer-related death or the time of last follow-up [The data were extracted from the SEER database using the SEER and LNR . The optP values <0.05 were considered statistically significant. The nomograms were internally and externally validated in training and test cohorts, respectively, Harrell's concordance index (C-index), receiver operating characteristic curve (ROC), and area under the ROC curve (AUC) were used to evaluate the exact prognostic performance of nomograms; the values of C-index and AUC closer to 1 implied a better predictive accuracy. The verification curve can reflect the consistency between the prediction and actual nomograms. The C-index, ROC, nomograms, and the verification curve of the training cohort were formulated and adjusted by using R version 3.6.3 in the RStudio environment, and the C-index and AUC were also calculated in the test cohort to further evaluate the performance of the nomogram.Summary statistics tables are used to describe demographic and clinical characteristics of the training cohort and test cohort, and data of continuous and categorical variables are presented as frequencies with percentages using Word 2016 for Windows. Survival time was defined as the time (in months) from surgery to death, last follow-up, or December 31, 2015. The optimal cutoff values of age, tumor size, the number of regional lymph nodes by pathological findings, pLNs, and LNR were evaluated by using the X-tile software as described previously. Univariate Cox regression analysis was applied to evaluate independent survival-related factors for overall survival (OS) and cancer-specific survival (CSS) in the retrospective data. In a multivariate analysis, the Cox proportional hazards regression model was applied for the categorical variables identified as significant in the univariate Cox regression analysis. The intensity of the association between each predicted categorical variables and survival was expressed as a hazard ratio (HR), and 95% confidence intervals (95% CI) were calculated at the same time. The Cox regression analysis was performed by using Statistical Product and Service Solutions (SPSS) version 26.0, and SEER data are deidentified before release and do not contain any personally identifying information. As the data are publicly available, no ethical approval is required. We received permission to access the research data file in the SEER program from the National Cancer Institute, USA (reference number 12151-Nov2019).n\u2009=\u2009867) and test group (n\u2009=\u2009370). Their basic information on demographic and clinical characteristics is listed in P < 0.05) in the univariate Cox regression analysis for OS. Categorical variables defined as having a significant association with OS on univariate analysis were studied in a Cox proportional hazards model, and there are four significant meaningful predictors: tumor size, age, metastasis status, and LNR in this multivariate Cox regression for OS (P < 0.05). In the multivariate Cox regression for CSS, the following 4 variables were identified to construct the Cox proportional hazards regression model, namely, tumor size, age, metastasis status, and LNR  is meaningless on account of medullary carcinoma cells that originated from C cells that do not absorb I131. A single-center experience indicates that adjuvant radioisotope therapy was not sensitive to the medullary carcinoma cells unless the MTC patient was accompanied by histological subtypes of papillary or follicular carcinoma [We construct the nomogram based on the four prognostic factors mentioned above. To our knowledge, this is the first nomogram with excellent predictive performances to investigate the prognostic value of MTC patients who underwent total thyroidectomy and cervical dissection. The utility of the nomograms was illustrated as follows: the point score of each factor can be calculated separately by reading the score above the factor vertically, for example, 65 points above the age of 48\u201371 and 45 points above M1 status in the OS nomogram. The total point score was derived and then read vertically downwards to the 3- or 5-year survival HR. Taking a hypothetical patient as an example, a 50-year-old patient with a tumor size of 30\u2009mm who underwent the standard surgical procedure was described in this article. His postoperative LNR was 50%, and there was no distant metastasis. It can be inferred that his 5-year OS is around 70% and 5-year CSS is close to 80% from the nomogram in the article. In our study, to build a refined nomogram, some controversial factors such as radiotherapy and chemotherapy were not taken into account. Treatment with radioactive iodine levels owing to SEER's lack of information regarding biochemical examination results, both of them are recognized as independent factors which predict the outcome. Meanwhile, our nomogram did not include calcitonin and CEA doubling times. Second, a classic surgical approach was used for cervical lymph nodes including unilateral or bilateral central lymphadenectomy, modified lymphadenectomy, and radical lymphadenectomy. We cannot evaluate the effectiveness of particular surgical techniques on account of this level of surgical detailed information that is not available in the SEER. Finally, familial MTC, multiple endocrine neoplasia (MEN)-2A or 2B, genetic RET mutation status, and other information about familial MTC cannot be obtained in the SEER database, which prevents us from independently assessing any predictor related to it. In addition, what we should know is SEER database derived from a variety of sites without a single standardised management protocol and from numerous surgeons with presumably varying skills, so that the outcome represents average but not necessarily optimal management. It should be noted that our nomograms have not been validated external datasets, and it is necessary to use other databases for calibration in the future. We anticipate physicians to use nomograms to evaluate prognosis, combined with clinical variables such as calcitonin, CEA, or the latest research results such as tumor necrosis and high mitotic rate  and expeIn summary, this study used routine clinical data to build the first nomogram model of 3- and 5-year CSS and OS among MTC patients who underwent total thyroidectomy and neck lymph nodes dissection. The nomogram provides a convenient prognostication model for the clinical practice of the surgeon and facilitates patient counseling on clinical prognosis and follow-up."}
+{"text": "Next-generation sequencing technologies revolutionized genomics by producing high-throughput reads at low cost, and this progress has prompted the recent development of de novo assemblers. Multiple assembly methods based on de Bruijn graph have been shown to be efficient for Illumina reads. However, the sequencing errors generated by the sequencer complicate analysis of de novo assembly and influence the quality of downstream genomic researches.k-mer clustering approach from the overlap-layout-consensus concept to deal with the sequencing errors generated by the Illumina platform. We further evaluate Clover\u2019s performance against several de Bruijn graph assemblers , overlap-layout-consensus assemblers  and string graph assembler (SGA) on three datasets . The results show that Clover achieves a superior assembly quality in terms of corrected N50 and E-size while remaining a significantly competitive in run time except SOAPdenovo. In addition, Clover was involved in the sequencing projects of bacterial genomes Acinetobacter baumannii TYTH-1 and Morganella morganii KT.In this paper, we develop a de Bruijn assembler, called Clover (clustering-oriented de novo assembler), that utilizes a novel https://oz.nthu.edu.tw/~d9562563/src.html.The marvel clustering-based approach of Clover that integrates the flexibility of the overlap-layout-consensus approach and the efficiency of the de Bruijn graph method has high potential on de novo assembly. Now, Clover is freely available as open source software from  Choosing the length of k is an important issue in the de Bruijn graph approach. Theoretically, for reads without sequence errors, smaller k-mers increase the connectivity of the graph and larger k-mers decrease the number of ambiguous repeats in the graph. There is therefore a balance between sensitivity and specificity determined by k [k-mers decrease the sensitivity and specificity further due to sequencing errors generated by the Illumina platform, in which the primary errors are substitution errors, at rates of 0.5\u20132.5% [Massively parallel DNA sequencing has become a prominent tool in biological research , 2. The ned by k . However0.5\u20132.5% .k-mers, and could the error allowance on k-mers improve the quality of de novo assembly. Therefore we developed a clustering-oriented approach, called Clover, to deal with those substitution errors, and use a new parameter p which describes the level of error allowance on k-mers. For example, setting k to 40 and p to 1 means that our algorithm uses each 40-mers in the input reads while allowing each of them to have the flexibility of 1 substitution error.In this study, we are trying to answer what happens if we design an approach to allow such errors on k-mers, Clover tries to cluster these k-mers together when their Hamming distance less than or equal to p, and merges each cluster of k-mers to a node by finding its consensus sequence. To avoid over-merging of clusters, which may occur on the boundary of repeat sequence, Clover will split each node into multiple nodes when the merged node has multiple major consensus sequences (see Implementation section for detail).With the flexibility of error allowance on Leptospira shermani assembly when using different level of error allowance . Setting p to 0 is equal to run our assembler with traditional de Bruijn-based approach, which does not have the flexibility of error allowance on k-mers. The assembly result shows that only setting p to 1 could dramatically increase the N50 both in contig and scaffold because it reduces the number of nodes to build the de Bruijn graph that increases the specificity. The result also shows that setting p to 2 only increases the N50 on contig whereas decreases on scaffold. In this case, reducing too many nodes could increase the specificity, but it seems losing some meaningful information, which decreases the sensitivity at the same time.After the steps mentioned above, the number of nodes in the graph will dramatically reduce, which therefore simplifies analysis of assembly. For example, Table p . The time cost is dropped when setting p to 1 due to the benefit of simplifying analysis, but setting p to 2 could not get the benefit more. Together with the phenomena described above, we should choose a suitable p, not as large as possible. In the case of L. shermani assembly, the suitable p is 1 and p/k is 2.5%, which is nearly the error rate of Illumina platform.The memory requirements, as shown in Table Clover proceeds through the following phases whose flowchart is shown in Fig.\u00a0k (k-mer) and p , Clover constructs a Hamming graph by extracting all the input k-mers as nodes. The graph\u2019s edges are created by the pairs of k-mers if the Hamming distance of the k-mers (or their reverse complements) is\u2009\u2264\u2009p. Figure\u00a0k-mers  can dramatically reduce the number of k-mers for constructing the de Bruijn graph later and accelerates the subsequent graph processing. In the implementation, Clover uses two steps to cluster the k-mers: Step 1 extracts all k-mers from the input reads. Step 2 constructs a Hamming graph of the k-mers and then performs a breadth-first search to find each component in the Hamming graph. If there is no error allowance needed (p\u2009=\u20090), Clover will omit the process of step 2.For given ers Fig.\u00a0b. Cloverk-mers  whose consensus sequence therefore is GGTCT as shown in Fig.\u00a0k-mers of the merged node, called v, have a nucleotide on a base pair, called x, whose occurrence is closed to that of the corresponding nucleotide, called y, in the consensus sequence, Clover splits the merged node into multiple nodes. More realistically, given a fractional threshold sp, if the occurrence of x is greater than or equal to sp times the occurrence of y, Clover collects all the k-mers with x into a new node, called v1, leaves the others into a new node, called v2, and recursively retries this splitting process on v1 and v2. Let q be the number of k-mers in the merged node. Then the consensus computing requires O(q\u2009\u00d7\u2009k) time, the condition checking requires O(k) time and collecting the k-mers into the new nodes requires O(q) time. Therefore, similar to the analysis of quick sort, the worst case of the time complexity on the whole process is O(q2\u2009\u00d7\u2009k). Figure\u00a0sp to 0.6. Finally, Clover collects all the resulting consensus sequences as the k-mer set for constructing de Bruijn graph in the next phase.Clover computes the consensus sequence for each merged node by selecting the nucleotide with the most occurrence on each base pair. For example, the fourth component of Fig.\u00a0k-mer set obtained in the previous phase, Clover constructs the de Bruijn graph by directly using the k-mer set as its node set. For any two nodes, it creates an edge between them if their corresponding k-mers have an overlapping of length k\u2009\u2212\u20091. Figure\u00a0For the k until the given minimum overlapping length m is achieved. Clover defaultly sets the parameter m to (k/2)\u2009+\u20091. For example, we compare the different settings of m on the Rhodobacter sphaeroides assemblies , Rhodobacter sphaeroides (4.6\u00a0Mb) and human chromosome 14 (88.3\u00a0Mb) [To evaluate the assembly correctness of Clover, we have tested three typical datasets in the GAGE study: roides 4.\u00a0Mb and hhttps://oz.nthu.edu.tw/~d9562563/src.html.We provide Clover source code with this submission  needed to cover 50% of the genome. The N50 statistics generated by Clover is the minimum contig length needed to cover 50% of all the sequence produced. However, the N50 statistics generated by the GAGE validation scripts is the minimum contig length needed to cover 50% of the reference genomic sequence provided in GAGE study. Therefore the N50 statistics of Clover in Additional file L. shermani seems to be poorer than S. aureus and R. sphaeroides. However, these two datasets in GAGE study have two libraries and the fragment size is up to 3500\u00a0bp [L. shermani dataset only has single library with fragment size 485\u00a0bp. The better assembly quality is caused by using more libraries. Practically, researchers usually use optical mapping to arrange scaffolds and then obtain the draft sequence [The result of  3500\u00a0bp , whereassequence .k-mer on single library or lower coverage dataset such as the L. shermani assembly, and larger k-mer on more complex genomes such as human chromosome 14. When using large k-mer, increasing the level of error allowance is especially needed even on ever error-corrected reads.In addition, our clustering approach can apply on ever error-corrected reads. For example, the assembly of human chromosome 14 is generated by clustering 80-mers while allowing 3 errors on Allpaths-LG corrected reads. Therefore Clover would not conflict with current error correction tools. In practice, we will apply smaller To assess Clover\u2019s run times and memory requirements, we have rerun above assemblers that follow the same processes and parameters of GAGE with their newest version on a 16-core AMD Opteron 6128 2\u00a0GHz server with 256\u00a0GB of RAM. The parameters of optimal result seem varying with the different version of assemblers and hence we only take their run times and memory requirements into comparisons. Because we don\u2019t have large-scale parallel environment, we only run ABySS on single-process version.Table k-mers clustering. In the k-mers clustering, Clover constructs a Hamming graph in which it links each pair of k-mers as an edge if the Hamming distance of the pair of k-mers is\u2009\u2264\u2009p. To accelerate the process, Clover utilizes the indexing technique that partitions a k-mer into (p\u2009+\u20091) substrings. If the Hamming distance of a pair of k-mers is\u2009\u2264\u2009p, there must exist a pair of substrings that are exactly the same. Therefore Clover uses (p\u2009+\u20091) hash tables which index each substring of all k-mers to find the candidate pairs of k-mers, and performs comparisons to check their real Hamming distances. Let n be the number of the reads. l be the length of the reads, k be the length of k-mers and p be the level of error allowance on the k-mers. Note that p can be 0 in this study. Then n\u2009\u00d7\u2009(l\u2009\u2212\u2009k\u2009+\u20091) is the number of k-mers within the reads, (p\u2009+\u20091) is the number of hash tables needed to find the candidate pairs of k-mers and each comparison for them requires O(k) time. Therefore, the worst case of the time complexity on k-mers clustering is O(n\u2009\u00d7\u2009(l\u2009\u2212\u2009k\u2009+\u20091)\u2009\u00d7\u2009k\u2009\u00d7\u2009(p\u2009+\u20091))\u2009\u2245\u2009O(n\u2009\u00d7\u2009(l\u2009\u2212\u2009k)\u2009\u00d7\u2009k\u2009\u00d7\u2009(p\u2009+\u20091)). Similarly, the memory needed to store all the sequences of the k-mers is O(n\u2009\u00d7\u2009(l\u2009\u2212\u2009k)\u2009\u00d7\u2009k), and the memory needed for all the k-mers on the hash tables is O(n\u2009\u00d7\u2009(l\u2009\u2212\u2009k)\u2009\u00d7\u2009(p\u2009+\u20091)). Since k is much larger than p, the worst case of the space complexity on k-mers clustering is O(n\u2009\u00d7\u2009(l\u2009\u2212\u2009k)\u2009\u00d7\u2009k).The major cost of Clover is the Acinetobacter baumannii TYTH-1 (4.0\u00a0Mb and 165 contigs) [Morganella morganii KT (3.8\u00a0Mb and 58 contigs) [A. baumannii TYTH-1 and M. morganii KT, 3682 and 3565 protein-coding sequences, 75 and 72 tRNA genes, and 6 and 10 rRNA genes were further predicted, respectively. Table It is worth mentioning that Clover was involved in two sequencing projects to respectively sequence bacterial genomes contigs)  and Morgcontigs) . The conThe limit of our current Clover is that it cannot apply on genomes with large size up to 250\u00a0Mb. This is caused by 256\u00a0GB of RAM in our server (see Run times and memory requirements section for detail). However, if the server has more RAM, the limitation could be eliminated. The memory requirement issue exists in many assemblers with which we compared in this study. As shown in Table We leave the parallelization of program as a future work that will further improve the performance of Clover. Besides, we leave the exploration of other possible clustering algorithms to further improve Clover as another future work.k-mers and the efficiency of the de Bruijn graph method, with which we improve the robustness with respect to sequencing error especially using large k-mers. We discovered the effect of our clustering approach that not only improves the assembly result but also accelerates the assembly process by simplifying analysis on the Leptospira shermani assembly. The evaluation of Clover on GAGE datasets finally shows that it achieves a superior assembly quality in terms of corrected N50 and E-size while remaining a significantly competitive in run time.In this study, we developed a new clustering-oriented de novo assembler, called Clover, that integrates the flexibility of the overlap-layout-consensus approach on clustering Project name Clover.Project home pagehttps://oz.nthu.edu.tw/~d9562563/Operating system(s) Linux.Programming language C, Python and Cython.Other requirements Python-devel to develop Python extensions.License GNU GPL2.Any restrictions to use by non-academics None.Additional file 1Clover source code for Linux. Please refer to \u2018Installation of Clover\u2019.Additional file 2Datasets and Installation of Clover. GAGE dataset list and locations, and build Clover\u2019s executing and programming environments.Additional file 3Leptospira shermani assembly statistics results. Section S2\u2014Clover assembly statistics results. Clover output screen text.Section S1\u2014Additional file 4Leptospira shermani assembly. Table S2\u2014Sensitivity comparison of different minimum overlapping lengths on Rhodobacter sphaeroides assembly. Supplemental analysis of Clover. Table S1\u2014Memory requirements (GB) of k versus p correlation on"}
+{"text": "Accurate 3D passive tracking of an underwater uncooperative target is of great significance to make use of the sea resources as well as to ensure the safety of our maritime areas. In this paper, a 3D passive underwater uncooperative target tracking problem for a time-varying non-Gaussian environment is studied. Aiming to overcome the low observability drawback inherent in the passive target tracking problem, a distributed passive underwater buoys observing system is considered and the optimal topology of the distributed measurement system is designed based on the nonlinear system observability analysis theory and the Cramer\u2013Rao lower bound (CRLB) analysis method. Then, considering the unknown underwater environment will lead to time-varying non-Gaussian disturbances for both the target\u2019s dynamics and the measurements, the robust optimal nonlinear estimator, namely the adaptive particle filter (APF), is proposed. Based on the Bayesian posterior probability and Monte Carlo techniques, the proposed algorithm utilizes the real-time optimal estimation technique to calculate the complex noise online and tackle the underwater uncooperative target tracking problem. Finally, the proposed algorithm is tested by simulated data and comprehensive comparisons along with detailed discussions that are made to demonstrate the effectiveness of the proposed APF. The marine resources have significant influences on human\u2019s living and social developments. Furthermore, the ocean plays an important role for national security and the underwater target tracking is one of the most significant research areas of marine science and underwater acoustic engineering nowadays. Therefore, to exploit the ocean resources and defend the national territory, the technology of accurately and reliably tracking an uncooperative underwater target is of vital importance.There are two categories of underwater target tracking techniques, namely the active underwater target tracking and the passive underwater target tracking. The former always utilizes high-power active sonar equipment to transmit active detection signals to track the target in real time ,2. This For the aspect of the passive sonar tracking system, two kinds of measurements are commonly utilized to track an uncooperative underwater target. The first one is the angle-based measurements. By collecting the passive signals from an underwater target, various kinds of DOA algorithms can be utilized to output both the azimuth angle and the elevation angle of the target ,7. Then,In order to build a robust and accurate underwater passive tracking system, the observability along with the accuracy analysis should be firstly studied comprehensively. Since the passive tracking system only utilizes the radiations from the interested underwater target to accomplish the tracking process and the frequency-based measurements are not available when the target moves at a slow speed far from the tracking system, only the angle information can be obtained and utilized. Therefore, the passive tracking process sometimes is called the bearing-only tracking in a 2D scenario and angle-only tracking in a 3D scenario. The observability analyses are comprehensively studied by several researchers ,10,11, aFirstly, the observability of a tracking system is not \u201cconstant\u201d. For the nonlinear system of underwater target tracking, even a full observable tracking system has different levels of observability for different configurations of the distributed underwater buoys that participate in the current tracking process. According to ref. , the obsIn addition, for a distributed system containing several underwater buoys, it is understandable that more measurements being utilized at the same tracking time can provide more accurate tracking results. However, since the underwater acoustic communicating resources are very limited, more underwater buoys will cost more underwater acoustic communicating resources which, as a result, will make the information fusion procedure much longer and decrease the tracking accuracy at its expense. Hence, the goal is to utilize an appropriate number of buoys to satisfy the accuracy requirements and save underwater acoustic communicating resources in the meantime. Therefore, the method of balancing the tracking accuracy and the system observability is crucial for the passive distributed underwater buoys system and has great researching significance. Although some researchers have developed the sensor scheduling and control principle for the distributed radar systems ,17,18, tBesides the considerations of the measurements, the target tracking algorithms also play an indispensable role in the tracking procedure. Since the underwater uncooperative target always stays in a certain moving mode for a long time and seldom makes maneuvering, the kinematics of the target can be modeled as the constant velocity (CV) model like many researchers supposed in ref. . Hence, By utilizing the CRLB and FIM analysis method along with the nonlinear observability analysis techniques, a real-time optimal topology design method of the distributed passive underwater buoys is proposed to balance the estimation robustness and accuracy dynamically.An online noise estimator is proposed based on the Sage-Husa estimating technique to estimate the 1st order and 2nd order momentum of the time-varying noise dynamically.An intelligent tracking algorithm for a time-varying non-Gaussian environment is proposed, namely the adaptive particle filter (APF). The proposed algorithm guarantees the convergence of underwater target tracking accuracy.Considering all the above-mentioned challenges and in order to track the underwater uncooperative target in a 3D real-time scenario under the time-varying non-Gaussian environment by distributed passive submerged buoys, this paper firstly proposed a real-time optimal topology-forming method of the distributed measurement system that balances the tracking robustness and accuracy. Then, a robust algorithm that combined the advantages of both the Bayesian posterior estimation techniques and the online noise estimation techniques is designed. The contributions can be summarized as follows:The rest of this paper is organized as follows. In In this section, the kinematics model of the underwater target and the measurement model of the distributed buoys system are proposed. Assuming the underwater uncooperative target performs a constant velocity (CV) mode, the underwater uncooperative target usually remains at the same speed to save energy. In addition, the measurements of the distributed underwater buoys are the passive angle-only information which can be divided into the azimuth angle information and the elevation angle information. The following section gives the details of the above-mentioned models, respectively.The main motive of the 3D tracking problem considered in this work is to estimate the position and velocity of an underwater uncooperative target with CV motion mode using noise-corrupted angle-only measurements from the passive distributed underwater buoys system. Consider  to ref. , the kinIt should be noted that, unlike many researches supposing te noise ,20,21,22The measurement model of the i-th buoy at tracking time k.Assuming the tracking procedure is performing in the free 3D space ) and the system state (From Equations (4)\u2013(6) and the time-updated state The maximum likelihood estimation-based underwater target tracking problem can be represented as maximizing the likelihood function Considering Hence, the FIM of the system formed by Equations (1) and (5) can be represented as:According to ref. , the minAdditionally, the CRLB can be represented by the inverse of the FIM, namely:Consequently, by minimizing the value computed by the above Equation (13), two out of three buoys of the distributed measuring system can be selected and the optimal topology based on estimating accuracy can be determined, namely:m is the discrete tracking step during the overall tracking procedure, Observability analysis of a nonlinear tracking system is the foundation of making reliable tracking results. From Equations (1) and (4), it can be found that the system to be analyzed is highly nonlinear. According to ref. , the GraAccording to the linear observability theory, the states can be estimated from the measurements at any biased initial guesses when a linear system is fully observable. Hence, in order to make the tracking procedure reliable and robust, it is essential to guarantee that the tracking system is fully observable especially when the target to be tracked is uncooperative, which means that the tracking process must have the ability of convergence at any random initial guesses. However, unlike the linear system, the theory of determining whether an arbitrarily nonlinear system is fully observable is still an open question. From Equation (15), it can be found that the OG matrix is time-varying which leads to the observability of a nonlinear system having local characteristics.According to the observability analysis criterion proposed in ref. , a nonlik.Since the conditional number can reflect whether a matrix is \u201chealthy\u201d, the index of observable degree is introduced to reveal whether an OG matrix is better under a certain topology of the distributed buoys. The observable degree is represented as follows:From the matrix analysis theory, the OG matrix is more robust to the disturbances when the value of According to the above analysis, if the tracking system is locally weak observable at tracking time k, the optimal topology design criterion based on robust tracking aspect can be represented as:In order to track the underwater uncooperative target as accurate as possible and consume the least acoustic communicating resources, an unity objective function considering both the tracking accuracy and the communication usage is designed as the following:Hence, by defining the adjust parameter From Equations (1) and (4), it can be found that the 3D passive tracking process is a high nonlinear problem with uncertain noise. Unlike many researches which regard the noise as the Gaussian white noise, we model the process noise and the measurement noise as the time-varying non-Gaussian noise since this assumption is closer to the reality. Hence, many of the existing nonlinear estimating algorithms cannot be directly adopted. To make the time-varying noise estimated online, several adaptive techniques have been developed ,28. HoweThe Sage-Husa online noise estimator is firstly introduced by ref.  for lineFrom Equation (21) to Equation (26), it can be found that the classic nonlinear Sage-Husa online noise estimator must utilize all the smooth values of the state within a certain tracking period, which leads to the total process hard to compute. According to ref. , the recFor time-varying noise, the latest measurement should be given more attention than the historical data. Therefore, the fading factor It can be found in Equation (36) that, when the index Since the passive underwater target tracking system is highly nonlinear and the disturbances are time-varying and non-Gaussian, the classic nonlinear estimators cannot be utilized. Here, we introduce the APF to make reliable estimations under the time-varying non-Gaussian environment.1.InitializationThe designed APF united the modified Sage-Husa online noise-estimating technique and the PF tracking technique to track an uncooperative underwater target by a certain topology of the distributed buoys system. Depending on the nonlinear tracking system represented by Equations (1) and (5), the APF can be divided into the following steps:2.Time updateAt tracking time 3.Measurement noise online estimationAccording to Equations (1) and (32), the time updated by every state particle under the time-varying non-Gaussian environment can be represented as:According to Equations (33) and (35), the measurement noise at time 4.Weight calculationFrom Equations (38)\u2013(41), the time-varying non-Gaussian measurement noise According to the theory of PF, the weight of every particle can be represented as:According to ref. , q(xki\u2223xFrom Equation (43), the weight of every particle can be computed by 5.Measurement UpdateFrom Equations (43) and (44), the weight of 6.ResamplingAccording to Equations (37) and (45), the measurement updated state based on Monte Carlo theory can be computed as:In order to avoid the particle degradation phenomenon and make the whole tracking process robust, the resampling method is adopted to generate new particles based on the weights calculated by Equation (45). The principle of the resampling method is to duplicate the more likely particles and cut off the less likely ones, which will lead to some same particles in the new particle system . Before 7.Process noise online estimationIf 8.Time propagationThe 1-st order momentum of the time-varying non-Gaussian noise at the current tracking time can be updated online by the modified Sage-Husa online noise estimator as:The tracking time will be propagated to Three-dimensional passive underwater target tracking algorithm for the time-varying non-Gaussian environment by distributed underwater buoysAlgorithm 1: 3D passive underwater target tracking algorithm for the time-varying non-Gaussian environment by distributed underwater buoys1. Optimal topology determination1 Use every two out of three measurements from the buoys to form the observable measurements sets as 2 Calculate the accuracy index 3 Calculate the robust index 4 Use the results from step 1 and 2 to calculate the overall index 5 With the preset index 2. Tracking the underwater uncooperative target by APF under the time-varying non-Gaussian environment1 Based on the determined sets of measurements at tracking time 2 Particles are time-updated by Equation (37);3 Online noise estimation by Equation (39);4 New weights are calculated by Equation (45);5 Resampling procedure are checked and performed by Equation (47) and resampling method introduced by ref. [6 Estimate the current process noise by Equation (48);7 Time propagation to run the whole algorithm at tracking time  by ref. ;6 EstimaConsidering both the optimal topology design criterion by In this section, comprehensive simulations are made to verify the proposed 3D underwater uncooperative target passive tracking algorithm. In addition, by setting the reference coordination, the coordinates of every buoy of the distributed system and the target are represented in the reference coordination. The detailed configuration of the distributed buoys is set out in In the simulations, the underwater target is supposed to perform in the CV motion mode with the actual initial state as It is also assumed that all the underwater buoys have the same stochastic process of the time-varying non-Gaussian measurement noise and can be modeled as:All the simulations in this paper are performed using MATLAB R2019b on the computer with the Microsoft Windows 10 system and the computer is configured with Intel(R) Core (TM) i7-9700k CPU @3.2 GHz. The simulation results are the average of 50 Monte Carlo experiments with the particle number of 2000 for every simulation. According to ref. , the totIn this subsection, the optimal topology design algorithm proposed by In the subsection, the APF algorithm proposed by From the simulation results, the estimating accuracies are enhanced by 92.4% and 83.4% by the proposed APF for The computational complexity of the APF, AEKF, and traditional PF are shown in The 3D passive tracking of the underwater uncooperative target using distributed underwater buoys for a time-varying non-Gaussian environment is studied in this paper. Firstly, for the sake of accurate tracking and minimum underwater acoustic resource utilization, the optimal topology of the distributed underwater buoys system is designed based on both accuracy theory and observability analysis. Then, based on the determined optimal topology of the distributed underwater buoys at current tracking time, an APF is proposed to tackle the nonlinear underwater target tracking problem with time-varying non-Gaussian noise. From the simulation results, the following conclusions can be drawn. Firstly, the optimal topology design method introduced by this paper can select proper sets of buoys dynamically and can provide the best estimations among all the possible configurations of the distributed buoys. Secondly, by adopting the online noise estimation technique, the APF has significantly higher accuracies compared to the AEKF and the traditional PF with very little extra computational load. It is believed that the APF proposed by this paper has great potential in real-time 3D underwater target passive tracking missions."}
+{"text": "Most models of neural responses are constructed to reproduce the average response to inputs but lack the flexibility to capture observed variability in responses. The origins and structure of this variability have significant implications for how information is encoded and processed in the nervous system, both by limiting information that can be conveyed and by determining processing strategies that are favorable for minimizing its negative effects. Here, we present a new modeling framework that incorporates multiple sources of noise to better capture observed features of neural response variability across stimulus conditions. We apply this model to retinal ganglion cells at two different ambient light levels and demonstrate that it captures the full distribution of responses. Further, the model reveals light level-dependent changes that could not be seen with previous models, showing both large changes in rectification of nonlinear circuit elements and systematic differences in the contributions of different noise sources under different conditions. Current models for neural responses typically focus on accurately estimating a neuron\u2019s average response to a stimulus but often fail to accurately reflect response variability. Such variability is central to the accuracy with which neural responses represent inputs and with which they can guide behavior. We present a new modeling framework that accurately captures observed variability in neural responses and find that multiple stochastic model elements are necessary to capture this variability. We show that model parameters can be accurately estimated using \u223c8\u2009min of data. We then apply the model to retinal ganglion cells, demonstrating light level-dependent changes in both deterministic and stochastic model elements changes that are either obscured or absent using more standard modeling approaches.Variability in neural responses can reveal aspects of circuit function that are not apparent from average responses alone. For example, identifying different sources of variability can guide the search into potential mechanisms that shape neural responses . VariabiIn order to accurately reflect neural variability, models must capture features that drive neural responses, as well as noise in the underlying mechanisms that produces true randomness. Ultimately, all these elements will be necessary to produce more complete models that disentangle the contributions of different mechanisms to neural responses. To date, much work has focused on incorporating into models additional stimulus features that drive spiking, dependence on response history, or modulation from other neurons . ComparaIn current models, the generation of spikes from a neuron\u2019s inputs is most commonly described as a Poisson process , potentiHere, we take the approach of incorporating stochastic model elements inspired by what is known about the biological circuitry and likely sources of noise. We present a model that includes multiple potential sources of noise, which may arise at different locations relative to a circuit nonlinearity and which may have distinct effects on the observed variability in responses. This model allows us to estimate the strengths of these individual noise sources and clarify the separate contributions of deterministic and stochastic model elements. We first demonstrate that this model can be tractably fit to a dataset of limited size and that we accurately recover model elements, both the shape of the nonlinearity and the strength of each noise source, in a simulated dataset with known model parameters. We then demonstrate an application of this model in retinal ganglion cells (RGCs). The model captures response variability under two different stimulus conditions and further reveals consistent changes in both the nonlinearities and inferred sources of noise that depend on stimulus condition. The model is suitable for a variety of systems and allows for comparisons across stimulus conditions, revealing changes that are either obscured or absent using more standard modeling approaches.\u2218C. Spike responses were recorded from RGCs using extracellular or loose-patch recordings with an Ames-filled pipette. Visual stimuli were presented on an OLED microdisplay monitor (eMagin) focused onto the photoreceptors. Stimuli were presented and data acquired using custom-written stimulation and acquisition software packages Stage (http://stage-vss.github.io) and Symphony (http://symphony-das.github.io). On-sustained and Off-sustained RGCs were targeted for recording based on their large soma size (>20\u2009\u03bcm in diameter) and responses to light increments and decrements. All recordings were from cells that responded reliably with more than five spikes to 100-\u03bcm diameter spots presented for 500\u2009ms at 20% contrast on a background of 10 R*/rod/s. For both mean light levels (10 and 1000 R*/rod/s), Gaussian noise stimuli were presented as spatially uniform spots 200\u2009\u03bcm in diameter at 50% contrast. The contrast of the spot was changed every 67\u2009ms (four frames at a monitor refresh rate of 60\u2009Hz). Noise stimuli that were modulated at higher temporal frequency did not robustly drive cells at 10 R*/rod/s. Cells were adapted to each new light level for at least 8\u2009min and until responses to flashed spots stabilized before recording. For On-sustained cells, five cells were recorded at both light levels, three cells at only low light, and one cell at only high light. For Off-sustained cells, two cells were recorded at both light levels.All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee at the University of Washington. Experiments were performed on whole mount preparations of retina from overnight dark-adapted C57/BL6 mice (ages 5\u201320\u2009weeks). All procedures were conducted under infrared illumination to preserve dark adaptation. Retinas were mounted ganglion cell-side up onto a poly-D-lysine-coated coverslip (BD Biosciences) before being placed in a recording dish that was continuously perfused at 7\u20139 ml/min with oxygenated Ames bicarbonate solution (Sigma) warmed to 31\u201334Linear filters were found by standard reverse-correlation methods: calculating the spike-triggered average and correcting for autocorrelation in the stimulus. Filters were smoothed by low-pass filtering with a frequency cutoff of 13\u2009Hz. For each cell, identical filters were used for the model with Poisson noise [linear-nonlinear-Poisson (LNP) model] and the multistage noise model. For cells in which data were collected at two different light levels, separate filters were calculated at each light level, with filters at higher light levels being faster and more biphasic than those at low light, consistent with previous work . ThroughBoth the filtered stimulus and responses were divided into time windows of \u223c60\u2013100\u2009ms, in which the average filtered stimulus was taken as the input to the model and the spike count was taken as the response. The exact length of the time window for a cell at a given light level was determined by the shape of the linear filter and corresponded to twice the width of the filter at half-max. This duration was chosen to produce minimal correlation between filtered stimulus values in neighboring bins. Bins of this length also minimize spike history effects because of refractoriness, which are expected on shorter timescales.tr is the neuron\u2019s response (spike count) in time bin t, tx is the average filtered stimulus value in time bin t, f is the nonlinearity, and Models with Poisson noise (LNP models) are given by:This is done for consistency with the multistage noise model presented below, in which the nonlinearity is parameterized this way. This function was chosen because it can capture the range of desired features in a nonlinearity, from highly rectified to effectively linear. We see little or no evidence of response saturation at high input values in our data and therefore did not choose a sigmoidal (saturating) nonlinearity. This parameterization does not reduce the model\u2019s ability to capture mean responses: on repeated stimulus presentations, the correlation coefficients between actual mean responses and predicted responses were 0.98 and 0.87 for the example cells shown at low and high light, respectively, regardless of whether the nonlinearity was a parameterized softplus function or was instead a nonparametric function found by locally estimated scatterplot smoothing (LOESS), a method of locally weighted regression. Model parameters for LNP models are found by maximum likelihood estimation, using the same routine described below for the multistage noise model.tr is the spike count in time bin t, and tx is the average filtered stimulus in time bin t. R rounds and rectifies to produce a spike count. The nonlinearity f is a softplus function, parameterized as in The model we present here A incorpoFor noise that fluctuates on fast timescales relative to data binning, noise is expected to follow Gaussian distributions because of the central limit theorem. When all sources of noise are Gaussian, the model has seven total parameters: four that determine the shape of the nonlinearity and three that determine the strength of the noise sources.Under some stimulus conditions , the multistage noise model with all Gaussian sources of noise did not accurately capture responses A. Such aWe therefore turned our attention to model elements that could alter the model\u2019s predicted variability. We tested whether our multistage noise model might be improved by adding response history dependence. In this model variant, input to the nonlinearity was given by the sum of the filtered stimulus value, an upstream noise value, and the spiking response in the previous time bin weighted by a factor determined by the optimization C, resultBecause the model predictions are not improved by incorporating response history dependence, we next considered changing the distributions of the noise sources directly. We used observed features of the responses to guide our choice of a new noise distribution. Specifically, at low input values, where the nonlinearity is flat and produces an output near zero, nearly all noise is expected to be contributed by the downstream noise source. Whereas response distributions for these low input values were approximately rectified Gaussian distributions at low light (corresponding to purely Gaussian downstream noise rectified by spike generation), response distributions at high light were well described by a mixture distribution:downp) this source of noise is present, while the remainder of the time it is absent. This might reflect the fact that this source of noise is itself engaged by a noisy process that takes effect randomly throughout stimulus presentation. Note that the original model, with purely Gaussian downstream noise, is a subset of this model. This modification adds one additional parameter to the model, for a total of eight parameters.This distribution can account for the large number of zero responses present at low input values B. The miIn brief, a combination of C++ and MATLAB code was used to find the maximum likelihood estimate of model parameters. What follows is the likelihood function for this model, broken down to reflect each step in the model for clarity. The full likelihood function can be found by plugging functions from preceding steps into upP, multP, and downP denote the probability distributions of each noise source. To calculate the likelihood of an observed spike count tr, first the distribution reflecting the input plus upstream noise is passed through the nonlinearity f. upP is shifted by  the observed input tx. The distribution of outputs from the nonlinearity \u03bb is:Let P\u039b is a distorted version of upP that spreads out where the derivative of the nonlinearity is large  and compresses where the derivative of the nonlinearity is small (derivative of the inverse is large). See The distribution After multiplicative noise is applied, the distribution is given by the following:multP increases with \u03bb. This has the effect of spreading the distribution more for larger values of \u03bb where the multiplicative noise distribution is wider.This is similar to a convolution, except that the standard deviation of The distribution after downstream noise is given by the following:YPwith the downstream noise distribution.This reflects a simple convolution of Finally, we integrate to obtain the probability of observing integer spike counts:tr as independent of responses at other times. The full likelihood RP  over all time points:We treat each observed response Note that the total recording time most strongly determines the number of data points available for fitting , rather than the number of spikes recorded, as is often the case for other models.The maximum likelihood estimate of parameters was found using the Nelder\u2013Mead method (implemented by MATLAB\u2019s fminsearch function). This provided the best performance among several optimization methods tested. A small modification to the fminsearch function allowed for bounds on parameters . Bounds were used only to ensure that impossible regions of parameter space  were not explored, rather than to constrain the optimization to a subset of desired parameter values. As this problem is not guaranteed to have a unique solution, for each dataset we began the optimization from 5 to 10 different randomized initial conditions. For noise parameters, initial conditions were drawn randomly from a uniform distribution of possible values. For nonlinearity parameters, parameters found by least-squares fitting were perturbed randomly by \u00b140% to set initial conditions. This amount of perturbation allowed for variability while ensuring that the initial conditions produced a plausible shape for the nonlinearity. The solution with the highest likelihood was reported. Although it is not necessary to perform this procedure to estimate parameters of the model with Poisson noise, the same procedure was used to make a fair comparison with the multistage noise model.https://www.gnu.org/software/gsl/). The full function of http://www.chebfun.org/). Machines running the Ubuntu operating system with multiple cores (16 or 40) were used to run the optimization in parallel with the Parallel Computing Toolbox in MATLAB.Several steps were taken to speed evaluation of the likelihood function. https://github.com/aiweber/Multistage_noise_model. The code is also available as The code described in the paper is freely available online at 10.1523/ENEURO.0191-21.2021.ed1Extended Data 1Extended Data 1, ZIP file.Code that fits the multistage noise model to data. See README file for additional details. Download In several figures, we indicate the level of each noise parameter that corresponds to an SNR of 0.5 to provide intuition for the strength of each noise source. Because the contribution of a single noise parameter to the overall SNR will depend on both the strength of other noise sources as well as the shape of the nonlinearity, here we calculate SNR for each noise source individually  and using either the true nonlinearity (in the case of simulated data) or the estimated nonlinearity .r, conditioned on the stimulus s. The outer variance (numerator) and expectation (denominator) are then taken over the stimulus distribution.We calculate the SNR as follows:KLD is the Kullback\u2013Leibler divergence:The JSD is a measure of similarity of probability distributions , which wNeural responses in many systems, including the retina, show non-Poisson variability and large changes in variability between stimulus conditions. Our goal is to understand the origins of this observed variability and how the contributions of different sources of variability change across conditions. Previous models typically capture mean responses well but do not accurately reflect response variability. We propose that variability can be better described by a model that incorporates multiple stochastic elements that represent plausible sources of noise in the biological circuit. Further, the relative contributions of these sources of noise suggest possible mechanisms giving rise to observed variability. We show that such a model can be tractably fit to data and then use the recorded responses of a well-studied neural population, retinal ganglion cells, as a benchmark for evaluating the model.Before proceeding, it is important to clarify the distinction between variability and noise. Noise refers to inconsistency in responses that arises because of stochastic processes and is considered to obscure the signal of interest. Noise is therefore generally  unfavorable from the perspective of neural coding. Noise can be considered a subset of variability, which more broadly refers to some inconsistency in a neuron\u2019s response and hence could include uncontrolled experimental variables, such as behavioral state or temperature. In the context of neural recordings, we refer to stochastic biological processes as producing noise but generally discuss variability in neural responses, as the sources of the variation are not fully known at the level of neural outputs. Variability in model responses arises entirely from stochastic model components, and hence we refer to it as noise.We observed that responses of RGCs exhibit variability that consistently deviates from Poisson variability and depends on light level. We first recorded responses from On-sustained RGCs of the mouse at two different levels of ambient illumination while presenting spatially uniform Gaussian noise stimuli , it does not accurately capture the response variability across trials, particularly at high light. We next found the nonlinear function that best predicts the neural response  as a function of the filtered stimulus. We parameterized the nonlinearity as a softplus function; this choice does not reduce the accuracy with which responses are predicted relative to a smoothed, nonparametric estimate of the nonlinearity . We first made use of the most common assumption for neural response variability: that spike responses follow Poisson distributions. Not only do response distributions show deviations from Poisson predictions at both light levels, but the deviations are qualitatively different in each of these cases. Comparing across the two light levels, it is apparent that the distribution of responses for a given filtered stimulus value is very different, even when the mean output of the nonlinearity is similar. Compare, for example, These observations indicate that variability in neural responses is driven by mechanisms that cannot be accurately modeled by simple Poisson noise. To help identify which mechanisms might account for this variability, we sought a model that would more accurately reflect the potential sources of variability at multiple circuit locations to account for observed response distributions. Because the linear-nonlinear framework accurately captures average responses to repeated white noise sequences, we built on these deterministic elements to allow for differences in response variability.One likely candidate for model improvement is dependence on response history. If response history is able to modify input to the nonlinearity, it can effectively alter the level of variability in responses such that a model with Poisson noise and response history dependence, such as Poisson GLMs, may exhibit sub-Poisson or super-Poisson variability in its outputs . HoweverPredicted response variability can also be altered by incorporating stochastic elements other than noise in spike generation. We sought to modify the linear-nonlinear model framework in this way, incorporating noise at different locations within the model. These stochastic model elements more accurately reflect that noise arises in several elements of neural circuits, rather than simply spike generation . In factIn our new model, we incorporated three potential sources of noise into a linear-nonlinear cascade framework to reflect the varied sources of noise present in neural circuits A. Noise upn) to indicate its position relative to the nonlinearity in the model. After the corrupted input is passed through the nonlinearity, it encounters multiplicative noise (multn), in which the output of the nonlinearity is multiplied by a random noise value. This is followed by another source of additive noise downstream of the nonlinearity (downn). The output of this step is a continuous, unbounded prediction of the response in the time window of interest. Rather than introducing an additional noisy spike generation step, we take the nearest nonnegative integer as the predicted spike count. This deterministic method of spike generation reflects the fact that spike generation itself accounts for little of the variability observed in neural responses  in the nonlinearity and eliminated by flat regions (far left). The effects of multiplicative noise scale with the output of the nonlinearity. Because the noise is multiplied by the output of the nonlinearity, larger output values result in greater variability. This is similar to Poisson noise, where output variance equals the mean output, although this multiplicative source of noise has greater flexibility in that output variance scales with the mean by a constant factor (not constrained to be 1). Both upstream and multiplicative noise therefore result in variability that depends on the input: response variability due to upstream noise scales with the derivative of the nonlinearity, and response variability due to multiplicative noise scales with the output value of the nonlinearity itself. Downstream noise is independent of the input and thus results in equally variable responses across all regions of the nonlinearity. In summary, the three sources of noise produce different signatures of variability in the responses that can be uniquely identified.To demonstrate the effects of each noise source in the multistage noise model, we simulated data from three models with identical deterministic properties but each with one of the non-Poisson noise sources described above. For comparison, we then fit a model that assumes Poisson noise to demonstrate the errors produced by a mismatch between the actual and assumed location of noise. Systematic errors are expected in this case, but we demonstrate the specific issues that arise for the three different types of noise in the multistage noise model to provide intuition for the results that follow.If only additive Gaussian noise upstream of the nonlinearity is present and an LNP model is fit to the resulting responses A\u2013C, the Note that for an LNP model, the nonlinearity in the model is identical to the mean predicted response, so the yellow lines in the left and right panels of If only multiplicative Gaussian noise is present, the inferred nonlinearities and response distributions may be well approximated by Poisson noise D\u2013F. In bIf only additive Gaussian downstream noise is present G\u2013I, the These examples demonstrate the impact that assumptions about noise can have on the inferred shape of the nonlinearity: incorrect assumptions can lead to strongly biased estimates of the nonlinearity operating in the circuit.One key feature of the linear-nonlinear model with Poisson noise is its simplicity to fit to data, requiring only standard reverse correlation methods to find the linear filter and least-squares estimate of the nonlinearity . Given tWe used a maximum likelihood approach to estimate model parameters. In order to reduce computation time, we first approximated the likelihood function and then used standard optimization methods to find the maximum of this function. Importantly, parameters for both the nonlinearity and noise are estimated simultaneously, as these interact to determine the likelihood. As demonstrated above, incorrect assumptions about the structure of noise in a circuit can bias estimates of the nonlinearity.Because the likelihood function is non-convex, optimization is not guaranteed to arrive at the maximum likelihood set of parameters. We therefore begin our optimization at several different initial conditions (5\u201310) and select the parameters that result in the overall greatest likelihood . In practice, initial conditions often converge to similar parameter estimates, suggesting that the likelihood does not have many deep local minima. Using this procedure, we find that we are able to estimate model parameters with a high degree of accuracy.We begin with a model in which all sources of noise are Gaussian distributed. Recall that the model still produces highly non-Gaussian spike counts in this case. To test the generality of these conclusions, we generated 30 simulated datasets with varying parameters, including both steep and shallow nonlinearities and different combinations of dominant noise sources. In these datasets, we can recover the nonlinearity that produced the data with a high degree of accuracy C. The erIn addition to recovering the nonlinearity, the multistage noise model estimates the strength of each noise source to within 20% of its true value for all noise sources that contribute meaningfully to the response D. Noise We next fit the model to ganglion cell responses at low light levels (10 R*/rod/s) to determine whether it accurately captures observed response variability. For the example cell shown in When we consider the full distribution of responses, however, we see that the multistage noise model outperforms a model with Poisson noise. More specifically, a Poisson approximation is somewhat suitable at higher and lower filtered stimulus values Ei,Eiii. n\u2009=\u20098), all exposed to the same level of ambient illumination (10 R*/rod/s). Results are summarized by plotting the JSD between the predicted and actual response distributions at three different input levels. JSD is a measure of difference between two probability distributions; lower JSD indicates better correspondence between two distributions. Across all filtered stimulus values, the JSD between the data and predictions from the multistage noise model is systematically lower than the data and the Poisson noise model  could also be recovered, we again generated simulated data from this model and used the same procedures to estimate model parameters.  Results for three example datasets and summary results across 12 simulated datasets are presented in We next fit the model to RGC responses at high light levels (1000 R*/rod/s). The nonlinearities inferred by the new model and a model with Poisson noise are markedly different, with the multistage noise model inferring a much more sharply rectified nonlinearity B. Again,n\u2009=\u20096), the multistage noise model predicts the distribution of responses better than the Poisson noise model across all input levels . Multiplicative noise, on the other hand, is lower at high light levels for all cells in which paired data are available . The inferred strength of the downstream noise source is higher at high light levels for all cells with paired data . The two Off-sustained cells did not show consistent changes in upstream or downstream noise parameters, but multiplicative noise increased strongly in both cells at high light .All three sources of noise present in the multistage noise model are needed to account for ganglion cell responses E. There A large body of work directly investigates the variability inherent in neural systems to inform our understanding of circuit function . MotivatThe changes we observe in nonlinearities across light levels are consistent with previous work, with increasing rectification at higher light levels . Yet theOur results show that all three noise sources in the model are required to account for ganglion cell response variability. A great deal of work points to a variety of origins of the noise in the retinal circuitry. Noise arising within the photoreceptors, and even in particular elements of the phototransduction cascade, has been studied extensively . Other wThe relative contributions of different noise sources in the retinal circuitry could change with ambient light level, and we indeed see that the strength of different noise sources in the model varies systematically with light level. The two light levels tested here engage different retinal circuits before convergence at the RGC, which may change the relative contributions of noise sources directly or by altering the location and degree of nonlinearities in the circuitry, thereby effectively changing the location of noise relative to the nonlinearity . AlthougLNP models and Poisson GLMs have gained widespread use in part because of their simplicity to fit to data. The model presented here is considerably more complex, although each of these additional components proved necessary to capture the full distribution of neural responses. Model parameters must be found via optimization on a relatively complex likelihood function and are not guaranteed to be unique. However, we find in practice that different initial conditions typically converge to the same set of parameters.The work presented here captures the responses of a neuron to only a single temporal feature of the stimulus, and a spatially uniform stimulus was chosen to eliminate the need for spatial selectivity in the model. Selectivity to multiple spatiotemporal stimulus features, as has been observed in several systems , could bA useful extension of this model would operate at finer timescales, predicting individual spikes rather than spike counts within a window of time (\u223c100\u2009ms). Predicting responses in longer windows reduced the need for incorporating response history dependence. Indeed, we found that incorporating dependence on the response in the previous time bin did not improve our model predictions. However, refractory and other spike history effects would be necessary to make predictions at finer timescales. History dependence has been shown to improve model accuracy in a number of contexts, including in the retina . In partIdeally, the model ought to optimize parameters of stimulus and spike history filters at the same time as nonlinearity and noise parameters. In the present study, we found linear filters using standard reverse correlation methods. Simultaneous optimization of these filters would require the addition of multiple new parameters, which could substantially slow optimization. Careful parameterization of these filters, incorporation of statistical priors, or additional simplifying assumptions may be required for this approach to be computationally tractable.The general framework presented here could be easily modified to make use of different distributions for each noise source. We presented two slightly different versions, which incorporated different but closely related distributions for downstream noise. One could similarly modify upstream or multiplicative noise distributions, as called for by different datasets. Parameter inference will to some extent depend on these choices in model selection. We find, however, for the two models presented here that inferred nonlinearities are generally robust to this distinction and that inferred noise parameters change in small but systematic ways.In conclusion, the model presented here holds several advantages over models that include a single source of variability. First, it is able to more accurately recover the nonlinearity in circuits in which noise is not dominated by a single source. Second, it provides better predictions of overall variability and has the ability to attribute variability to different sources. Given the importance of noise in shaping the flow of information through a circuit, it is important that a model capture features of this variability in the neural responses. This work opens the door to two potentially fruitful lines of future work: (1) extending the model to include additional features of stimulus and history dependence, and (2) conducting additional experiments to more closely link the sources of variability in the model to features of the biological circuit."}
+{"text": "Metabolic reprogramming and cellular plasticity drive tumorigenesis. However, how these cellular events collectively contribute to the oncogenic process is poorly understood. Epithelial-mesenchymal transition (EMT), a fundamental mechanism of cellular plasticity, is governed by the EMT transcription repressors such as Snail. In the present study, through establishment and characterization of inducible overexpression of Snail in A549 lung cancer cells, we aim to define the metabolic reprogramming in response to Snail in the EMT of lung cancer cells.Our metabolomic analysis suggests that forced expression of Snail accompanied reduced diversion of glycolytic metabolites to the serine/glycine metabolic shunt, a critical metabolic branch that distributes glucose catabolic intermediates to the major anabolic pathways. Our gene expression profiling and molecular characterization revealed that Snail actively suppressed the expression of glycine decarboxylase (GLDC), a key enzyme on the serine/glycine metabolic shunt, through binding to an evolutionarily conserved E-box motif and thereby inhibiting the promoter of the GLDC gene. Besides, knockdown of GLDC led to a cellular function shift from proliferation to migration.This study has revealed a novel molecular link that integrates the serine/glycine metabolism with the Snail-mediated EMT program in cancer cells. For example, the elevation of Snail has been shown to augment differential responses in the glycolysis, the primary catabolic pathway of cancer cells, with the upregulated and downregulated glycolysis seen in the breast and lung cancer, respectively. Therefore, it is imperative to elucidate the molecular relationship between the EMT transcription factor and the metabolic target for a better understanding of the metabolic basis for EMT.Interestingly, while gaining migratory capacity during EMT, the cells often slow down proliferation, implying that significant rearrangement of cellular states including cellular metabolism would occur in the EMT processes.,14 Synthesized from the serine that is derived from the glycolytic catabolites, the glycine is broken down to form the folate-mediated one-carbon unit, supplying key anabolic intermediates for supporting cell proliferation.,14 Therefore, high turnover of glycine is a unique metabolic feature associated with rapidly proliferating cancer and embryonic stem cells. Consistently, the key enzymes of this high-flux serine/glycine metabolic shunt including phosphoglycerate dehydrogenase, serine hydroxymethyltransferase, and glycine decarboxylase (GLDC) which are responsible for the synthesis and catabolism of serine and glycine, respectively, are all found to be significantly elevated to drive cancer proliferation.\u201319 However, given a clear functional shift from proliferation to migration during EMT, it is not known whether the serine/glycine metabolic shunt is reprogrammed at the molecular level in this important cellular process.The small amino acid glycine is a key metabolic intermediate that connects the major catabolic and anabolic pathways in cancer cells.GLDC gene and thereby suppresses the GLDC promoter. Thus, these observations have revealed a novel molecular link that integrates the serine/glycine metabolic rearrangement with the Snail-induced EMT program.In the current study, through characterization of an EMT tissue culture model, we identified that GLDC, the glycine catabolic enzyme is a novel direct target of Snail. We found that Snail binds to an evolutionarily conserved proximal E-box motif in the Human lung cancer A549 and H1299 (ATCC) and human embryonic kidney 293FT (Invitrogen) cells were maintained in Dulbecco\u2019s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 0.1 ng of streptomycin/ml. and pcDNA3.1 (Invitrogen) to yield pInducer20-Snail-HA and pcDNA3.1-Snail-HA for doxycycline-inducible and constitutive overexpression of wild-type Snail, respectively. For construction of the expression vector for the transcriptionally inactive Snail mutant that lacks SNAG domain, the sequence encoding the amino acids at the positions of 2\u20138 at the N-terminal of Snail protein, was deleted in pcDNA3.1-Snail-HA to yield pcDNA3.1-Snail\u0394SNAG-HA using a site-directed mutagenesis kit (Stratagene). For construction of the luciferase reporter for CDH1 promoter, a ~400 bp sequence spanning the nucleotide positions-365 to +48 of CDH1 gene was amplified from human genomic DNA and cloned into pGL3-basic (Promega). For construction of the luciferase reporters for GLDC promoter, a ~1 kb sequence spanning the nucleotide positions-962 to +55 of GLDC gene was amplified from human genomic DNA and cloned into pGL3-basic (Promega) to yield the wild-type reporter GLDC-luc. The mutated reporter constructs with targeted deletion of either the proximal or the distal E-box motif were generated using site-directed mutagenesis and designated as E1 mut-luc and E2 mut-luc, respectively.For construction of the Snail expression vectors, the full-length human Snail cDNA was fused with a 3\u2019 in-frame HA epitope tag sequence and was cloned into pInducer20For establishment of A549-Snail cell line, the lentiviral particles were produced by cotransfection of pInducer20-Snail-HA with pMD2.G and psPAX2 packaging vectors into 293FT cells, and were then infected into A549 cells. The infectants were selected in the media containing 500 \u03bcg/ml of G418, and the drug-resistant cell clones were screened for doxycycline-inducible overexpression of Snail-HA protein using Western blotting.For knockdown of GLDC, the siRNA oligos (Origene) was transfected into A549 cells using Lipofectamine RNAiMAX reagent according to the vendor\u2019s specifications.Cells were washed twice in phosphate-buffered saline (PBS) and lysed in the hypotonic lysis buffer . Protein concentrations were determined with the Bradford reagent (Bio-Rad). Cell lysates (40 \u03bcg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose filters. The blots were saturated with 5% nonfat milk and probed with antibodies against GLDC , AMT , DLD , GAPDH , E-Cadherin , N-Cadherin , Snail , or HA . Following a wash with PBST (PBS containing 0.1% Tween 20), the blots were incubated with peroxidase-coupled goat anti-rabbit immunoglobulin G . The immunolabeled protein bands were detected by enhanced chemiluminescence method (Perkin Elmer).Cells grown in the chamber slides were fixated with 4% paraformaldehyde, and subsequently permeabilized with 0.2% Triton X-100. After saturation with 3% Donkey serum, cells were incubated with the primary antibody against HA  or E-Cadherin , and then incubated with the TRITC-conjugated Donkey anti-rabbit  or FITC-conjugated Donkey anti-mouse  secondary antibody. The immunofluorescence was visualized in the cells using confocal microscopy (Olympus).,23 To measure cell proliferation, equal number of cells were seeded on 6-cm plates, and were subjected the indicated treatment. The number of cells was then counted using a hemocytometer at the indicated time. To measure cellular capacity for migration, the Boyden chamber matrigel migration assay was performed. In brief, cells were seeded in the serum-free media in 8 \u03bcm Boyden chamber (Corning) coated with Matrigel (BD Bioscience), and the chamber was placed inside a bottom plate containing complete growth media as migratory attractants. Following 24 h incubation, invasive cells retained on the filter were fixed with 1% paraformaldehyde and stained with 1% crystal violet, and quantified under a microscope.Cell proliferation and migration were examined as previously described. Data are presented as the relative abundance of each individual metabolite in reference to the level of the control cells without Snail induction.Cell metabolism was quenched by flash freezing of the cells in liquid nitrogen. Cellular metabolites were extracted in 80% aqueous methanol, and then quantitated using the LC-MS/MS methods as previously described.Total cellular RNA was isolated using the Trizol reagent (Invitrogen). To profile the differential expression of mRNAs in the A549-Snail cells with or without Snail induction, cDNA libraries compatible for Illumina sequencing were prepared by using the QuantSeq 3\u2019 mRNA-seq Reverse (REV) Library Prep Kit (Lexogen) according to the manufacturer\u2019s instruction. The resultant cDNA libraries were assessed using a TapeStation (Agilent), and subjected to 100 bp single-end sequencing using the Illumina HiSeq 2500 system at the Wayne State University Applied Genomics Technology Center. The sequencing reads were mapped to the human reference genome, and the differential gene expression was determined.T), and normalized to PPIA mRNA level of PPIA or \u03b2-actin housekeeping gene. The results were expressed as the fold changes relative to the untreated control, and the standard deviation (SD) bar graphs were plotted. The PCR primers used are listed in To determine the mRNA expression using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cDNA libraries were constructed by random priming using the SuperScript III First-Strand Synthesis System (Invitrogen). The synthesized cDNA was used as a template for qRT-PCR with SYBR Green qPCR Master Mixes (Thermo Scientific) on an Mx3000P cycler (Stratagene). All mRNA levels were determined as the delta-delta threshold cycle (\u0394\u0394CRenilla reniformis luciferase control vector Prl-CMV (Promega) into 293FT or A549-Snail cells using Lipofectamine 2000 (Invitrogen). The levels of Snail proteins were increased either by cotransfection of the 293FT cells with pcDNA3.1-Snail-HA or pcDNA3.1-SnailDN7-HA vector, or treatment of A549-Snail cells with doxycycline. Dual luciferase assays were performed according to the manufacturer\u2019s instructions (Promega).Firefly luciferase reporter plasmids (2 \u03bcg each) were cotransfected with 0.04 mg of A549-Snail cells were treated with doxycycline to induce the expression of Snail-HA protein for 2 days, and were then cross-linked in formaldehyde. The nuclei were isolated and sonicated using conditions resulting in prominent DNA fragments between 500 and 750 bp. Chromatin samples were immunoprecipitated with anti-HA antibody (Santa Cruz) and Protein G sepharose . DNA samples purified from the precipitated chromatin were used as PCR template. Primers for PCR reactions were: GLDC promoter: 5\u2019-GCTGGGAAGAGGGTAGGAAG-3\u2019 (forward) and 5\u2019-GCGCTCAACCAAGACACTC-3\u2019 (reverse); CDH1 promoter: 5\u2019-GTGAACCCTCAGCCAATCAG-3\u2019 (forward) and 5\u2019-TCACAGGTGCTTTGCAGTTC-3\u2019 (reverse); and GAPDH promoter: 5\u2019-CGGCTACTAGCGGTTTTACG-3\u2019 (forward) and 5\u2019-GCTGCGGGCTCAATTTATAG-3\u2019.t-test.Statistical analyses were performed with GraphPad Prism 6 software . Data are presented as means \u00b1 SD statistical significance between two groups was determined by unpaired in vitro model for EMT. As shown in To recapitulate the EMT process in tissue culture, we engineered the lentiviral-mediated, doxycycline (Dox)-inducible overexpression of Snail in the A549 cells, a lung carcinoma cell line that has been widely used as  We observed that Snail induction had negligible effects on the levels of the metabolites of the tricarboxylic acid (TCA) cycle [Wondering if Snail induction triggers metabolic alteration, we measured the levels of numerous metabolites in the A549-Snail cells using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis. We observed that Snail induction reduced the levels of the nucleotide biosynthetic intermediates . Warburg As GLDC is a metabolic oncogene previously reported in lung cancer, and it locates on the serine/glycine metabolic shunt that diverts the glycolytic metabolites to the major anabolic pathways, we characterized how Snail regulates the expression of GLDC in detail. We first examined the effects of Snail overexpression on the levels of the transcripts of each GCS components in two clones of the A549-Snail cells. We observed that overexpression of Snail significantly reduced the level of the GLDC transcript without affecting those of the other GCS components including AMT, GCSH, and DLD indicating that Snail selectively suppresses GLDC, the rate-limiting component of GCS [GLDC gene in lung cancer cells.We then investigated the molecular basis for which Snail induces metabolic changes. As Snail is a transcription repressor, we performed gene expression profiling to identify the metabolic enzyme transcripts that decreased in the Snail-overexpressing cells. We found that the transcript encoding GLDC was among the top hits that decreased in response to Snail induction in the A549-Snail cells. GLDC is the rate-limiting component of the glycine cleavage system (GCS), a mitochondrial multimeric enzyme complex that breaks down glycine and concomitantly synthesizes a one-carbon unit, which is a crucial anabolic intermediate required for cell proliferation.t of GCS . Consistt of GCS . Thus, o We, therefore, tested whether an inverse relationship between Snail and GLDC expression exists in the cells exposed to TGF-\u03b2. As expected, treating the cells with increasing amounts of TGF-\u03b2 led to concomitant upregulation of Snail and downregulation of GLDC at both the transcript and protein levels in dose-dependent manners [Transforming Growth Factor (TGF)-\u03b2 is a cytokine that actively induces Snail and thus promotes EMT in A549 cells. manners . As cytoGLDC. As Snail belongs to a family of sequence-specific transcription repressor that recognizes the E-box motif with a consensus of 5\u2019-CAGGTG-3\u2019,,6 we searched for the possible Snail binding site(s) in the sequence of human GLDC gene. We found two perfectly matched E-box motifs in the promoter of GLDC that located at nucleotide positions \u221223 to \u221218 and \u2212303 to \u2212298, respectively, relative to the transcription start site. Multi-sequence alignment demonstrated that the proximal motif (positioned at \u221223 to \u221218) but not the distal one (positioned at \u2212303 to \u2212298) conserved in two rodent species [in vivo, we induced the expression of the Snail-HA protein in A549-Snail cells and performed the chromatin immunoprecipitation (ChIP) assay using anti-HA antibody. The ChIP analyses demonstrated that Snail interacted with the promoter of GLDC as well as the promoter of CDH1, a bona fide Snail target, and not with the promoter of GAPDH, an irrelevant negative control gene [GLDC E-box motifs only has 278 nucleotides that were not separable in the ChIP assay, we conclude that Snail interacts with at least one of the E-box motifs in the promoter of GLDC in A549 cells.We then investigated the molecular mechanism of how Snail represses the expression of  species . To testrol gene . As the GLDC promoter, we generated a set of luciferase reporters that carry either the wild-type or the mutated GLDC promoter [\u0394SNAG, inhibited the promoter of GLDC as well as the promoter of CDH1 in a dose-dependent manner [GLDC promoter to Snail [GLDC.To examine whether and how Snail represses the activity of promoter . The luct manner . In addito Snail . TogetheGLDC reduced the expression of GLDC protein by ~ 80%, and significantly decreased the proliferation and increased the migration of A549 cells [To examine how reduced expression of GLDC influences the cell behavior, we knocked down GLDC in A549 cells using small interference RNA (siRNA). The data demonstrated that transfection of the siRNA oligos specific for 49 cells . Thus, r\u20139 it is interesting to address whether these events are functionally coordinated. Concerning to tumor biology, it is mechanistically insightful and therapeutically useful to elucidate how metabolic reprogramming couples with the EMT program, which could help for developing novel therapeutic approaches against metastasis and chemoresistance through disrupting the metabolic basis for tumor plasticity.,29 In this study, we examined how gain of Snail, a prototypic EMT transcription factor and an oncogenic protein, affects the fluctuation of the backbone metabolic pathways in lung cancer cells, and further identified that GLDC is a novel metabolic component integral to the Snail-induced EMT program.As metabolic reprogramming and cellular plasticity are common features for the adaption of cells to the environment, We predict that the sharp divergence of the alterations of the glycolytic metabolites under the condition of Snail induction may reflect a significant rearrangement of a metabolic branch that connects with the glycolytic pathway at the interface of GAP and 3PG. Indeed, the serine/glycine metabolic shunt, a vital connector between glycolysis and key anabolic pathways, is right started using 3PG as metabolic substrate. Thus, our metabolite analysis predicted that Snail significantly rearranges the serine/glycine metabolism in the EMT commitment of the lung cancer cells.To robustly examine the metabolic fluctuation in the process of EMT, we established an inducible EMT tissue culture model through ectopic expression of Snail under the control of doxycycline-inducible promoter in the lung cancer A549 cells. We demonstrated that induction of Snail robustly activated EMT, as manifested by the appearance of the critical molecular and functional signatures of EMT, including concomitant reduction of E-cadherin and elevation of N-cadherin, as well as increase of migration and decrease of proliferation in the Snail-expressing cells. Using targeted metabolomics analysis, we probed the influence of Snail overexpression on the metabolite fluctuation of the backbone metabolic pathways, those including nucleotide synthesis, glycolysis, and TCA cycle. We observed that the levels of nucleotide biosynthetic intermediates were uniformly decreased in the Snail-expressing cells, suggesting the metabolic demand for DNA synthesis possibly decreased when the cells commit to EMT and slow down proliferation. In contrast, the levels of TCA cycle metabolites remained unchanged in the Snail-expressing cells, indicating that mitochondrial oxidation is likely bioenergetically necessary for supporting the EMT-committed cells. Interestingly, a biphasic response in the levels of the glycolytic metabolites to the Snail induction was observed. After Snail induction, dividing at the interface between GAP and 3PG, the levels of the upstream glycolytic metabolites were decreased, yet the levels of the downstream ones remained unchanged. At present, we do not fully understand the logic behind this clear split in the alterations of the glycolytic metabolites in response to Snail induction. However, a clue may lie in the fact that in addition to feeding the TCA cycle, glycolysis diverts its metabolic flux to specific key branching pathways including the pentose phosphate pathway and the serine/glycine metabolic shunt.GLDC gene mediates the binding and thereby the inhibition of Snail to the GLDC promoter. Also, we demonstrated that treatment of cells with TGF\u03b2, a canonical EMT-inducing cytokine, led to concomitant induction of Snail and suppression of GLDC in dose-dependent manners. Thus, we demonstrated that this newly identified Snail/GLDC axis is an integral component of the EMT program. GLDC is the rate-limiting enzyme of GCS that catabolizes glycine for the synthesis of critical anabolic substrate \u2013 the folate-mediated one-carbon unit. GLDC is a metabolic oncogene sufficient to drive the proliferation of cancer cells. For metabolic networking, GLDC, as a bottleneck enzyme, is essential for cancer cells to sustain the metabolic flux through the serine/glycine shunt. In this sense, it is plausible that GLDC functions as a regulatory checkpoint that influences the activity of the serine/glycine branch and in turn, the distribution of glycolytic flux in response to Snail induction. Indeed, the functional significance of this newly identified Snail/GLDC axis is further supported by the observation that reduction of GLDC is sufficient to induce a switch of cellular phenotype from proliferation to migration in both the present [ studies.Interestingly, our gene expression profiling identified that the expression of GLDC, a key enzyme of the serine/glycine metabolic branch was significantly reduced in response to Snail induction. Our molecular characterization further defined that an evolutionarily conserved proximal E-box motif in  present  and prevIn summary, the present study has identified that GLDC is a novel metabolic target directly controlled by Snail. It further suggests that the Snail/GLDC regulatory axis, as an integral part of the EMT program, poses a significant impact on the diversion of glycolytic flux to biosynthetic pathways, and thereby the arrangement of global anabolic/catabolic activities in the process of EMT. However, the functional importance of this novel regulatory link in the context of tumor biology needs further investigation.1"}
+{"text": "African Americans and Appalachians experience greater incidence and mortality rates of colorectal cancer due to factors, such as reduced prevalence of screening. An educational session (the Screen to Save Initiative) was conducted to increase intent to screen for colorectal cancer among African Americans and Appalachians in Ohio. Using a community-based approach, from April to September 2017, 85 eligible participants were recruited in Franklin County and Appalachia Ohio. Participants completed a knowledge assessment on colorectal cancer before and after participating in either an educational PowerPoint session or a guided tour through an Inflatable Colon. Logistic regression models were used to determine what factors were associated with changes in colorectal cancer knowledge and intent to screen for colorectal cancer. The majority (71.79%) of participants gained knowledge about colorectal cancer after the intervention. Multivariate results showed that race  and intervention type  were associated with a change in knowledge. The association between education and intent to screen was marginally statistically significant . A change in colorectal cancer knowledge was not associated with intent to screen. Future educational interventions should be modified to increase intent to screen and screening for colorectal cancer. Further research with these modified interventions should aim to reduce disparities in CRC among underserved populations while listening to the voices of the communities. For both men and women combined, colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States . HoweverThe CRC burden is unevenly distributed along factors, such as race, socioeconomic status (SES), and geography \u20139. In paA multitude of factors contribute to the differential risk of CRC among AA. For example, there are biological elements, such as single nucleotide polymorphisms that may partially explain the differences in CRC incidence rates , 16. HowThe APP populations experience a high risk of CRC as well. CRC incidence and mortality rates among APP men and women in Ohio are greater than those of non-APP residents of Ohio , 26. TheTheoretical models can be used to predict behavior. For example, the theory of planned behavior may provide a framework for understanding this effort. The theory is centered on individual intention to perform a behavior, which is determined by the independent elements of attitude towards the behavior, subjective norms, and perceived behavioral control. The theory of planned behavior model has practical applications for increasing cancer screening among people with insufficient or limited knowledge , 34; incTherefore, an intervention was conducted to increase CRC knowledge to improve CRC screening rates in AA and APP populations. The primary aim was to determine if a change in knowledge of CRC is associated with intent to screen for CRC. The secondary aims were to elucidate what factors were associated with a change in CRC knowledge and intent to screen for CRC.The Screen to Save (S2S) initiative was a national effort funded by the Center to Reduce Cancer Health Disparities (CRCHD) and National Cancer Institute (NCI) aimed at enhancing knowledge and promoting healthy behaviors to address health disparities in CRC for at-risk groups . As partParticipants came from five locations in Franklin County, a metropolitan county of over 1.3 million residents with more racial diversity than most other Ohio counties, with 33.2% non-White and 23.8% AA residents, and from two Appalachian counties in Ohio\u2014Jackson County, with approximately 32,000 residents and little racial heterogeneity (3.2% non-White and 0.7% AA), and Scioto County, with approximately 75,000 residents and 5.6% non-White residents and 2.7% AA. The Appalachian region of Ohio includes 32 counties of the Southern and Eastern region. These locations were chosen to target AA and APP, respectively, but participation was open to men and women of all races and ethnicities who were 50\u201374\u00a0years old. To use a diverse array of community outreach efforts, participants were recruited using posted flyers, personal communications, emails through listservs, and church bulletins. Community health educators (CHE) of various backgrounds, including a registered nurse, who were employees of The Ohio State University, conducted educational sessions with participants. The Ohio State University Comprehensive Cancer Center is deeply connected to communities in both Central Ohio (Franklin County) and Appalachia Ohio (Jackson and Scioto Counties) through outreach and education efforts \u201343. CHEsIn total, surveys (described below) were collected from 104 participants across community centers and local health fairs, but only 85 participants met the age eligibility criterion (50\u201374\u00a0years). All participants received a study information sheet prior to providing verbal consent. Prior to receiving either intervention (described below), participants were given a short survey and assessment. The survey, which was developed by the CRCHD National Outreach Network (NON), obtained demographic information, such as age, gender, race, language, education level, and health insurance coverage. Specific information related to CRC, such as screening and family history were collected as well. Survey questions included those modified from the existing instruments from peer-reviewed literature and those constructed to reflect topics not included in the existing surveys . To valiThe type of education session intervention varied based on the site location. One intervention consisted of a PowerPoint presentation for a group led exclusively by a CHE who covered basic knowledge about CRC and screening . This intervention follows a traditional template of using small media as an effective educational tool . The othi \u03f5 \u2124 \u2229 , with negative values indicating a reduction in knowledge after the intervention and positive scores representing an increase in knowledge after the intervention. The standardized change in knowledge (Sk) was calculated for each participant to account for the variability in preintervention scores. The measure is derived from the following:I \u03f5 \u2124 \u2229  and the range for Sk is [\u2212\u00a01,0) The primary outcomes were change in CRC knowledge and intent to screen for CRC. Change in knowledge (\u0394K) was measured by subtracting the participant\u2019s score on the preintervention assessment from his or her score on the postintervention assessment. The domain for \u0394K is Screening intent was determined from the postintervention question assessing likelihood to \u201cget screened for colorectal cancer\u201d. Intent to screen was measured on a 4-point Likert-type scale for each question with answers ranging from 1 to 4. Responses were categorized as \u201cStrongly Agree\u201d if participants recorded a score of 4 and categorized as \u201cDo Not Strongly Agree\u201d if a combined score of 1\u20133 was measured.The preintervention survey collected the primary independent variables on demographic information which included: age (50\u201374\u00a0years old), gender , race , education , and insurance . To allow for sufficient sample sizes, age was categorized as 50\u201365 or 66\u201374\u00a0years old, gender as either male or female, race as White or AA, and education as either college graduate or not a college graduate. In addition, insurance was categorized as private only, Medicare only, or other.The preintervention survey asked participants if they have ever been screened for CRC. In addition, they could indicate whether they had an at-home stool test like FOBT/FIT, a colonoscopy, and/or used another method of screening. The date of the most recent screening was obtained for each method as well. Using the CRC screening guidelines set forth by the USPSTF, a participant was categorized as \u201cwithin guidelines\u201d if he or she had an at-home stool test within the past year, a colonoscopy within the past 10\u00a0years, or a flexible sigmoidoscopy within the past 5\u00a0years [In the preintervention survey, participants were asked if they ever had a father, mother, brother, sister, child, or niece/nephew diagnosed with CRC. They were asked for the same information about both their paternal and maternal grandfather, grandmother, aunt, and uncle. An additional section for each relative allowed participants to indicate if the CRC diagnosis occurred before age 50. Any responses marked \u201cYes\u201d were categorized as having a family history of CRC, while those marked \u201cNo\u201d or \u201cDon\u2019t Know\u201d were categorized as not having a family history of CRC.Descriptive statistics  were used to\u00a0describe demographic and additional characteristics of participants\u00a0and to compare factors of interest according to standardized change in knowledge and in intent to screen for CRC. Logistic regression was used to obtain odds ratios to examine potential associations between factors of interest and both standardized change in knowledge and intent to screen for CRC. Multivariate logistic regression was used to build two models of factors associated with each outcome separately. Models were built backwards, initially including all potentially important factors of interest and, one by one, removing factors that were not associated with outcomes. Removal of factors stopped when all factors in the final model were at least marginally statistically significant. Then, factors which were removed earlier in the model building process were reconsidered for model inclusion, to be certain that removal from the model had not been based only on order of removal.\u00a0The p values were 2-sided, with alpha set at 0.05 for hypothesis tests. Statistical analyses were conducted using SAS\u00a0Software version 9.4.Demographic and CRC characteristics of the participants are listed in Table k to avoid an indeterminate result. Furthermore, these participants all scored a 14 on the postintervention assessment, so the intervention had neither a beneficial nor deleterious effect on them.There were 7 participants with a preintervention score of 14; these participants were removed for any analysis involving STable Table The multivariate logistic regression model for change in knowledge about CRC is displayed in Table pThe univariate logistic regressions for intent to screen for CRC are presented in Table This study utilized two community-based educational interventions to increase CRC knowledge and intention to screen for AA and APP populations in Ohio. The interventions successfully increased CRC knowledge for participants. However, the findings revealed that a standardized change in knowledge was not associated with an intent to screen for CRC. Level of education attained was the only factor found to have a marginally statistically significant association with an intention to screen for CRC, with college graduates, counterintuitively, reporting a lower intention to screen. In contrast, a standardized change in knowledge about CRC was associated with race, intervention type, and insurance. Participants who participated in the Inflatable Colon tour and AA participants had lower standardized changes in knowledge. Insurance confounded associations between standardized change in knowledge and both race and intervention type.It was surprising to discover that a standardized change in knowledge was not associated with an intent to screen because the results do not align with the predominant findings in the literature , 36. HowHigher education levels are commonly associated with higher participation in CRC screening \u201355. TherIn this study, race, intervention type, and insurance through Medicare only were independently associated with a standardized change in knowledge. Findings pertaining to race were similar to those previously reported , 46, 48.Neither intervention was associated with increasing intent to screen for CRC, but participants receiving the PowerPoint session had a greater standardized change in knowledge. However, it is important to consider the utility of each intervention for a variety of circumstances. For example, the Inflatable Colon is more attractive for drawing in community members with families to learn about CRC at community fairs than a 50-min PowerPoint session on the same topic. On the other hand, transporting the Inflatable Colon to a sufficiently large space and then assembling and disassembling it requires much more effort and real estate than hosting a PowerPoint session in a small classroom. Furthermore, while the PowerPoint session was associated with a greater standardized change in knowledge, both interventions increased the change in knowledge for the majority of participants; therefore, both interventions can be used as an effective teaching tool. The pros and cons of the various educational modalities should be strongly considered in the planning stage of future community-based interventions.The impact of the interventions should not be diminished despite the finding that a standardized change in knowledge was not associated with intent to screen for CRC. The majority of participants gained knowledge about CRC from the interventions. Knowledge is a critical component that patients must possess in order to participate in shared decision making with their healthcare team. Therefore, promoting patient autonomy through education is imperative, whether the informed patient elects to undergo screening or not. Because vulnerable populations, such as AA and APP experience lower knowledge about CRC and screening, providing community interventions like the ones utilized in this study will help boost patient autonomy by enabling them to become more involved in their medical decisions with their healthcare providers , 58.This study benefited from several strengths in its design. In specific, the interventions were aimed at helping AA and AP, which are vulnerable populations for CRC. In addition, the study was strengthened by examining standardized change in knowledge, rather than simply change in knowledge. Participants arrived to the study relatively knowledgeable, so there was little room for improvement for many participants. In contrast to change in knowledge, standardized change in knowledge better accounts for marginal gains made by high preintervention participants, although it does so at the expense of discounting marginal gains made by those with low preintervention scores. Overall, standardized change in knowledge measures how much participants were able to learn based on the possible remaining knowledge available to them, thus creating a more equitable metric to contextualize knowledge gains.This study was not without limitations. The sample size was relatively small, participants were not randomized to receive an intervention , and some additional potentially confounding demographic factors were not collected in this study. Also, the study focused on intent to screen rather than observing screening behavior; follow-up of participants to verify screening outcomes would provide important information about the relationships between knowledge gains and hard screening outcomes. In addition, there may have been some misclassification of CRC screening history for those who did not provide the date of their last screening. Furthermore, all participants were considered to have average risk of CRC, although more rigorous screening regimens are recommended for individuals with elevated risk factors for CRC . Lastly,This study focused on identifying factors associated with a standardized change in knowledge about CRC and intent to screen for CRC for AA and APP in Ohio. These findings may be useful in designing interventions to assist populations experiencing disparities in CRC across the United States. Knowledge about factors associated with change in CRC knowledge and intent to screen for CRC may help delineate potential future audiences that could benefit the most from these interventions. Furthermore, resources that facilitate screening may need to be offered in conjunction with a change in knowledge to modify screening behaviors since a change in knowledge alone is not sufficient to generate intent to screen. For example, in addition to utilizing the Inflatable Colon, providing onsite screening resources at health fairs, such as an FOBT/FIT, may be beneficial . Future"}
+{"text": "Individual-level geographic information about malaria cases, such as the GPS coordinates of residence or health facility, is often collected as part of surveillance in near-elimination settings, but could be more effectively utilised to infer transmission dynamics, in conjunction with additional information such as symptom onset time and genetic distance. However, in the absence of data about the flow of parasites between populations, the spatial scale of malaria transmission is often not clear. As a result, it is important to understand the impact of varying assumptions about the spatial scale of transmission on key metrics of malaria transmission, such as reproduction numbers. We developed a method which allows the flexible integration of distance metrics  with temporal information into a single inference framework to infer malaria reproduction numbers. Twelve scenarios were defined, representing different assumptions about the likelihood of transmission occurring over different geographic distances and likelihood of missing infections (as well as high and low amounts of uncertainty in this estimate). These scenarios were applied to four individual level datasets from malaria eliminating contexts to estimate individual reproduction numbers and how they varied over space and time. Model comparison suggested that including spatial information improved models as measured by second order AIC (\u0394AICc), compared to time only results. Across scenarios and across datasets, including spatial information tended to increase the seasonality of temporal patterns in reproduction numbers and reduced noise in the temporal distribution of reproduction numbers. The best performing parameterisations assumed long-range transmission (>\u2009200\u00a0km) was possible. Our approach is flexible and provides the potential to incorporate other sources of information which can be converted into distance or adjacency matrices such as travel times or molecular markers. Geo-located health facility or residence data are increasingly collected as part of surveillance for diseases such as malaria2, but could be more effectively utilised to infer transmission dynamics, in conjunction with additional information such as symptom onset time and genetic distance3. However, challenges exist in making use of these diverse data sources and leveraging the information they contain within a single inference framework. This is particularly true of endemic diseases such as malaria, where individual level data are increasingly collected in moderate-low transmission or elimination settings, but unlike many epidemic viral diseases8, the development of approaches to analyse such data has been relatively recent11.Individual-level disease surveillance data, collected routinely and as part of outbreak response, capture a wealth of information which could improve measurements of transmission and its spatiotemporal variation, in turn informing the design of epidemiological interventions15. As a result, the spatial location of individual cases can provide useful information in inferring transmission dynamics when combined with additional forms of information, such as temporal and molecular data. Furthermore, in elimination settings malaria transmission is thought to take on epidemic dynamics16, meaning the importance of space and highly dynamic factors such as human movement patterns becomes more relevant. However, in the absence of data about the flow of parasites between populations, the spatial scale of malaria transmission (here meaning the distance between locations where one person was infected or detected and the locations where another person received an infective bite) is often not clear. As a result, it is important to understand the impact of varying assumptions about the spatial scale of transmission on key metrics of malaria transmission, such as reproduction numbers. In many contexts, not all cases may be observed within a surveillance system. Missing cases further complicate inference, as without information about how likely cases are to be missing, ambiguity can exist as to whether long-range transmission occurred or whether cases were infected by a closer, unobserved source of infection.Malaria transmission is shaped by processes occurring on a wide range of spatial scales. In the absence of human mobility, transmission is limited to the range of the mosquito vector, however human movement, ranging from regular commutes to seasonal or one-off migration events across longer distances can import parasites into new areas provided competent vectors are present11 to estimate individual reproduction numbers (Rc or case reproduction numbers) and explore the impact of varying assumptions about the likelihood of a case having a source of infection not observed in the dataset and the spatial kernel (the function describing the relationship between distance metric and likelihood of transmission occurring) on results, as well as determining the feasibility of inferring the distance kernel and amount of missing cases from surveillance data. Briefly, the model builds upon previous diffusion network approaches20 which estimate the likelihood of transmission networks using known or inferred times of infection, by also allowing additional distance information to inform inference. They provide a flexible framework to integrate multiple data types, and have been evaluated using real and simulated transmission processes at multiple scales and under varying network structures21.To explore the impact of including distance measures and assumptions about their relationship to transmission likelihood, we developed a flexible framework to incorporate pairwise distances  into our previously published inference framework22 have weakly informative priors on the likelihood of unobserved sources of infection and assume that cases can be linked by transmission across larger spatial scales (over 200\u00a0km). However, we find there can be issues of parameter identifiability, which become increasingly relevant when there are not enough data available about key parameters in the model.We defined twelve scenarios representing different assumptions about the likelihood of transmission occurring over different geographic distances and likelihood of missing infections (as well as high and low amounts of uncertainty in this estimate). These scenarios were applied to four individual level datasets from malaria eliminating contexts to estimate individual reproduction numbers and how they varied over space and time. We used two simple spatial kernels describing the relationship between Euclidian distance between residences and likelihood of transmission occurring, to explore various assumptions about the relationship between locations of cases and likelihood of transmission occurring between them, as well as the impact of unobserved cases. We find the best performing models by second order AIC (\u0394AICc)11 which uses the time of symptom onset to infer reproduction numbers and their spatiotemporal variation. We then tested the impact of varying assumptions about the relationship between location of cases and the likelihood of transmission as well as the impact of unobserved infection as modelled by competing edges, \u03b5, considering twelve scenarios , El Salvador (P. vivax) and Eswatini  using Exponential and Gaussian spatial kernels, described in the methods section of this paper.We developed a framework to integrate distance information into a previously published inference frameworkP. vivax and P. falciparum). Both scenarios assume longer range human movement is likely and impose a smaller penalty on cases occurring larger distances. These scenarios also allow variation in epsilon edge values and use a very weakly informative prior on epsilon edges, but with a different mean . These results also return smaller mean Rc results than time-only versions of the model  \u0394AICc scores than models which included only time Table . In addiel Figs. , 3, 4.TaRc estimates were found depending on boths \u03b5 and \u03b2 parameters. When \u03b2 is higher, the assumption is that there is little movement of parasites within the country and therefore cases with residential addresses which are far away are unlikely to have infected each other. When this is the case and we assume there are unobserved sources of infection , then Rc values are very low. However if we assume there are little or no unobserved sources of infection, but continue to make restrictive assumptions about space, then most Rc values are very low, but in the localities where there are cases, we estimate much higher Rc values as there are no other possible infectors within a reasonable time and/or spatial area. This is illustrated in Figs. Across all datasets, large differences in Rc estimates under different scenarios several trends are seen across all datasets  and \u03b2 (distance) was seen. When \u03b2 is low, although lower values of \u03b5 produce slightly higher mean Rc values, the difference in Rc estimates with varying prior values for \u03b5 is much smaller than when \u03b2 is a higher value. In other words, when the prior for \u03b5 is low, 1e\u221210, Rc estimates do not vary as \u03b2 changes, however when the prior for \u03b5 is much higher, then increasing \u03b2 from 1e\u22124 to 0.1 reduces Rc estimates (from 0.21 to 0.01 for P. vivax).For both as Figs. , 12, sugRc values, their spatial and temporal distribution and the \u0394AICc/Akaike weights for each model were compared alongside a time only model. These results suggest that including spatial information improved models as measured by AIC, compared to time only results. The prior values for both the distance function and epsilon value have very strong impacts on the estimated Rc, although relative temporal trends tend to stay consistent.We developed an approach to estimate malaria transmission network properties, which allows the flexible integration of distance metrics, such as Euclidian distances or travel times, with temporal information within a single inference framework. Twelve scenarios and corresponding parameter values were defined which represented (a) varying likelihoods of transmission over different distances and (b) varying likelihoods of missing infections (as well as high and low confidence in this estimate). These scenarios were applied to four individual level datasets from malaria eliminating contexts and using two different spatial kernels. The estimated 11.Approaches such as the one presented here can be useful tools for making the most of available surveillance data in near elimination and elimination settings. As more countries collect detailed individual level data, and reach lower case counts which make measures such as parasite prevalence less informative, there is more potential for approaches which leverage the detailed information contained in individual level data. Reproduction numbers can be used to measure the impact of elimination interventions, seasonality and identify areas of higher receptivity to transmission, if importation were to occur there, and when incorporated into geostatistical models with spatially resolved covariates can be used to create maps of receptivityFor all datasets considered, all model versions which used geographic information had lower \u0394AICc values than the time only model. Based on the Akaike Weights and \u0394AICc values for each model, large differences in \u0394AICc were seen between different scenarios. Scenarios 9 and 11 produced the lowest \u0394AICc values. These were parameterisations which penalised long range transmission the least where and the prior on epsilon edges was only weakly informative. These parameterisations also return much lower reproduction numbers than using time alone.23. This \u201cfatter-tail\u201d in the exponential can be seen in Figs. Exponential Kernels consistently outperformed Gaussian kernels as measured by \u0394AICc. Although classic models of dispersion are as a diffusion process with Gaussian displacement, more leptokurtic or \u201cfatter-tailed\u201d probability distributions, where more of the probability density is concentrated in the tails of the function, are often found to better represent empirical dispersal patterns than traditional Gaussian kernels\u03b1ij nor do we fix epsilon, but we do define priors and maximise the posterior rather than the log likelihood. Therefore, we are comparing negative log likelihoods from a maximised posterior, meaning we are not considering the information included in the prior. In addition, many \u03b1ij values shrink to zero, however are still counted as parameters in the AIC estimation. Therefore, there is no recognition of which versions of the model produce fewer non-zero parameters. Whilst this difference in AIC is interesting to note, we would argue the broader trends in how Rc varies over time and space with different assumptions about both the spatial kernel and the number of unobserved sources of infection are more important to consider.However, there are many limitations to using \u0394AICc in model comparison, particularly when estimation of some of the parameters are being carried out within a Bayesian context. We do not fix An interesting pattern which was noted across scenarios and across datasets was how including spatial information in the likelihood tended to increase the seasonality of temporal patterns in reproduction numbers and reduced noise in the temporal distribution of reproduction numbers. This could be suggestive of importation events leading to localised infections. Scenario 4 is also an interesting set of assumptions to consider as it assumes cases generally only infect cases near them and that unobserved cases of infection are unlikely. Under this assumption foci of infection are very clear and clear \u201csources\u201d of infection.2 and Eswatini 17,364\u00a0km2), at higher mean priors for \u03b2, the model converged on an estimate for \u03b2 which was informed by the data. This was not the case for the dataset from China, which represents a much larger area geographically and where dynamics are likely to be strongly driven by importation. Given that for the kernels we used in this analysis, increasing values of \u03b2 lead to more restrictive assumptions about the scale of transmission, perhaps this difference is due to the different spatial scales at which the analysis was being carried out.The results of the sensitivity analysis reveal interesting differences between the different datasets and contexts contained in this dataset. For both El Salvador and Eswatini, which are both small countries  or whether there are unobserved source(s) of infection leading to both cases. This is also exemplified in the results of the sensitivity analysis, where the mean of the prior for beta strongly shapes the final estimate of beta, and the epsilon value also shapes beta.17, and indeed approaches from others have also noted problems with unconstrained distance kernels in space\u2013time diffusion modelling . One potential way to address this is to divide the epsilon edge by the distance parameter \u03b2.In the absence of reliable information about either of these values, strong assumptions must be made about either/both the likelihood of cases being infected by unobserved sources of infection and the relationship between distance and. Similar approaches analysing the diffusion of twitter hashtags, it was recommended to fix the parameter beta, and the authors acknowledged potential challenges in estimating this parameter24 using temporal, spatial, and travel history to infer malaria reproduction numbers also found that travel history must be highly accurate or infections must be focal in time to be able to accurately estimate reproduction numbers.Whilst the temporal aspect is not fixed, we view the utility in this method in excluding or penalising improbable transmission links between far away cases, rather than as a way of trying to determine what the spatial relationship between cases is for malaria transmission, or determining the relative contribution of space to malaria transmission. Recent simulationsP. vivax, and prevalence of asymptomatic infection).An additional approach which could alleviate this problem is to collect internal travel history as part of surveillance in future data collection efforts. This may help tease apart the relationship between space and transmission. There also may be regions where there is more information to parameterise both the spatial scales of transmission and the likelihood of cases being unobserved  between January 2010 and June 2014. For each case detected during this time (N\u2009=\u20091373), case investigation was carried out. For each case the following were collected: GPS coordinates of household location, demographic information , use of malaria prevention interventions such as long-lasting insecticide treated bednets (LLINs), and date of symptom onset, diagnosis and treatment, as well as travel history. Based on travel history cases were defined as locally acquired, imported. For a small number of cases (N\u2009=\u200958) the local/imported status was determined \u201cunknown\u201d. For the purposes of this analysis, these cases were treated the same as local cases, i.e. they were assumed to have potentially been infected by other cases in the dataset and/or been infectors themselves.This dataset, previously analysed by Reiner et al.32  and P. ovale (N\u2009=\u2009822) were reported but excluded from the analysis due to the lower public health concern of these species.This dataset consists of individual-level case data for all confirmed and probable cases reported in China between 2011 and 20162 Tables , 5. The 10 . For each case, the date of symptom onset was recorded. Residential address was available for all but two cases. For these cases, the location was available at the municipio, or municipality level, and the coordinates of the centroid of the municipality (which for both were cities) were used as the geo-location. Two cases had addresses listed outside of El Salvador, both of which were located in Guatemala. All cases within El Salvador with full addresses (N\u2009=\u200985) were georeferenced by latitude and longitude to caser\u00edo/lotificaci\u00f3n level, which is approximately neighbourhood or hamlet level.This dataset consists of all confirmed cases of malaria recorded by the Ministry of Health in El Salvador between 2010 and the first two months of 201611 by introducing a second function, f2, which describes the relationship between space (or distance of any kind) and likelihood of transmission. An appropriate function such as a Gaussian kernel is defined and the parameter(s) shaping that distribution, \u03b2, are either fixed, or given a prior distribution and estimated from the data. Multiplied, together, this returns a single function:t, spatial locations x, transmission rates \u03b1, spatial parameter(s) \u03b2.In order to incorporate pairwise distance metrics, we extended our previously published algorithm applied to Yunnan Province, ChinaAs before, the hazard is defined as the pairwise likelihood divided by the survival term:To illustrate this approach by applying to several malaria line-lists, we used a shifted Rayleigh distribution to model serial interval distributions, ti and at residence location xi being infected by a case showing symptoms at time tj and at residence location xj, becomesti and at residence location xi being infected by a case showing symptoms at time tj and at residence location xj isUsing a shifted Rayleigh distribution and an exponential kernel the pairwise likelihood of a case showing symptoms at The vast majority of disease surveillance and outbreak response datasets will not be able to capture all cases due to asymptomatic infection, underreporting and movement of people in/out of the surveillance area. Therefore, it is important to consider the impact of missing information on results and identify potential missing sources of infection. We use Epsilon edges, Adding epsilon, \u03b5, as a competing hazard and survival returns:rTensorflow package33. A prior probability was defined for the parameter shaping the serial interval of malaria, informed by previous characterisations of the serial interval of malaria34. Because data about how likely the cases were to have moved long distances or the likelihood of a case has been infected by an unobserved source of infection were not available for the contexts explored here, several different parameterisations of the model were used to represent different scenarios , and the corresponding Akaike Weights.The algorithm was written in TensorFlow, implemented in R via the os Table  and a deTwelve scenarios Table  were conRc and its spatial patterns were illustrated for each dataset and parameter combination and compared to the results of the time-only version of the algorithm. The results were also mapped to compare how spatial patterns in Rc were affected by assumptions about space and unobserved infections. The maps shown in Figs. www.openstreetmap.org).The timeseries of c) was calculated using the equation f(x) is the model likelihood, K is the number of parameters estimated and n is the sample size of the data used to fit the parameters. The AIC35 is used in model comparison, by creating a comparison of negative log likelihood that penalises increases in model parameters, to prevent overfitting. AICc is recommended for use with smaller datasets with larger numbers of parameters, and as the sample size n increases AICc converges to AIC22. The differences in AICc for each model, known as \u0394AICc, were calculated to compare models. Typically, a \u0394AICc of greater than 10 is considered strong evidence that that model performs worse than the model it is being compared to.In order to compare models quantifiably, the second order Akaike Information Criterion  prior choice for \u03b2 on both the maxmum-a-posteriori estimates for \u03b2 and the estimated mean Rc.In the scenario analysis above the distance shaping parameter is fixed. However due to the uncertainties in the relationship between distance and likelihood of transmission, in many contexts it may be useful to estimate \u03b2, the mean for a Gaussian Kernel was varied between 0.00001 and 0.01 and for an exponential kernel the means considered ranged between 0.0001 and 0.1. For both the standard deviations varied between 0.0001 and 0.1 (table). Every possible combination of the parameters were run for each dataset and both Gaussian and exponential spatial kernels, giving a total of 2400 parameter combinations tested per kernel, per dataset.To consider the effect of varying parameter values and explore their interactions, a range of distance and epsilon edge priors were considered. A truncated normal prior was used for both parameters, and the mean and standard deviation were varied. For Supplementary Information 1.Supplementary Table 1."}
+{"text": "Pdgfr\u03b1 conventional knockout mice reported hypoplasia of the sinus venous myocardium including the sinoatrial node (SAN) accompanied by increased expression of Nkx2.5. This mouse line embryos die by E11.5 due to embryonic lethality, rendering them difficult to investigate the details. To elucidate the underlying mechanism, in this study, we revisited this observation by generation of specific ablation of Pdgfr\u03b1 in the SAN by Shox2-Cre at E9.5, using a Shox2-Cre;flox/floxPdgfr\u03b1 conditional mouse line. Surprisingly, we found that resultant homozygous mutant mice did not exhibit any malformation in SAN morphology as compared to their wild-type littermates. Further analysis revealed the normal cardiac function in adult mutant mice assessed by the record of heart rate and electrocardiogram and unaltered expression of Nkx2.5 in the E13.5 SAN of Pdgfr\u03b1 conditional knockout mice. Our results unambiguously demonstrate that Pdgfr\u03b1 is dispensable for SAN development after its fate commitment in mice.Palate-derived growth factor receptor \u03b1 (Pdgfr\u03b1) signaling has been reported to play important roles in the cardiac development. A previous study utilizing  Nkx2.5\u2013 head and an Nkx2.5+ sinoatrial junction or tail, is a group of specialized cells with obvious heterogeneity located on the dorsal wall of the right atrium close to the entrance of superior vena cava, functioning as the primary cardiac pacemaker that regulate the rhythm of the heartbeat by spontaneously producing electrical impulses , composed of an impulses . The firimpulses , then thimpulses . These pimpulses . Deficieimpulses .Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel subtype 4), Tbx3, Shox2, and Nkx2.5, work as cardiac markers and are critical for the development of SAN. For instance, mutation in Shox2 results in a severely hypoplastic SAN due to ectopic Nkx2.5 expression in the head of SAN , located on the surface of a wide range of cell types, is bound by certain isoforms of PDGF ligands and thereby becomes active in stimulating cell signaling pathways that elicit responses including growth, development, and differentiation. There are two types of PDGFR, \u03b1 and \u03b2, functioning in the form of homodimer, \u03b1-\u03b1 (termed as Pdgfr\u03b1) and \u03b2-\u03b2 (termed as Pdgfr\u03b2), or heterodimer, \u03b1-\u03b2 (termed as Pdgfr\u03b1-\u03b2) . Each tyry veins .GFPPdgfr\u03b1) reported that Pdgfr\u03b1 is markedly expressed at the venous pole in the mesocardium, the myocardium of the sinus venosus, the proepicardial organ, and the coelomic mesothelium in the E9.5 heart, as well as in the developing SAN at E10.5. Several cardiac malformations including the significantly smaller SAN accompanied by increased expression of Nkx2.5 were further observed in Pdgfr\u03b1 conventional knockout mouse line  as a reporter in a knock-in mouse line . Antibodies used for this study include anti-Pdgfr\u03b1 , anti-Hcn4 , anti-Nkx2.5 , and anti-GFP .Mouse embryos were harvested from timed pregnant females. Bodies of collected embryos were fixed in 4% Paraformaldehyde at 4\u00b0C for 24 h and further processed for paraffin embedding and sectioning at 7 \u03bcm. Slides were subjected to Hematoxylin/eosin staining for histology analysis or immunofluorescent staining as described previously . The meaImages of consecutive H&E stained sections (7 \u03bcm) of SAN and surrounding tissues were captured, stacked, and loaded into Amira 6.0.1, in which subsequent alignment, segmentation, and 3D model generation were performed.flox/floxShox2-Cre;Pdgfr\u03b1 mice and their wild-type littermates were anesthetized with 1.5% isoflurane supplemented with O2 and placed in prone position on a Mouse Monitor S (Indus Instruments) to be recorded as reported previously  embedding.The protocol of the E13.5 SAN scRNA-seq was described in a previous study  and thist test. All data of the volume measurements are presented as mean \u00b1 SEM. Graphics of statistical analysis were performed with GraphPad Prism 5.0 software. P < 0.05 was considered statistically significant.Statistical analysis on the volume measurement were performed with independent unpaired Student\u2019s Pdgfr\u03b1 is expressed as early as E7.5 in several cardiac progenitor tissues including SAN during early heart development , this pattern of Pdgfr\u03b1 expression was confirmed  in adult flox/floxShox2-Cre;Pdgfr\u03b1 mice showed that the electrocardiograms are normal with the P waves corresponding to atrial depolarization clearly identical to their adult wild-type littermates and each P wave followed by a normal QRS complex corresponding to ventricular depolarization (N = 6/each group). This normal electrocardiogram from flox/floxShox2-Cre;Pdgfr\u03b1 mice indicated that cardiac function, particularly the pacemaking function of the SAN, was normal in the Pdgfr\u03b1 conditional knockout mice. Given the fact that a smaller SAN was acquired with 37% reduction in volume at E13.5 in Pdgfr\u03b1 conventional knockout mice in the previous study (N = 3/each group). Therefore, our results provided compelling evidence that Pdgfr\u03b1 is dispensable for SAN morphogenesis and physiological function in mice when it is ablated with Shox2-Cre at E9.5.To further investigate the role of  as E9.5 . To vali embryos  but abse embryos , confirmus study , we furt\u2013 head and an Nkx2.5+ junction , mesenchymal cells (MC), endothelial cells (EC) and cardiomyocytes (CM), but Nkx2.5 was only restricted in CM and appeared not overlapped with Pdgfr\u03b1  progenitors and dorsal mesocardium at E8.0  at E9.5, extends to the sinus valves originating from the SAN at E10.5, and becomes restricted to the entire SAN including the head and tail regions at E11.5 and later  can be found in the article/supplementary material.The animal study was reviewed and approved by The Animal Ethical and Welfare Committee of Fujian Normal University.XuH designed the study and administrated the whole study. XZ and FW conducted experiments and collected data and drafted the manuscript. HL provided the E13.5 SAN scRNA-seq raw data. XiH analyzed the scRNA-seq data. ZG involved in methodology and statistical analysis. YZ and XH performed manuscript reviewing and polishing. All authors gave final approval of the version to be published, and agreed to be accountable for all aspects of this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Mycoplasma hominis (M. hominis) colonization of low abundance on female fecundability.To explore the impact of pre-pregnancy vaginal M. hominis colonization was confirmed by qPCR. Cox models were used to estimate the fecundability odds ratio (FOR) for women with M. hominis.In total, 89 females participating in a pre-pregnancy health examination program were included, and their pregnancy outcomes were followed up for 1 year. Vaginal swabs were collected, 16S rRNA genes were sequenced, and M. hominis was 22.47% (20/89), and the abundance was relatively low . In terms of the vaginal microbiome, the Simpson index of the positive group was significantly lower than that of the negative group (P\u2009=\u20090.003), which means that the microbiome diversity appeared to increase with M. hominis positivity. The relative abundance of M. hominis was negatively correlated with Lactobacillus crispatus , but positively correlated with Gardnerella vaginalis, Atopobium vaginae and Prevotella bivia . The cumulative one-year pregnancy rate for the M. hominis positive group was lower than that in the negative group . After controlling for potential confounders, the risk of pregnancy in the M. hominis positive group was reduced by 38% when compared with the positive group .The prevalence of M. hominis at a low level in pre-pregnant women is negatively correlated with female fecundability.The vaginal colonization of The online version contains supplementary material available at 10.1186/s12866-022-02545-7. The reduction of female fertility is a complex problem of public health; its potential causes include social, environmental and physiological factors , 2. A meMycoplasma are prevalent, and are the smallest genital Mollicutes in the vaginal microbiome; they are unique genera with no cell wall around the cell membrane [Mycoplasma hominis (M. hominis) is the most common Mycoplasma species in the vagina. Some studies have found that the colonization rate of M. hominis is 3.1\u201315% [M. hominis infection is associated with BV [M. hominis was associated with the structure of the vaginal microbiota, [M. hominis might play an important role in female health. Inconsistent findings suggest that vaginal M. hominis infection is a potential reason for female infertility, [M. hominis might affect the female TTP. Therefore we used the data from a pregnancy-planning cohort to explore, among asymptomatic pregnancy-planning females, whether a low abundance of M. hominis colonization can impact the TTP, in order to provide an innovative perspective for female infertility prevention.membrane . Mycopla 3.1\u201315% . Althoug with BV  and pret with BV , it is s with BV  because robiota,  which surtility,  but no sTreponema pallidum, cytomegalovirus or Toxoplasma gondii; (4) those who did not collect the vaginal swabs because of a menstrual period; (5) women who were diagnosed with BV or colpomycosis, which required medical treatment. A total of 102 women met the criteria and were recruited, after giving informed consent. The Ethics Committee of Zhongda Hospital approved this study (2018ZDSYLL072-P01), all methods were performed in accordance with the declarations of Helsinki. In the follow-up phase, 13 females who gave up attempting pregnancy within 3\u2009months and refused to tell us the exact time they had have kept the desire of pregnancy were excluded. Thus, 89 females were analyzed in this study.A pregnancy-planning cohort study was conducted at the Maternal and Child Health Center of Gulou district, Nanjing, China, from September 2018 to June 2019. All the women who participated to the free pre-pregnancy health examination program and stated that they were ready to attempt pregnancy were included. This program was supported by Chinese government from 2010, which aimed to reduce birth defects rate among population. The original design, organisation and implementation of the program have been described previously . With thGynecologists used two sterile swabs and a speculum to collect vaginal secretions. The swabs were transferred to a\u2009\u2212\u200980\u2009\u00b0C refrigerator, and saved for bacterial nucleic acid extraction. Following the standard procedures in our laboratory,  swabs wewww.biocloud.org). Paired-end reads were merged by FLASH . Some low quality tags were removed using Trimmomatic (v0.33). Denoised sequences were clustered using USEARCH (version 10.0), and tags with similarity \u226597% were regarded as operational taxonomic units (OTUs). Subsequently, the NCBI database was used to annotate taxonomic information with QIIME. The information on OTU relative abundance was normalized using a standard sequence number corresponding to the sample with the fewest sequences. The samples with representative sequences of M. hominis were judged as the positive group, and the qPCR method was used to confirm the abundance by cycle threshold value (CT). Considering that the study participants were all asymptomatic, CT\u2009<\u200935 was regarded as positive. The primers (5\u2032-TCACTAAACCGGGTATTTTCTAACAA-3\u2032 and 5\u2032-TTGGCATATATTGCGATAGTGCTT-3\u2032) were used. The ChamQ SYBR qPCR Master Mix  was used for reaction preparation (20\u2009\u03bcl), and a standard amplification program was set up in the Real-Time PCR System . There were 3 replicates for each sample, at least two consistent CT values were considered credible results, otherwise, we would repeat the detection.The V3\u2013V4 hypervariable regions of the DNA samples were amplified by polymerase chain reaction (PCR) using universal primers (338F: 5\u2032-ACTCCTACGGGAGGCAGC3\u2019 and 806R: 5\u2032-GGACTACHVGGGTWTCTAAT-3\u2032). The PCR products were sequenced on an IlluminaHiseq 2500 platform . Sequencing data was processed on the Biomarker biocloud platform  were recorded. The TTP was calculated as the interval between the date of participation in the study and the date of the LMP obtained at follow-up (if pregnant within 1\u2009year) or last follow-up date (if not pregnant).M. hominis and other species were evaluated using the Spearman correlation coefficient. Linear discriminant analysis effect sizes (Lefse) were calculated to determine biomarkers between the groups. Kaplan\u2013Meier plots and the log-rank test were used to present the cumulative pregnancy rate among different groups. In order to adjust for some potential confounders, Cox models were used to estimate the fecundability odds ratio (FOR) and the 95% confidence interval (CI). The FOR estimated the odds of becoming pregnant in the current month for participants with or without M. hominis infection, conditional on not being pregnant in the previous month, thus FOR <\u20091 indicated a reduction in fecundability. Model A was adjusted by age, pregnancy history and menstruation regularity and couples\u2019 age differences; Model B was additionally adjusted by vaginal cleanness grading and the Shannon and Simpson index to control for the impact of overall vaginal microenvironment. A two-sided P value \u22640.05 was deemed statistically significant.The continuous variables were described as mean\u2009\u00b1\u2009standard deviation , or median with interquartile range ; comparison among groups was assessed by t test  or Kruskal\u2013Wallis test . The chi-square test was used to test differences in frequency distribution among groups. Alpha diversity indices, including Simpson and Chao1 indices, were calculated by mothur software (version 1.30). A higher Chao1 index and lower Simpson index mean a more diversity for microbiome, the details are shown in the Supplementary text. The binary Jaccard distance, one of the \u03b2-diversity indices that focuses on differences in taxonomic abundance profiles from multiple samples, was assessed with QIIME software, and following principal coordinate analyses (PCoAs), and permutational multivariate analysis of variance (PERMANOVA) were used to compare the differences between groups. PCoA is a non-constrained data dimension reduction analysis method, which is also a visualization method to study the similarity or difference of data. Differences between individuals or groups can be observed through PCoA plot, thus it was widely used in microbiome data analysis . The corM. hominis positive rate was 22.47% (20/89). Among these samples, all the CT values were more than 25 (28.32\u2009\u00b1\u20092.31). The woman\u2019s age, age differences within couples, pregnancy history and menstruation situation were comparable between the M. hominis positive and negative groups .Among the 89 women, the average age was 28.60\u2009years. After qPCR confirmation, the P\u2009=\u20090.003, Fig. M. hominis positivity. However, the Chao1 indices were not significantly different between groups .In terms of \u03b1 diversity indices, the Simpson index of the positive group was significantly lower than that of the negative group  and positively correlated with Gardnerella vaginalis, Atopobium vaginae and Prevotella bivia , but it was not associated with Ureaplasma parvum.Further, we explored the potential M. hominis positive group was 6\u2009months; that in the negative group was 11\u2009months. The Kaplan\u2013Meier method showed that the cumulative pregnancy rate for the M. hominis positive group was lower than that of the negative group . After adjusting for the indices reflecting the vaginal microenvironment, including vaginal cleanliness grading and Shannon and Simpson index, this association remained . Previous studies have always used a case\u2013control design, which leads to difficulty in making a causal inference because we cannot infer whether the M. hominis infection was earlier than the infertility. Thus, our study provides more convincing evidence that M. hominis infection reduces female fecundability, by using a prospective design.Many epidemiological studies have found that vaginal ic women . The bacis of BV,, 16 but iagnosis , 18. Miyiagnosis  found th section . The ass hominis\u00a0. Ma et a hominis\u00a0 reportedM. hominis. In sequenced amniotic fluid/placental isolates, Allen [M. honimis that encode surface-located membrane proteins, which is associated with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth. Additionally, a surface lipoprotein, MHO_0730, has been found to have effects on promotion of infection and modulation and evasion of innate immunity [M. hominis can also cause direct tubal damage or altered ciliary activity within the fallopian tubes [M.hominis would impact female fertility through above three pathways, these hypotheses are also reasonable for M.hominis. Additionally, M.hominis had been found to be associated with local inflammatory level, which might participate in the regulation of female fertility [M. hominis colonization and female TTP.The development of genomics has increased the understanding of the pathogenic mechanisms of s, Allen  identifiimmunity . Previouimmunity . M. homian tubes . Howeveran tubes  bioactivan tubes  and the an tubes . Althougertility . FurtherM. hominis is meaningful for evaluation of fecundability, because this association is independent from the vaginal microenvironment.This study is strengthened by its prospective design, and the TTP was used to evaluate fecundability. According to clinical practice guidelines, infertility should be diagnosed when a women has not become pregnant after having 12\u2009months of regular, unprotected intercourse . This meM. hominis would not be accurately identified by culture. Therefore, our results lack comparability with other studies. Second, some researchers think that the vaginal microbiome shows dynamic change within one cycle, so our one-time assessment may not reflect the vaginal microenvironment in its entirety. Third, like all other prospective TTP related studies, there is inevitably selection bias [M. hominis information could not be get. The future work should require the participants to collect vaginal swabs at a same fixed time in their menstrual cycle. Additionally, 13 women who refused to continue the follow-up without exact TTP in the first 3\u2009months were excluded, which might expand selection bias. Fifth, our findings needs to be further validated because many potential confounding factors were not collected and did not be taken into account, such as male partners\u2019 sperm quality, M. hominis infection status, and the couples\u2019 regularity of sexual relations. In addition, couples\u2019 educational levels is also important to be considered, because it can indirectly reflect the ability of couples to identify the fecundity period in the female menstrual cycle. Lastly, our findings should be interpreted cautiously, because all the participants were from Nanjing, China, and the sample size is small. The vaginal microenvironments ae diverse among females with different ethnicity, [There are several inevitable limitations to this study. First, although the high-throughput sequencing method we used is a frequently used method of bacterial detection, it is not yet popular in clinics. Some samples with low abundance of ion bias . Becausehnicity,  and therM. hominis in women pre-pregnancy is negatively correlated with female fecundability. M. hominis screening is necessary for pre-pregnancy health examination.In summary, vaginal colonization by Additional file 1 Supplementary text. The formula about Chao1 and Simpson indices"}
+{"text": "This study aims to investigate the role of sex and ovarian hormones in hippocampal damage and cognitive deficits and behavioral dysfunction in rats induced by chronic exposure to hypobaric hypoxia.Six-week-old male and female SD rats were housed for 3 months either in a real altitude  environment as the model of chronic hypobaric-hypoxia (CHH) or in a plain as controls. The animal behavioral and hippocampal neurons at subcellular, molecular, and ultrastructural levels were characterized after CHH exposure.N-methyl-D-aspartate receptor NR2A and NR2B subunits in male rats\u2019 hippocampus. In addition, compared with the sham CHH group, the expression level of PON2 protein decreased in the OVX-CHH group\u2019s hippocampus whereas oxidative stress, neuroinflammation, and degeneration of hippocampal neurons increased in the OVX-CHH group\u2019s hippocampus.After 3 months of CHH exposure, (1) male CHH rats\u2019 serum testosterone level was lower than male controls\u2019 whereas female CHH rats\u2019 serum estradiol level was higher than female controls\u2019; (2) Morris water maze test finds that male rats showed more learning and spatial memory deficits than female rats; (3) male rats showed more severe hippocampal damage, hippocampal inflammation, oxidative stress and decreased hippocampal integrity (neurogenesis and dendritic spine density) than female rats; (4) Western blot analysis shows that, compared with the male control group, in male CHH group\u2019s hippocampus, expression of nNOS, HO-1, and Bax protein increased whereas that of Bcl-2 protein decreased; (5) Expression of PON2 protein in male rats (CHH and controls) was lower than female rats (CHH and controls). In addition, CHH exposure decreased the expression of PON2 protein in both male and female rats; (6) qPCR analysis reveals that CHH exposure reduced the gene expression of After CHH exposure, male rats were significantly more likely than female rats to develop hippocampal damage, hippocampal neuroinflammation, and cognitive decline and deficits, suggesting that sex and ovarian hormones were significantly involved in regulating the rats\u2019 susceptibility to CHH exposure-induced hippocampal damage. Among the numerous people living in high-altitude areas for a long time all over the world, about 12 million people live in the Qinghai-Tibet Plateau . In receCognitive deficits induced by chronic exposure to hypobaric-hypoxia are mostly maladaptive responses associated with oxidative stress and inflammation , blood-bParaoxonase 2 (PON2) \u2248 43 kDa, as a widely expressed intracellular enzyme (absent in the serum), belongs to a gene family composed of PON1, PON2, and PON3 . PON2, aIt has been reported that women and female animals cope better than men and male animals with exposure to decreased oxygen partial pressure at high altitudes . Women aGiven that postmenopausal women have a higher level of oxidative stress and that estradiol is neuroprotective by suppressing oxidative stress , we hypoThis study was approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University, Chengdu, China. All the experiments were carried out in compliance with the relevant ethical guidelines, regulations and protocols.n = 52) and female (n = 52) rats were randomized into either the chronic hypobaric-hypoxia (CHH) group or the control group. The latter was reared in Chengdu, China, at an altitude of 500 m above sea level (a.s.l.) for 3 months, while the CHH group was transported from Chengdu, China to Yushu, a city in the Qinghai-Tibet Plateau of China at an altitude of 4,250 m a.s.l. and also reared there for 3 months. The CHH rats were transported by a special experimental animal transport vehicle equipped with a constant temperature system that kept the in-vehicle temperature at 21\u201324\u00b0C constantly. All the animals had access to standard rodent chow and tap water ad libitum in all cages (with six rats in each cage), with an environmental temperature of 22\u201324\u00b0C and humidity of 50\u201360%. Three months later, the CHH group was transported back to Chengdu from the laboratory animal room at Yushu in the Qinghai- Tibet Plateau.A total of 104 6-week-old male and female Sprague-Dawley rats weighing 160\u2013170 g  were used in the present study. Male (n = 8 of each sex per group). From the first day of 2 weeks before the end of 3 months of CHH exposure, the selected 32 rats were intraperitoneally injected once a day with BrdU  solution prepared with normal saline at a dose of 100 mg/kg for 7 consecutive days (5-bromodeoxyuridine (BrdU) was used as an exogenous proliferation marker. In the S phase of cell division, the marker was integrated into a genetic material. A total of 32 rats in the CHH and control groups were randomly selected (ive days . Two ween = 8 of each sex per group randomly chosen). For routine blood analysis, the coccygeal vein blood  was collected with a Mindray automatic hematology analyzer . The analysis parameters included white blood corpuscles (WBC), red blood corpuscles (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and mean platelet volume (MPV). The analysis was carried out with the Auto Hematology Analyzer .After 3 months, blood samples from the CHH and control groups were collected from the tail vein under isoflurane anesthesia . The blood samples were centrifuged at 4\u00b0C  for 10 min to separate the plasma from the whole blood. The supernatant was taken, with the samples with severe hemolysis discarded, and then frozen at \u201380\u00b0C and stored for subsequent analysis. The intact hippocampal tissues were immediately collected into labeled tubes and stored at \u201380\u00b0C until analysis.Serum concentrations of estradiol (cat. no.: E-EL-0152c) and testosterone (E-EL-0155c) were tested using enzyme-linked immunosorbent assay (ELISA) by rat commercial kits  according to the manufacturer\u2019s instructions.Inflammatory markers in the hippocampus were assayed by ELISA, using commercial assay kits. The concentration of IL-1\u03b2 (cat. no.: ERC007), IL-6 (ERC003), and TNF-\u03b1 (ERC102a) levels were assessed by using rat ELISA kits .According to the manufacturer\u2019s instructions, we measured the levels of superoxide dismutase (SOD) (cat. no.: KTB1030), malondialdehyde (MDA) (KTB1050), glutathione peroxidase (GSH-Px) (KTB0640), glutathione (GSH) (KTB1600), and catalase (CAT) (KTB1040) in the hippocampus with commercial assay kits . The total protein concentration of the supernatant was detected by the BCA kit.2mmol\u20131) was used to estimate the formed MDA, and the resultant value was expressed in nanomoles/mg protein.The final product malondialdehyde (MDA) was determined by spectrophotometry, as described by The reduced glutathione (GSH) levels were measured from the crude homogenate by fluorescence method according to the method of With the assay kit of GSH-Px, CAT, and SOD, the enzyme activities of GSH-Px, CAT, and SOD were measured at 340, 540, and 450 nm by spectrophotometry, respectively.Several experimental studies have reported that simulated CHH exposure contributes to emotional behavioral changes, such as anxiety-like behavior . The hipn = 8/per group) (Morris water maze (MWM) test is a hippocampal-dependent assessment of spatial learning by rodents (r group) . In the n = 8/group). All MRI examinations were performed in a 7.0 T magnetic resonance imaging system . For the rat studies, a 72-mm open birdcage quadrature volume resonator was utilized for excitation, and a four-channel (2 \u00d7 2) phased array coil was used as a receiver. Anesthesia was initially induced with 2.5% isoflurane and maintained by 1\u20131.5% isoflurane (2 L/min oxygen flow) during MRI scanning. The rat was placed in the prone position with the head immobilized with a toothed rod and two ear pins. A respiratory rhythm sensor was installed under the abdomen to observe respiration and monitored at 50-80 breaths per minute. Also, the rats were kept on an animal heating pad connected with hot circulating water, which was maintained at 37 \u00b1 1.0\u00b0C during scans to prevent the body temperature from dropping.Imaging experiments were conducted after behavior assessment (2 \u00d7 0.137 mm2 (matrix size = 256 \u00d7 256), and total scan time of approximately 38 min. A prior method  sequence. These T2-weighted images (T2WI) were acquired using a 2D RARE sequence with the following parameters: repetition time (TR) = 11,400 ms, echo time (TE) = 48 ms, number of excitations (NEX) = 6, slice thickness = 0.35 mm, slice gap = 0 mm, field of view (FOV) = 35 mm \u00d7 35 mm, in-plane resolution of 0.137 mmr method  for VBM \u20131. After the perfusion, the brains were extracted and fixed in 40 ml of 4% paraformaldehyde. After sufficient post-fixation, the brains were embedded in paraffin. A total of 4-\u03bcm-thick coronal sections were consecutively taken from about bregma \u20133.11 mm by sled microtome.Following behavior assessment, the brains of all the rats were collected and underwent morphologic studies. The animals were euthanized with an overdose of isoflurane and perfused transcardially with 250 ml of ice-cold saline, followed by 300 ml of 4% paraformaldehyde (PFA) in phosphate-buffered saline  for 30 min at 12 ml\u22c5minn = 6 rats/group) and each area in 3\u20135 sections using \u201cImage-Pro Plus 6.0\u201d software . The slides were independently assessed by two observers blinded to the status of the rats, and the average results of their assessment were regarded as the final data.For the neurodegeneration study, the brain sections of the rats were stained with Fluoro Jade-B (FJB) dye, which is a fluorescent substance that binds sensitively and specifically to degenerating neurons. The staining was performed by referring to +/NeuN+ cells were counted and quantified by Image-Pro Plus 6.0 software . About 3\u20135 sections per area per rat (n = 8 rats/group) were analyzed, and the mean number of positive cells in the hippocampus was counted in each image.After the paraffinized brain slides were dewaxed and rehydrated, the sections were incubated with one of the following two primary antibodies, anti-NeuN /anti-Iba1  and anti-NeuN /anti-BrdU antibody  in blocking serum at 4\u00b0C overnight. The following day, the sections were incubated with secondary antibodies consisting of Alexa Fluor 488 goat antimouse and Cy3 goat antirabbit for 1 h in the dark at room temperature. The cell nucleus was counterstained with DAPI for 10 min in the dark. The sections were covered with anti-fade mounting medium. Each of the above was washed three times for 5 min with PBS. Iba1-labeled cells were counted in the hippocampal subregions of interest  under an immunofluorescence microscope . For double labeling of BrdU and NeuN, the images were observed by a confocal microscope , and co-labeled BrdU\u00ae kit . The brain was impregnated in a Golgi-Cox mixture of solution (provided by the kit producer) for 2 weeks in the dark at 37\u00b0C and then transferred into a protectant solution prior to paraffin embedding for 48 h according to the manufacturer\u2019s instructions. The brains were coronally sectioned into slabs of 100 \u03bcm using an oscillating microtome  and the slabs were collected on 5% gelatin-coated microscope slides. The slides were then stained in concentrated ammonia, fixed with sodium thiosulfate, dehydrated in gradient ethanol, and cleared in xylene until mounting.The dendrites and the spines in the hippocampus are critical carriers for learning and memory function. To evaluate the effect of plateau hypoxic exposure on them, the neural morphology in the hippocampus was assessed. A modified Golgi-Cox staining method was used for the analysis of hippocampal dendrites and spines as previously described . After bN = 5 rats per group and n = 4\u20135 neurons per region in each hemisphere  selected randomly per rat] were imaged at 200-fold magnification, and the spines were imaged at 1000-fold magnification with a Nikon wide-field microscope (Eclipse Ci-L). The total dendritic lengths were quantified by the Neuron J Plugin for ImageJ/Fiji software . Sholl analysis with the ImageJ program was used to analyze the dendritic complexity by counting the number of intersections across dendrites with an overlaid concentric sphere at 10-\u03bcm intervals from the cell soma (n = 5 rats per group) were examined for dendritic spine density.The hippocampal neurons in CA1 and CA3 regions . Serum testosterone level in the male CHH group was significantly lower than the male control group (p = 0.017) whereas there was no significant difference between the female CHH group and the control group (p > 0.05) .Serum sex hormone level obtained by ELISA is shown in p = 0.015), IL-6 (p = 0.027), and TNF-\u03b1 (p < 0.001) in the male CHH group were significantly higher than the male control group. IL-1\u03b2 level in the hippocampus of the female CHH group was significantly higher than the female control group (p = 0.025). There were no significant differences in IL-6 (p > 0.999) and TNF-\u03b1 (p = 0.315) in the hippocampus between the CHH group and the control group. The above statistical results indicate that the male CHH rats showed higher levels of inflammatory factors than the control group.Hippocampal ELISA results  reveal tp = 0.004) whereas the content of SOD (p = 0.004) and the activities of antioxidant enzymes such as GSH (p = 0.017), CAT (p = 0.002) and GSH-Px (p = 0.002) decreased significantly. CAT enzyme activity in the female CHH group was significantly lower than in the female control group (p = 0.001). Compared with the female control group, the differences in the contents of MDA (p = 0.734) and SOD (p = 0.1), the activities of GSH (p = 0.122) and GSH-Px (p = 0.947) in the female CHH group were not statistically significant between the groups. The above statistical results indicate that oxidative stress levels were more significant in the male but not in the female CHH rats when compared with the corresponding control group  = 29.39, p < 0.001] and main effect of sex  but no interaction between group and sex . Post hoc analysis shows that the percentage of time spent in the central arena was significantly reduced in both male and female CHH groups compared with the corresponding male and female control groups  = 2.63, p = 0.116; sex: F = 1.04, p = 0.316; the interaction F = 0.52; p = 0.478]  = 122.9, p < 0.001]. The main effect of the group (with and without CHH exposure) was significant , indicating that CHH exposure increased the escape latency of the CHH rats compared with the control group. The main effect of sex was significant , indicating that the escape latency of the male rats was longer than that of the female rats ; however, on days 1\u20134, the escape latency was not significantly different between CHH-exposed female rats and the control female rats (p > 0.05)  = 287.6, p < 0.001]. The interaction between training days and the group was significant , indicating that CHH exposure\u2019s effect was significant ; on days 1\u20134, there was no significant difference in swimming distance between CHH-exposed female rats and the control female rats (p > 0.05)  . The swinificant . Sidak\u2019s > 0.05) . OverallF = 12.51, p = 0.001, indicating that CHH exposure affected the number of crossings across the platform; main effect of sex was significant F = 18.17, p < 0.001; sex and group interaction was significant F = 5.14, p = 0.031]. The percentage of time spent in the target quadrant decreased by the male CHH rats . In the interaction between sex differences, the male rats showed more deficits in spatial memory ability, indicating that CHH exposure varied in its effects on cognitive function between the two sexes.In the probe trials , the malFWE corrections but when displayed with an uncorrected p < 0.0001. The macro-structural alterations showed several regional volume reductions prominently in the hippocampus  between male groups (CHH vs. control). There was no significant difference between the female groups (CHH vs. control) (data not shown).Voxel-based morphometry reveals that the t-maps for gray matter volume (GMV) were generated after the total brain volumes were controlled for. All macro-structural alterations did not survive pocampus . In otheF = 72.13, p < 0.001; CA3: F = 84.68, p < 0.001; DG: F = 81.94, p < 0.001]. The main effect of sex  was significant in different hippocampal subregions , and the interaction between the two sexes was significant in different hippocampal regions . CA3: F = 37.6; p < 0.001; DG: F = 22.03, p < 0.001. Compared with the control group, the number of FJB positive cells in the CHH groups  was higher than that in the control group  (p < 0.05). Moreover, the male CHH group showed more FJB-positive cells than the female CHH group (p < 0.05)  . HoweverF = 29.02, p < 0.001; CA3: F = 100.7, p < 0.001; DG: F = 46.09, p < 0.001]. The main effect of sex was significant . There was a significant interaction between group and sex . Compared with the control group, the number of Iba-1 positive cells in the male CHH group\u2019s hippocampus significantly increased (p < 0.001). The number of microglia in the male CHH group was significantly higher than the female CHH group (p < 0.01), whereas no significant difference was observed in the number of microglia between the female CHH group and the female control group (p > 0.05) and there was no significant difference between the male and female control groups (p > 0.05)  .p < 0.05) whereas the difference between the female CHH and the female control groups was not significant (p > 0.05)   = 93.69, p < 0.001; sex: F = 5.71, p = 0.03; the interaction between group and sex: F = 11.59, p = 0.004. Post hoc analysis finds that the total length of hippocampal dendrites in the CHH group was significantly shorter than the control male and female rats  = 2.22, p > 0.05; To further explore sex differences in the effects of CHH exposure on neuronal morphology, we examined dendritic morphology and synaptic ultrastructure . Golgi-CF = 44.44, p < 0.001] and main effect of sex , and the interaction between group and sex . Post hoc analysis reveals that, in the CA1 region, the dendritic spine density of hippocampal neurons in the male CHH group was significantly lower than in the male control group . There was no sex difference between CHH and control groups in the dendritic spine density of hippocampal neurons in the CA3 region  , indicating that CHH exposure raised HO-1 protein expression level in male and female rats  . After 3 months of CHH exposure, the expression level of PON2 protein decreased in the hippocampus of both female and male CHH rats (p < 0.05).   was significantly higher than male rats (CHH group and control group) (= 0.008] .P < 0.05) whereas no significant difference was found between the female CHH group and the control group (p > 0.05).   whereas no significant difference was observed between the female CHH group and the control group (p > 0.05).  , but not in female rats (p > 0.05)  , and the hippocampal level of NR2B of the female rats showed a downward trend. Nevertheless, there was no statistical difference compared with the female control group (p > 0.05).  , but not in the female CHH group (p > 0.05)  (p < 0.05) but not in the female CHH group (p > 0.05)   were observed at the ultrastructural level. The male CHH group showed severe apoptotic death of neuronal cells . Howeverp > 0.05). The weight of the uterus of female rats in the OVX-CHH group decreased significantly, and the level of the serum estradiol decreased significantly, which evidences the effectiveness of ovariectomy in this experiment. Compared with the sham-CHH group, there was no significant difference in hematological indexes between the OVX-CHH group and the sham-CHH group (p > 0.05). Compared with the sham CHH-group, the proinflammatory cytokines  in the hippocampus of female OVX-CHH rats increased significantly (p < 0.05). Compared with the sham-CHH group, the content of MDA in the hippocampus of female rats in the OVX-CHH group increased and the activity of antioxidant enzymes  decreased (p < 0.05)  see .p < 0.001)  of the OVX-CHH group increased significantly (< 0.001) . These rThe results of this study show that after ovarian hormone deficiency, the expression of nNOS, HO-1, and Bax protein increased whereas the expression of PON2 and Bcl-2 protein decreased in the hippocampus of female rats .Chronic hypobaric-hypoxia exposure can lead to hippocampal neuron damage and cognitive decline, which has been widely reported. However, there are few studies on the vulnerability of sex differences to hypoxia at high altitudes. In this experiment, we have made a major finding as regards such sex differences. In this study, in the animal model raised at a natural high altitude for 3 months, differences were found between the two sexes in hippocampal damage and cognitive deficits, which has not been reported previously. Compared with its respective control group, the male CHH group, rather than the female CHH group, showed a more significant decline in cognitive ability, more obvious neuronal injury, microgliosis, neuroinflammation, and oxidative stress in the hippocampus, and more severe decreased hippocampal integrity (such as neurogenesis and dendritic spine density), which are closely related to neurological deficits. Male rats seemed to be more vulnerable than female rats to hippocampal injury and cognitive impairment induced by CHH, possibly due to the low expression level of PON2 protein in males.It has been reported that the hypobaric-hypoxia environment may be deleterious to male reproductive function, which is accompanied by a decrease in serum testosterone level . It has It has been reported that oxidative stress and decreased antioxidant capacity play an important mediating role in the neurophysiological disorders of memory impairment caused by hypobaric\u2013hypoxia . In the Inflammation-induced by hypobaric-hypoxia may lead to brain injury and neuronal loss with long-lasting effects on the regional or global volume of the hippocampus. Moreover, inflammation also inhibits hippocampal neurogenesis . The redHippocampal neurons in male rats are more susceptible to CHH than in female rats. FJB staining shows that, after 3 months of CHH exposure, the neurodegeneration of male CHH-exposed rats was significantly higher than that of female CHH rats. The total dendritic length and dendritic spine density of hippocampal neurons in male CHH-exposed rats were significantly lower than in female CHH-exposed rats. This finding morphologically evidences that CHH exposure can induce more hippocampus-dependent spatial memory impairments in male CHH rats than in their female counterpart.+ cells can be expressed in neurons 21 days after BrdU incorporation . In both female and male rats, CHH exposure can reduce PON2 protein expression. Nevertheless, PON2 protein expression in the female CHH group was significantly higher than in the male CHH group (P < 0.05). Clinical and experimental evidence shows that oxidative stress and inflammation are the most important contributors to cognitive impairment and neuronal damage at high altitudes  receptors  of CHH-exposed rats. HO-1 is particularly sensitive to the induction of cytokines, oxidants, and other stressors  and nNOSeceptors . NMDA reeceptors . Elevateeceptors .As an anti-apoptotic protein located in the outer membrane of mitochondria, Bcl-2\u2019s overexpression in neurons can inhibit neuronal apoptosis by maintaining mitochondrial integrity . In ratsNMDA is composed of different subunits such as NR1, NR2, and NR3, while NR1, NR2A, and NR2B are highly expressed in the hippocampus . NR2A orCompared with sham-CHH rats, OVX-CHH rats showed higher levels of oxidative stress and proinflammatory cytokines. Compared with sham-CHH rats, the expression of nNOS, HO-1, and Bax protein increased whereas the expression of PON2 and Bcl-2 protein decreased. The expression level of PON2 in women is usually significantly higher than that in men , and theThis study is not exempt from limitations of its own. First, the animals housed at high altitudes were transported back to standard conditions at plain to perform euthanasia rather than being euthanized at high altitudes. Moving the rats to standard conditions may likely affect the gene expression in these animals. Second, due to the lack of clearly defined specific substrates and standardized measurement techniques of these enzymes, the tissue distribution of PON2 could not be displayed by histopathological methods such as immunohistochemistry . FinallyIn this study, male rats seem to be more vulnerable to the effect of hippocampal damage induced by chronic exposure to hypobaric hypoxia, which might be related to the low level of paraoxonase 2 (PON2) expression. Studying the sex differences in hippocampal damage and cognitive impairment caused by hypobaric-hypoxia may help explore the mechanism underlying the sex differences that hypobaric-hypoxia destroys hippocampal integrity and leads to cognitive impairment, which may be very important for the understanding of precision medicine.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University, Chengdu, China. Written informed consent was obtained from the owners for the participation of their animals in this study.FG conceptualized the project. FG and DZ designed the study and drafted the manuscript. DZ, MZ, BH, YW, and LW contributed to the literature search, data collection and analysis, and data interpretation. FG critically revised the manuscript. All authors approved the final version of the manuscript."}
+{"text": "Facility cultivation has been evolved from greenhouses to smart farms using artificial intelligence (AI) that simulates big data to maximize production. However, the big data for AI in smart farm is not studied well; the effect of differences among varieties within a crop remains unclear. Therefore, the response of two varieties of blueberry, \u2018Suziblue\u2019 and \u2018Star\u2019, to light was tested using SAPD meter in order to demonstrate the environmental responses could be different among varieties within the same species. The results showed that those two varieties had significant differences in SPAD values based on the leaf\u2019s position and time, whereas \u2018Star\u2019 did not. This indicates that the effect of light depends on the variety, which implies that other traits and other crops may show similar differences. These results are based on a simple experiment. However, it is enough to elucidate that it is extremely important to characterize responses to the environment not only for each crop but also for each variety to collect data for smart farming to increase accuracy for modeling; consequently, to maximize the efficiency of these facilities. Smart farming is a farm management concept developed to overcome the current challenges of food production caused by the lack of an adequate workforce in rural areas owing to the urban concentration of the population . The focThe light was selected from among the many environmental factors because it is one of the most variable and significant factors that affects crop growth and yield ,10. BlueBlueberry \u2018Star\u2019 and \u2018Suziblue\u2019 were cultivated in Gosan, Jeju Island. Those two were chosen because they have different genetic background. These cultivars were grown in a rain-shelter house for five years. The SPAD value was measured in 15 replicates at sunrise (8 AM), full sun (1 PM), and sunset (6 PM) for the two cultivars planted in the houses. In addition, SPAD value was measured depending on the position of the foliage directly in contact with sunlight. Leaf position was divided into three classes as high (tree height ratio > 0.9), middle (> 0.5), and low (> 0.1).The SPAD-502 chlorophyll meter  can measure light intensity transmitted through the leaves at two wavelengths (650 and 940 nm) in a non-destructive manner. The SPAD reading value is calculated from the logarithm of the transmittance at 650 nm related to that at 940 nm .Data analysis was performed with the R programming language was used for performing these tests . The data sets were checked for normality using the Shapiro-Wilk test . BecauseSignificant differences were observed in the SPAD value of the leaves at different times and positions in each cultivar . NotablyLeaves of the shrub-like blueberry can be divided into sun leaves, and shade leaves based on the position and distribution of the leaves. In general, shade leaves tend to be thick with a thin cuticle and short palisade cells, whereas sun leaves are thin and have a thick cuticle and long palisade cells or form several layers of palisade cells . In the SPAD value is based on two factors which are the transmission of red light at 650\u2009nm and the transmission of infrared light at 940\u2009nm . Since rVaccinium darrowii [V. darrowii, an evergreen tree in the genome of \u2018Suziblue\u2019.The experiment in the current study had 15 replications per cultivar with no significant difference among those replications based on ANOVA . Based odarrowii . Unlike darrowii . This apThe fact that SAPD values in two different cultivars are different has the following implications for the smart farm system. First, the results can contribute to the decision-making for light addition in the smart farming system. Additional light is necessary for Suziblue, whereas it is not in \u2018Star\u2019. This information could be very helpful in reducing costs for setting up a smart farm system. Second, this hypothesis can be applied to other traits in other crops.Smart farm is evolving with development of artificial intelligence (AI). When AI is applied, it needs big data. However, big data could be useless if it does not fit to statistical analysis which is fundamental of AI; mixture of various data results in low R-squares. This would lead not accurate and not precise results for controlling environments. In the current study, light is one of the most important factors for photosynthesis which is directly associated with yield. The other parameter, time, has different value of light intensity which may result in different responses in two different cultivars. If those factors affect the SPAD value, it could be assumed that it would affect photosynthetic efficacy. Thus, it is here presented that the light responses of different cultivars are different using SAPD values although they are the very same species. There could be due to several reasons such as different genetic background of parents for each cultivar. No matter why it is so, if there is a difference between two varieties within the same species, it should be carefully treated no matter why it is so. Based on this, we suggest that variety-specific studies not crop-specific ones are necessary for crop modeling for smart farming. It would be great that this study will encourage various variety-specific studies for crop modeling to provide the proper big data sets to assist AI operation in a smart farm system."}
+{"text": "To explore the potential mechanism of triptolide in diabetic nephropathy (DN) treatment using network pharmacology.The main targets of triptolide were screened using the TCMSP, DrugBank, and NCBI databases, and gene targets of DN were searched using the DrugBank, DisGeNET, TTD, and OMIM databases. All of the above targets were normalized using the UniProt database to obtain the co-acting genes. The co-acting genes were uploaded to the STRING platform to build a protein-protein interaction network and screen the core acting targets. Gene ontology and Kyoto encyclopedia of genes and genomes analyses of the core targets were performed using Metascape. Molecular docking validation of triptolide with the co-acting genes was performed using the Swiss Dock platform.We identified 76 potential target points for triptolide, 693 target points for DN-related diseases, and 24 co-acting genes. The main pathways and biological processes involved are lipids and atherosclerosis, IL-18 signaling pathway, TWEAK signaling pathway, response to oxidative stress, hematopoietic function, and negative regulation of cell differentiation. Both triptolide and the active site of the core target genes can form more than 2 hydrogen bonds, and the bond energy is less than -5kJ/mol. Bioinformatics analysis showed that triptolide had a regulatory effect on most of the core target genes that are aberrantly expressed in DKD.Triptolide may regulate the body\u2019s response to cytokines, hormones, oxidative stress, and apoptosis signaling pathways in DN treatment by down-regulating Casp3, Casp8, PTEN, GSA3B and up-regulating ESR1, and so forth. DN is characterized by varying degrees of proteinuria and a rapid decline in renal function in the later stages, and is currently treated mainly with symptomatic treatments such as glycemic control, lipid regulation, and edema reduction.Diabetic nephropathy (DN) is a major microvascular complication of diabetes mellitus (DM) and the leading cause of end-stage renal disease worldwide. Clinical experience has shown that TP could reduce proteinuria in DN, stabilize renal function, and have better efficacy than renin angiotensin blockers. However, the pathogenesis of DN is complex, and there are many targets of TP action; therefore, the specific mechanism and drug mechanism of TP in treating DN are not explicit.Tripterygium wilfordii hook f., a traditional Chinese herbal medicine, can dispel wind and dampness, promote blood circulation, relieve pain, and activate blood circulation to reduce swelling. Triptolide (TP) is an epoxidized diterpene lactone compound isolated from Tripterygium wilfordii, also known as triptolide and triptolide alcohol, and is one of the main active ingredients of Tripterygium wilfordii and its toxic component. Recent studies have found that TP has anti-inflammatory, anti-tumor, and immunosuppressive effects and is widely used in tumor, rheumatological, cardiovascular, and renal diseases. and Hopkins formally proposed the definition of network pharmacology in 2007. By 2020, the total number of relevant articles published on the China Knowledge Network and PubMed reached 1944 in 1 year, which has become a popular tool for research on the basis and mechanism of traditional Chinese medicine pharmacology. This study aimed to analyze the targets and mechanisms of the action of TP in treating DN by network pharmacology and conduct molecular docking validation to provide a theoretical basis and experimental direction for subsequent cell and molecular experiments and promote its clinical application.Network pharmacology is a new discipline that selects a specific signal and designs multitarget drug molecules. Barab\u00e1si first proposed the concept of network biology in 2004,TCMSP, NCBI, DrugBank, DisGeNET, TTD, OMIM, Uniprot, Venny 2.1, Cytoscape 3.9, String platform, Metascape, Auto Dock Vina, GEO database, and so forth.The targets of TP were searched in the TCMSP, DrugBank, and NCBI databases with \u201cTriptolide\u201d as the search term, and the species was limited to \u201cHomo sapiens.\u201d The targets of DN were searched in the DrugBank, DisGeNET, TTD, and OMIM databases with \u201cdiabetic nephropathy\u201d as the search term. All the above targets were gene normalized using the UniProt database, and the drug targets and disease targets were integrated and de-weighted to obtain the TP and DN target sets.A PPI network was built using the string platform. The co-acting targets of TP and DN were collected using Venny 2.1 software. These were imported into the String platform to work a PPI network, set the protein species to \u201cHomo sapiens,\u201d hide the isolated proteins in the network, and the other parameters were kept at the default settings. The PPI network graph data were exported in TSV format. The data were imported into Cytoscape, and a network analyzer was used to analyze and calculate the network topology characteristic value of the intersection PPI and filter the data to obtain the core acting targets. Simultaneously, the protein functional sub-modules were determined by further analysis of the PPI network using the MCODE plug-in.The Metascape platform integrates multiple authoritative databases and supports annotation, enrichment analysis, and construction of PPI networks for batch genes or proteins, with more powerful functions, timely updates, and reliable data. The core acting targets were analyzed using the Metascape database for gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The results were saved and passed through R software for visualization.Molecular docking is an effective method to verify the relationship between molecules and targets. Molecular docking was performed using TP as the ligand and the core targets of TP for DN treatment as the receptor. The 3-dimensional structure of TP was downloaded from PubChem CID, and the core acting target structures were downloaded from the PDB database and routinely processed using Chem-Bio Drew. Then, Pymol software was used to remove redundant protein structures, remove other irrelevant ligands such as water molecules, and import Auto Dock Vina software to select the semi-flexible docking mode with docking parameters as default. A binding energy of less than 0 kJ/mol indicated that the ligand molecule could spontaneously bind to the receptor protein. And the binding energy less than \u20135.0 kJ/mol indicates good binding, and the smaller the binding energy, the better the docking.Most of the therapeutic effects of drugs on diseases are achieved by directly or indirectly regulating the genes that are aberrantly expressed in the disease. Diabetic Kidney Disease and Triptolide were used as the key words to search in the GEO database, and the species was limited to \u201cHomo sapiens.\u201d Appropriate datasets were selected for bioinformatics analysis to clarify the abnormal expression of each co-acting target in DKD, as well as to clarify the effect of TP intervention on gene expression of co-acting targets. If the expression changes of the same gene are opposite in these 2 cases, there is a high degree of certainty that TP will work through that gene; the opposite is less certain.A total of 76 drug target genes, 693 disease target genes, and 24 target gene intersections were identified Fig. .The co-acting targets were uploaded to the STRING platform to obtain the PPI network map. The results showed that there were 24 nodes in the network, with 220 interactions, and the average degree value was 18.3. Seven genes, including vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF), Interleukin 6 (IL6), signal transducer and activator of transcription 3 (STAT3), cellular tumor antigen p53 (TP53), estrogen receptor (ESR1), and Transcription factor AP-1 (JUN), were associated with the highest degree value of 22; the C3 gene had the lowest degree value of 4. By combining the average degree value and median degree value, genes with degree values greater than 15 were selected as the core acting targets of TP for DN treatment. The intersection network diagram is shown in Figure Gene enrichment analysis was performed using the Metascape platform, including KEGG pathway analysis, biological process, cellular component, and molecular function GO analysis.R. The bubbles from red to green represent the -Log10 (P) values from large to small; the bubble area represents the number of genes in the pathway, and the horizontal axis represents the ratio of genes in the pathway to all genes. The results are shown in Figure After KEGG pathway analysis, the top 20 genes of Homo sapiens were sorted according to the number of enriched genes. The bubbles were plotted in After GO analysis, the top 20 results with the -Log10(P) value were selected for visualization, and biological process, cellular component, and molecular function were plotted in bar graphs with the bar color from dark to light representing the -Log10(P) values from large to small and the bar length representing the gene count of that pathway. The results are shown in Figure The active sites of the core acting targets with a higher degree were able to form more than 2 hydrogen bonds, and the binding energies were all less than -5 kJ/mol, indicating good binding activity. Among them, the binding energy of tretinoin with signal transducer and activator of transcription1 was the lowest, at -7.83 kJ/mol, with the most active binding ability. Details are presented in Table 2FC from large to small, that is the expression difference is from large to small. The results are shown in Figure The GSE30122 dataset was selected for bioinformatics analysis to clarify the aberrant expression of co-acting targets in DKD, and the GSE93206 dataset was selected for bioinformatics analysis to determine the effect of TP intervention on the expression of co-acting targets by condition qualification. Red indicates gene up-regulation, blue indicates gene down-regulation, and the color from dark to light represents the absolute value of Log However, the pathogenesis of DN is not yet clear, and the treatment is not comprehensive enough.By 2019, 463 million adults aged 20 to 79 years worldwide had DM, approximately 15% to 25% of patients with type I diabetes and 30% to 40% of patients with type II diabetes developed renal complications and may progress to end-stage renal disease.,8 It has been found that VEGF expression levels are significantly higher in renal epithelial cells and distal tubular epithelial cells in DN patients compared to the general population, which may be associated with increased renal vascular permeability leading to proteinuria. TNF is produced by monocytes and macrophages during acute immune responses and can promote cell proliferation, differentiation, cytokine production, apoptosis, and necrosis; a close relationship between TNF and the development of DM has been demonstrated. IL6 is one of the most important inflammatory cytokines, while IL6 can bind to specific receptors to stimulate the Janus kinase/signal transducer and activator of transcription signaling pathway, plays a vital role in peripheral metabolic organs such as the adipose, pancreas, and immune system, and is a new potential therapeutic target for diabetes. STAT3 can affect cellular autophagy through different subcellular localization patterns. For example, cytoplasmic STAT3 inhibits cellular autophagy by sequestering eukaryotic initiation factor 2-\u03b1 kinase 2 and other signaling molecules associated with autophagy.\u201316 In addition, mitochondrial translocation of STAT3 inhibits oxidative stress-induced autophagy and may effectively protect the mitochondria from mitotic degradation.\u201319 TP53 encodes proteins that respond to various cellular stresses and regulate the expression of target genes to induce cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes\u201322; for example, by downregulating the anti-apoptotic gene product Bcl-2, upregulates the pro-apoptotic gene product Bax, and other pathways to participate in the regulation of the apoptosis pathway. The ESR1 inhibits nuclear factor kappa B (NF-\u03baB) -mediated transcription of the IL6 promoter by reducing NF-\u03baB DNA-binding activity and displaces RELA/p65 and associated co-regulators from the promoter, thereby regulating inflammation, immunity, and stress responses. AP-1 (AP1), a transcription factor involved in JUN, is a recognized integrator of extracellular signals,26 and plays a vital role in the treatment of various inflammatory lesions, transplant rejection, fibrosis, and organ damage. The remaining core target genes were mostly associated with inflammatory responses, cellular autophagy, immunity, oxidative stress, apoptosis, cellular senescence, and tumors.In the PPI network analysis in this study, VEGFA, TNF, IL6, STAT3, TP53, ESR1, and JUN were tied for first place with a degree value of 22. VEGFA regulates vascular permeability and it is a key regulator of angiogenesis during the growth of solid tumors.A total of 109 KEGG pathways were obtained in this study, and the enrichment results showed that TP treatment of DN mainly involved the following types of signaling pathways: DM-related, immune-related, and cell survival-related pathways.\u03baB and causes the expression and release of large amounts of IL6 and TNF, which eventually causes chronic cell activation and tissue damage.,29 In podocytes, the AGE-RAGE signaling pathway can stimulate VEGF, which increases vascular permeability and causes proteinuria. It also stimulates the production of TGF-\u03b21, which leads to glomerular extracellular matrix production and tubular epithelial mesenchymalization\u201333; induces enhanced MCP-1 expression and promotes glomerulosclerosis and tubulointerstitial fibrosis; stimulates angiotensin II production or overexpression at renal component AT1 receptors to accelerate the progression of DM; AGE-RAGE signaling pathway activates nicotinamide adenine dinucleotide phosphate oxidase, which in turn activates mitogen-activated protein kinase, extracellular regulated protein kinases, extracellular signal-regulated kinase [ERK1/2] ERK1/2 and P38 and other mediated signaling pathways via reactive oxygen species, resulting in the activation of NF-\u03baB by phosphorylation.\u201338 TP may reduce renal damage and decrease urinary protein levels in DN patients through the AGE-RAGE signaling pathway and has a positive effect on blood glucose in DN patients through insulin resistanceDM-related pathways include the AGE-RAGE signaling pathway in diabetic complications and insulin resistance. The AGE-RAGE signaling pathway activates NF-\u03baB signaling pathway. The IL-17 signaling pathway can activate NF-\u03baB and induce NF-\u03baB-dependent cytokines to upregulate inflammatory gene expression; IL-17 has pathogenic functions in immune-mediated glomerular diseases. IL-17 has also been found to limit the growth of fungi in the kidney, prevent kidney tissue damage, and protect kidney function during the transmission of candidiasis through the kallikrein (Klk) Kallikrein- Kinin system (KKS). The Toll-like receptor signaling pathway  is one of the earliest determinants of immune activation, which can activate TGF-\u03b21, TNF, AT1, NF-\u03baB, etc, through cellular exogenous, intrinsic, and specific responses,43 to induce a series of immune responses and thus have an impact on DN. Th1 and Th2 cell differentiation is influenced by various factors, such as antigen type and concentration, co-stimulatory molecules, cytokine concentration, immunoreactive hormones, transcription factors, and type of antigen-presenting cells. Disruption of the Th1/ Th2 balance can induce a range of autoimmune diseases and adversely affect pancreatic \u03b2-cells, thereby aggravating the condition of DN patients. The NF-\u03baB signaling pathway includes 3 main pathways: the canonical, alternative, and atypical pathways. After activation, it can combine with other pathways, such as the TILs signaling pathway, to cause inflammation and fibrosis in the kidneys.,46 TP may treat DN by inhibiting local inflammation and excessive immunity in the kidney through the IL-17 signaling pathway, Toll-like receptor signaling pathway, Th1 and Th2 cell differentiation, NF-\u03baB signaling pathway, etc.Immune-related pathways include the Interleukin 17 (IL-17) signaling pathway, the Toll-like receptor signaling pathway, Th1 and Th2 cell differentiation, and the NF-\u03baB, PI3K-Akt, mTOR, and IL-17 signaling pathways. The TNF signaling pathway induces podocyte apoptosis by inducing overexpression of retinoic acid receptor responder 1 in podocytes. While mediating renal inflammation and fibrosis, the NF-\u03baB signaling pathway also aggravates renal injury and proteinuria by inducing apoptosis through oxidative stress. The PI3K/AKT signaling pathway is related to proliferation, differentiation, and apoptosis. It connects downstream mTOR signaling to the PI3K-AKT-mTOR pathway, which can induce autophagy and increase the renal oxidative stress response.,50 The mTOR pathway can also exacerbate glomerulosclerosis by promoting DN mouse thylakoid proliferation in combination with other factors and pathways, which has been confirmed by in vitro experiments on human renal thylakoid cells. In addition, the IL-17 signaling pathway activates the mitogen-activated protein kinase pathway, which includes extracellular signal-regulated kinase, p38, and JUN N-terminal kinase, to induce apoptosis. TP may reduce renal injury and decrease proteinuria by reducing apoptosis and autophagy in renal cells by inhibiting the TNF, NF-\u03baB, PI3K-Akt, mTOR, and other signaling pathways.Cell survival-related pathways include the TNF, apoptosis, NF-Analysis of the retrieved datasets found that TP inhibited the overexpressed CASP3, CASP8, CCNE1, CXCR4, GSK3B, HSPA5, Janus kinase 2, NFE2L2, PLAU, PTEN and PTGS2 in DKD, while upregulating ESR1 and TGFB1, which were repressed in DKD, promoting each gene restores its proper expression level, thereby treating DKD. The expression trends of some other genes under TP intervention were consistent with the trends in DKD, suggesting that TP is less likely to treat DKD through these genes. However, human is a complex whole, and the pathways and organismal responses associated with each gene are complex and variable. Therefore, a consistent trend does not mean that regulating the expression of these genes is ineffective, and further experimental verification is still needed. In addition, different disease stages, disease states, and individual responses to gene expression may also vary widely, and the effects of drugs on them will also be different. Perhaps this can be used to explain the poor efficacy of some patients in clinical practice.At present, the construction of a drug-disease molecular network is rarely reported in DN. The application of molecular network science can help to further reveal the mechanism of TP for DN treatment and provide direction for further research on pharmacological, toxicological, and pharmacodynamic substance bases. In this study, the mechanism of TP in the treatment of DN was predicted based on network pharmacology and molecular docking, and it was found that the mechanism was mainly related to the reduction in the inflammatory response, immune suppression, inhibition of apoptosis, and inhibition of glomerulosclerosis. In addition, TP may have ameliorating effects on insulin resistance. However, this study only predicted the mechanism of TP for DN from the available internet data, lacking experimental and clinical validation, and failed to clarify the treatment protocol for the prescribed dose and duration of TP for DN. Therefore, further experimental validation and validation in a large sample, multicenter, randomized, controlled clinical trials are still needed.YG designed the study and wrote the paper. YL and ZG performed the research and analyzed the data.Conceptualization: Ying Gao.Data curation: Yingying liu.Formal analysis: Ying Gao, Zhaoan Guo.Writing \u2013 original draft: Ying Gao, Zhaoan Guo."}
+{"text": "Mammalian adipose tissues are broadly divided into white adipose tissue (WAT) and thermogenic fat tissue (brown adipose tissue and beige adipose tissue). Uncoupling protein 1 (UCP1) is the central protein in thermogenesis, and cells that exhibit induced UCP1 expression and appear scattered throughout WAT are called beige adipocytes, and their induction in WAT is referred to as \u201cbeiging\u201d. Beige adipocytes can differentiate from preadipocytes or convert from mature adipocytes. UCP1 was thought to contribute to non-shivering thermogenesis; however, recent studies demonstrated the presence of UCP1-independent thermogenic mechanisms. There is evidence that thermogenic fat tissue contributes to systemic energy expenditure even in human beings. This review discusses the roles that thermogenic fat tissue plays in energy consumption and offers insight into the possibility and challenges associated with its application in the treatment of obesity and type 2 diabetes. Mammalian adipose tissues are broadly divided into two types: white adipose tissues (WAT) and thermogenic fat tissue, the latter of which consists of brown adipose tissue (BAT) and beige adipose tissue. WAT comprises white adipocytes that possess a large unilocular lipid droplet, and its role is to store excess energy in the body in the form of neutral fat (triglyceride) . By contUncoupling protein 1 (UCP1), which is expressed in the inner mitochondrial membrane, plays a pivotal role in the thermogenic mechanism of brown adipocytes. UCP1 is activated through stimulation of the sympathetic nervous system, dissipates the proton gradient generated by the electron-transport chain, and releases energy as heat . This th18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT), it was revealed that human adults also possess physiologically active brown adipocytes in the neck region [In human beings, brown adipocytes were previously detected only in neonates, resulting in the assumption that they undergo atrophy or have no physiological significance in adulthood . Howeverk region ,8. Throuk region ,10, sugg2 [Obesity is defined as excessive fat accumulation and is diagnosed at a body mass index \u226530 kg/m2 . Obesity2 . Type 2 2 ,13,14. TThis review discusses the roles that thermogenic fat tissue plays in energy consumption and offers insight into the challenges associated with its application in the treatment of obesity and type 2 diabetes.Classical brown adipocytes differentiate from muscle progenitor cells expressing myogenic factor 5 (MYF5). Progenitors expressing paired box 7 (PAX7) and engrailed-1 also differentiate into either brown adipocytes or muscle cells. A previous study reported that these progenitors occur at embryonic days 9.5 to 11.5 in mouse embryos . While c+ Sca1+ Cd81+ cells have been identified as beige progenitor adipocytes [Beige adipocytes differentiate from MYF5-negative cells. There are two hypotheses on the origin of beige adipocytes . One is ipocytes . Moreoveipocytes . SMA is ipocytes , this in3-adrenergic receptor agonists are said to be the external stimuli, and when mice were exposed to 6 \u00b0C, approximately 50% of beige adipocytes were derived from existing adipocytes [On the other hand, an in vitro study reported that mature adipocytes can undergo conversion to beige adipocytes . Cold exipocytes . It is sipocytes . This isipocytes .A recent study using single-cell RNA-seq of adipose tissue revealed dipeptidyl peptidase 4 (DPP4) as an adipose stem cell marker . DPP4-poA well-known regulatory factor of beige-adipocyte differentiation is the transcription factor PR domain-containing 16 (PRDM16), which was previously known as a factor that regulates brown-adipocyte differentiation . PRDM16 In the late 1970s, a study demonstrated that cell respiration  in mitochondria is the main determinant of non-shivering thermogenesis . Subsequ3-adrenergic receptor activates adenylate cyclase, resulting in elevation of cyclic adenosine monophosphate levels and activation of protein kinase A (PKA) [UCP1-dependent thermogenesis occurs when the sympathetic nervous system is activated, which in turn results in norepinephrine secretion . Norepin A (PKA) . PKA act A (PKA) ,35.Several studies reported an association between UCP1-dependent thermogenesis and glucose metabolism. BAT-deficient mice created by transgenic expression of diphtheria toxin under Ucp1-promoter regulation showed obese and diabetic phenotypes . Additio3 adrenergic receptor stimulates insulin secretion and contributes to the facilitation of lipid uptake into activated brown adipocytes [Glucose is taken up into the cells through GLUT1 or GLUT4. This glucose is used for glycolysis via glucose-6-phosphate, and pyruvate subsequently moves into mitochondria . Citrateipocytes . Of noteipocytes . This stUcp1-knockout mice created using pure C57BL/6J mice can also maintain body temperature under long-term cold stimulation [Until recently, only UCP1 was thought to contribute to non-shivering thermogenesis; however, UCP1-independent thermogenic mechanisms have subsequently been demonstrated. A report suggested that Ucp1-knockout F1 mice created by crossing C57BL/6J mice with 129/SvlmJ mice were able to maintain body temperature under cold stimulation . Additiomulation . Therefo2+ cycling mediated by sarco-endoplasmic reticulum ATPase (SERCA) (2+ in the cell. Ca2+ flows into the cytoplasm through the ryanodine receptor (RyR) and inositol 1,4,5-triphosphate receptor (IP3R) located on the endoplasmic reticulum membrane. Subsequently, Ca2+ uptake into the endoplasmic reticulum again by SERCA results in production of adenosine diphosphate (ADP). Skeletal muscles are reported to have the ability of non-shivering thermogenesis by Ca2+ cycling [2+ cycling, particularly in thermogenesis in beige adipocytes. SERCA activity contributes to thermogenesis [One UCP1-independent thermogenic mechanism is thought to involve Ca (SERCA) . The end cycling , and an  cycling . Recentl cycling . Further cycling , suggestogenesis , so SERC2+ cycling and binds to SERCA to facilitate ATP hydrolysis. In contrast to mice capable of maintaining body temperature under cold stimulation following the removal of brown adipocytes, Sln-deficient mice were unable to maintain body temperature when brown adipocytes were removed [Sln-deficient mice become obese when fed a high-fat diet [2+ cycling regulators in beige adipocytes.Sarcolipin (SLN) is potentially involved in Ca removed . Based ofat diet , these ffat diet . Future Creatine cycling represents a different type of UCP1-independent thermogenic mechanism. Creatine is involved in energy production, particularly in muscles, during rapid movement. Specifically, phosphocreatine reacts with ADP to synthesize creatine and ATP. A previous study showed that internal respiration in mitochondria mediated by creatine cycling was facilitated in the beige adipocytes of mice reared under cold stimulation . Additio+) outflow from mitochondria is considered another UCP1-independent thermogenic mechanism and was suggested by the observation that increased H+ outflow increases oxygen consumption by mitochondria [+ outflow in UCP1-deficient mitochondria [The influence of proton (Hchondria . It is cchondria .Ucp1-knockout mice with enhanced function of AMP-activated protein kinase (AMPK), which is involved in energy production [Another study reported beiging in subcutaneous adipose tissue of oduction . Interes3-adrenergic receptor agonists [3-adrenergic receptor following administration of PPAR\u03b3 agonists during the first three days rather than across the entire period of adipose-tissue differentiation (seven days) [In parallel with the elucidation of both UCP1-dependent and -independent mechanisms for energy consumption, many factors have been reported that induce thermogenic adipocytes. PPAR\u03b3 agonists  induce beige adipocytes. Rosiglitazone stabilizes PRDM16 expression , and an agonists , suggesten days) , indicatFibroblast growth factor 21 (FGF21) also induces beige adipocytes. Although FGF21 is a hormone secreted by the liver during hunger, it is also secreted by brown and beige adipocytes under cold stimulation or \u03b2 adrenergic receptor stimulation, which induces UCP1 expression via activation of PGC1\u03b1 . FGF21 wIrisin, a myokine secreted by skeletal muscles, reportedly induced beige adipocytes during exercise. Irisin acts on white adipocytes and increases UCP1 expression through p38 mitogen-activated protein kinase and extracellular-signal regulated kinase signaling . GLUT4 iUCP1 transcription [Il6-knockout mice show decreases in STAT3 activity and the beiging potential of white adipocytes, this suggests that IL-6/STAT3 signaling might be a target for beiging.The cytokine interleukin-6 (IL-6) is also secreted by skeletal muscles during exercise, with a recent study showing increased IL-6 secretion from beige adipocytes rather than white adipocytes in human beings . This stcription . Given tGroup 2 innate lymphoid cells (ILC2s), which work in response to tissue damage and allergen exposure, also play a potential role in beige-adipocyte biogenesis. IL-33 reportedly stimulates ILC2s, resulting in the secretion of IL-5 and IL-13 , which pBmp7-knockout embryos do not express UCP1, and BMP7 reportedly promotes the beiging of WAT by increasing PGC1\u03b1 expression [Bone morphogenetic proteins (BMPs) may also play a role in inducing thermogenic adipocytes. pression . Anotherpression . Additiopression . Furtherpression . TherefoThe average mass of BAT is 50\u201370 g . BAT is Several studies have investigated the changes in glucose uptake activity in human BAT triggered by exposure to cold conditions. When young healthy individuals were exposed to a room temperature of 17 \u00b0C for 2 h per day over a period of six weeks, significant reduction in total fat mass, which correlated with glucose uptake in BAT, was observed . AnotherStudies have also shown the effect of cold-induced BAT activation on glucose metabolism. A study conducted in the U.S. showed that cold-induced BAT activation increased the clearance of plasma glucose . A crossThe next step is to determine the mechanism of BAT activation in human beings, since oral or intravenous medication would be easier for clinical use than exposure to cold conditions. However, the clinical use of most substances that activate thermogenic adipose tissue is controversial. The effect of FGF21 and its analogs on glucose metabolism has been investigated, but a recent report suggested that beiging was not influenced by the concentration of FGF21 circulating in the blood . MoreoveIrisin is reported to stimulate beiging in specific types of human adipocytes , and a pAlthough IL-6 reportedly contributes to beiging , a recenAnother theory is that food intake increases the body\u2019s total energy consumption, a phenomenon known as diet-induced thermogenesis; however, this was disproved by a recent study that did not observe an association between BAT activity and calorie intake in 102 human adults . ResearcThere are currently no drugs capable of inducing beiging available for the treatment of type 2 diabetes or obesity. Currently, PPAR\u03b3 agonists, specifically thiazolidine drugs represented by pioglitazone, are used for the treatment of type 2 diabetes. These drugs improve insulin resistance through two pathways. One is PPAR\u03b3 activation strongly facilitating fat accumulation in WAT, resulting in improved levels of ectopic fat in skeletal muscles and the liver under obese conditions. The other involves decreased secretion of tumor necrosis factor-\u03b1 and other inflammatory cytokines, the levels of which are elevated during obesity, resulting in the facilitation of adiponectin secretion. Although the administration of pioglitazone to human adipocytes facilitates beiging in vitro, to the best of our knowledge, there are no reports that oral administration of pioglitazone facilitates glucose uptake by human brown adipocytes. By contrast, a study reported decreased brown-adipocyte activity under cold stimulation in a group administered pioglitazone . Therefo3-adrenergic receptor stimulation, which strongly induces beige adipocytes, numerous studies have evaluated the use of mirabegron. One report from a study of women in their 20s with no underlying disease showed that long-term administration of mirabegron (4 weeks) increased the metabolic activity of brown adipocytes [With respect to \u03b2ipocytes . Anotheripocytes . It is cIn terms of associations with FGF21, sodium-glucose transport protein-2 (SGLT-2) inhibitor is being used for the treatment of type 2 diabetes, with the associated mechanisms involving inhibition of glucose absorption in renal tubules. Part of this mechanism also involves hypoglycemic effects that facilitate the breakdown of fat and reduce adipose tissue. The possible involvement of FGF21 in these activities was reported in a study using a mouse model . RecentlAdditionally, other reports suggest an association between glucagon-like peptide 1 agonists and beiging. Injection of liraglutide into rats fed a high-fat diet for 12 weeks induced the expression of markers of beige adipocytes in subcutaneous adipose tissue . MoreoveIt is also important to consider the side effects of these drugs. Specifically, PPAR\u03b3 agonists can cause fluid retention, and their use is not recommended in patients who have concomitant heart failure. Additionally, in the case of mirabegron, its use should be limited for patients with urinary retention and hypertension, and risks associated with dehydration and urinary tract infection need to be considered for SGLT-2 inhibitors. Future studies are required to identify drugs with higher efficacy and lower risks of adverse side effects.3-adrenergic receptor stimulation, with evidence that it improves blood glucose levels in human beings [18FDG-PET/CT also supports its feasibility for clinical application. It has been shown by more than 130,000 scans from nearly 50,000 patients that BAT activity detected by 18FDG-PET/CT is independently correlated with lower incidence of type 2 diabetes [18FDG-PET/CT remains the gold standard to reflect BAT activity.In this review, we discussed the role of thermogenic adipose tissue in increasing energy consumption and the factors potentially involved. Among the factors mentioned, the most applicable for clinical usage is \u03b2n beings . The facdiabetes . Howeverdiabetes , but sindiabetes , we thin18FDG-PET/CT has accelerated BAT studies in human beings, this imaging tool reportedly underestimates the actual content of BAT; the influence of BAT on energy consumption may therefore be larger than that reported [This review has limitations. First, many of the results are based on animal models. Regarding in vivo human studies, some reports showed a correlation between BAT activation and increased glucose uptake ,14; howereported . Moreovereported . There a3-adrenergic receptor stimulation currently has the highest potential for clinical usage because of evidence from human studies showing that activation of thermogenic adipose tissue leads to energy consumption. As a measurement, 18FDG-PET/CT is the best method to reflect the effect of energy consumption of thermogenesis fat. 18FDG-PET/CT is not perfect in terms of radiation exposure and underestimation, so it is necessary to identify other therapeutically applicable molecules. Recent studies showed that beige adipocytes are diverse in origin and thermogenic mechanisms, including UCP1-independent mechanisms. Future studies of this diversity in human beings may provide insights to improve clinical approaches.Among the factors that have been previously reported to induce thermogenesis, \u03b2"}
+{"text": "Amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) are neurodegenerations with evolutionary underpinnings, expansive clinical presentations, and multiple genetic risk factors involving a complex network of pathways. This perspective considers the complex cellular pathology of aging motoneuronal and frontal/prefrontal cortical networks in the context of evolutionary, clinical, and biochemical features of the disease. We emphasize the importance of evolution in the development of the higher cortical function, within the influence of increasing lifespan. Particularly, the role of aging on the metabolic competence of delicately optimized neurons, age-related increased proteostatic costs, and specific genetic risk factors that gradually reduce the energy available for neuronal function leading to neuronal failure and disease. Finely controlled fractionated muscle movement enables humans to perform complex activities that require precise voluntary execution of force and speed of movement . The anaBoth advanced cognition and a versatile motor repertoire were critical to the success of human evolution, which involved a rapid expansion of cerebral network connectivity occurring within the constraints of a bony cranium . The relSince the mid-nineteenth century, recognition of devastating diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), have emerged. These predominantly involve frontal and prefrontal neurons, but their pathology extends beyond these regions. Clinical, genetical, and biochemical features of these diseases converge on protein misfolding and metabolic dysfunction as common end points associated with impaired neuronal dysfunction .This article briefly (1) outlines the evolution of the frontal and prefrontal cortex with respect to communication and language, and motor function specific to humans; (2) considers the roles played by evolutionary changes, cerebral metabolism, and senescence in an era of increasing lifespan; (3) raises the concept of possible neuron exhaustion, with proteomic cost minimization as a selective force challenged with increasing age; and (4) links this idea to ALS and FTD.We hypothesize that these disorders and other neurodegenerations reflect in part a mismatch between evolved neocortical cellular and metabolic processes at a protein level, in the context of rapid and ever-increasing complexity of human interaction, and the relatively recent increased human lifespan. For conciseness, we do not attempt to detail genetic components of these diseases , nor do A unified etiology of ALS/FTD is proposed that implicates evolutionary optimized neurons, metabolically challenged by RNA/protein turnover in certain risk phenotypes leading to neuronal exhaustion and disease. We suggest that the hypothesis can be expanded with more data and point toward metabolism and protein turnover as potentially key targets for efficient treatment paradigms.The evolution of cognitive function and brain development has resulted from the complex interplay of nature and nurture, where development seems to be driven by genes and shaped by the environment , 10. ModLanguage probably evolved out of gesture as a protolanguage \u201320. GestComplex forms of communication, especially human language, defines one of the most difficult problems for evolutionary biology \u201323. LangThe basic layout of the larynx and vocal tract is highly conserved and virtually homologous in both form and function among all terrestrial mammals, including humans . Indeed,The greatly expanded corticomotoneuronal system with associated neo-cortical networks underlies finely tuned motor control of hand function , the ability to traverse uneven terrain, and for example, to skate, ski, and play professional football, and employ diversified vocalization enabling variable pitch, tone, velocity of speech and loudness, in a complex association with respiratory function , 30.Vocal cues are a rich source of information about a speaker's emotional state. The term \u201cprosody\u201d refers to the changes in pitch, loudness, rhythm, and voice quality corresponding to a person's emotional state \u201333. The The motor and premotor areas of the human and non-human primate cortex are engaged not only in preparation and execution of voluntary movement but also perform fundamental computations associated with executive function and other cognitive aspects of behavior . FurtherHumans have evolved a finely tuned pincer grip, utilizing the thumb and index finger. Impairment of this can be an early, unique feature of ALS, referred to as the split hand . SimilarIt has been hypothesized that the evolution of cognition increased the returns from cooperating to the point where the benefits to self were sufficient for cooperation to remain stable as the group size increased and the relatedness decreased , with thThrough evolution, hominin brain sizes smaller than homo sapiens remained stable at 400\u2013500 cc until about 2 million years ago . The humAt a micro level, the functional capacity of a neuronal structure is inherently limited by its neural architecture and signal processing time . An impoAs modern humans migrated out of the African sub-continent into the colder habitats, there was pressure to modify cerebral energy metabolism in a brain that was progressively increasing its metabolic demands in comparison to other body organs . Within Complex variations in the dietary intake associated with the habitat's wildlife and foraging options and diverse cultural and technological impacts  contribute to this evolutionary history , 53, 54.via various selection and dietary restriction experiments and endocrine pathway mutations  leading to aggregates , 67. MutThese cellular processes that maintain normal neuronal physiology throughout life are diverse, and exponentially fail with increasing age . Increasvia some specific molecular toxicity as larger aggregates  or as more recently accepted, in the intracellular oligomeric state, with suggestions including interaction with other proteins and cell membranes , and outside the cells, these deposits will be less likely to interfere with cellular functions, and also less costly as they would be less targeted (and less accessible) to the intracellular proteases . In studper se\u2014could be pathogenic. This is particularly relevant in the context of the most energy-demanding cells such as neurons, where the ATP costs of proteostasis would be first felt due to the large energy demands for inter-neuronal signaling via ion pumps (perhaps 50% of energy budget)  of misfolded protein turnover within the cells . This ef budget) .via reduced foraging needs and cognitive capacity.The energy costs of maintaining the proteome  defines 20\u201370% of the cellular energy budget of various organisms and thus has probably been under heavy selection pressure for minimization . First ivia a repeat expansion or an unstable protein product, could be envisioned to contribute ATP costs to neurons operating near maximal capacity, and possibly accelerating aging-induced deterioration of involved networks  was associated with aggravation of disease .At the clinical level, ALS has been associated with a hypermetabolic presentation that could suggest elevated metabolic costs during pathogenesis , 94, 101Frontal and prefrontal lobe diseases are predominantly disorders of the aging nervous system. With the recent increase in longevity, largely determined by adequate shelter, good nutrition, medical advances, and reduced mortality in early life, the incidence of these neurodegenerations has increased. Extended longevity in the recent generations is unlikely to simply reflect the Darwinian natural selection, nor the Hamiltonian inclusive fitness . As humaWe propose that protein aggregation, the hallmark of neurodegenerations such as ALS and FTD, occurs because of the increasing metabolic burden accompanying neuronal proteostasis. This in turn is a consequence of the intersection of the evolving human brain in response to evolutionary and environmental pressures, and increasing age, which over time leads to metabolic exhaustion of energy-demanding neocortical neurons .A possible mechanism for the protein aggregation lies in the energy costs of misfolded protein turnover, but other possibilities exist. While general proteasome inhibition is not a valid therapeutic strategy for these diseases, it does suggest that the burden of RNA/protein turnover could be a contributing factor in the etiology, consistent with the perspective provided earlier.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RH conceived the concept. KK created the Figure. All authors contributed to writing and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "COVID-19, the disease caused by SARS-CoV-2, has claimed approximately 5 million lives and 257 million cases reported globally. This virus and disease have significantly affected people worldwide, whether directly and/or indirectly, with a virulent pathogen that continues to evolve as we race to learn how to prevent, control, or cure COVID-19. The focus of this review is on the SARS-CoV-2 virus\u2019 mechanism of infection and its proclivity at adapting and restructuring the intracellular environment to support viral replication. We highlight current knowledge and how scientific communities with expertize in viral, cellular, and clinical biology have contributed to increase our understanding of SARS-CoV-2, and how these findings may help explain the widely varied clinical observations of COVID-19 patients. As of November 21st, 2021, over 257 million cases of coronavirus disease 2019 (COVID-19) have been reported, and more than 5 million lives claimed globally [Coronaviruses are named as such because the S proteins resemble a halo or corona on scanning electron microscope imagery . SARS-CoThe main virus-specific functions of the SARS-CoV-2 NSPs are: NSP1 - cellular mRNA degradation, global translation inhibition; NSP2 - cell cycle progression disruption; NSP3 - formation of double-membrane vesicles ; NSP4 - formation of DMVs; NSP5 - main SARS-CoV-2 protease; NSP6 - formation of DMVs, NSP7 - replication complex; NSP8 \u2013 primase; NSP9 - RNA binding protein; NSP10 - cofactor of NSP14 & NSP16; NSP11 - unknown, NSP12 - RNA-dependent RNA polymerase; NSP13 - RNA helicase, 5\u02b9 phosphatase, NSP14 - N7-MTase, 3\u02b9-5\u02b9 exonuclease; NSP15 \u2013 endonuclease; and NSP16\u20132\u02b9-O-MTase, mRNA capping.The remaining 1/3 of the SARS-CoV-2 genome encodes for the structural proteins S (spike), E (envelope), M (membrane), and N (nucleocapsid), and several open reading frames  [There are two notable differences between SARS-CoV-1 and SARS-CoV-2. First is the presence of the ORF8 polypeptide found in SARS-CoV-2 but not in SARS-CoV-1. SARS-CoV-1 has a 29 nucleotide (nt) deletion (del) which splits it into ORF8a and ORF8b. Second, SARS-CoV-2 contains a gene encoding a novel orphan protein, ORF10, which is not present in SARS-CoV-1 .\u22124 substitutions per site per year [SARS-CoV-2\u2019s evolutionary rate has been estimated to be around 9\u2019\u00d7\u201910per year , while aper year , and on-Mutations have been detected in all parts of the viral genome, including in the leader 5\u02b9 untranslated region (UTR), orf1ab , spike, ORF3a, ORF8, nucleocapsid, and ORF10 [Mutations associated with the spike are of particular interest, as they influence human-to-human transmission, as well as human-to-animal passage. Within the spike, mutations tend to fall into four general classes, those that affect the receptor-binding domain (RBD), which are of importance because some may provide both immune escape or a fitness advantage, as well as facilitate reverse zoonotic events. There are some mutations that occur in the N-terminal domain (NTD), which is the portion most exposed on the virus surface. There is evidence for immune selection in this region, and preliminary evidence that at least one of these changes (delH69/delV70) could improve fitness . MutatioThis large SARS-CoV-2 genome diversity has been categorized by different nomenclature systems, describing variants of varied public health interest or concern. Pango lineages B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) have been classified as \u201cvariants of concern\u201d (VOC) because they present mutations that have been shown to impact diagnostics, treatments, or vaccines, conferring increased transmissibility and increased disease severity. The impact of these mutations highlights the need for further research not only on the mechanisms of SARS-CoV-2 intracellular processes, but also how the extracellular environment may lead to further spread of the virus and subsequent public health burden.The SARS-CoV-2 virus can exert physiological effects by directly infecting cells and via intercellular signaling by the infected cells. In this review, we provide insight into SARS-CoV-2 infection and intracellular host responses  to viral invasion. We emphasize a canonical set of reactions induced by SARS-CoV-2, which we have organized for the reader\u2019s consideration. However, there is tremendous variation in cellular responses to SARS-CoV-2, depending on factors including the cell type, organ type, metabolic and physiological context, patient genetics, individual clinical characteristics , and stage/severity in the COVID-19 disease.This is the first of a three-part comprehensive series of linked reviews on SARS-CoV-2 covering: intracellular effects (present study); extracellular consequences (review 2); and current and potential therapeutics (review 3). This review and the two that will follow aim to provide a foundational understanding of the current knowledge on SARS-CoV-2, from basic biology to clinical outcome and therapy avenues, that highlight future areas of research and could help inform public health interventions across the world.SARS-CoV-2 targets the nasal cavity and lungs; however, the detailed cellular tropism remains unclear, and likely varies among individuals. Furthermore, there is increased variability of viral cellular tropism with the emergent SARS-CoV-2 variants, which include Alpha, Beta, Gamma, Epsilon, Eta, Iota, Kappa, 1.617.3, Mu, Zeta, and in particular, Delta and Omicron, as well as the various lineages of each variant . This isSeveral investigations have employed 3D organoid cultures to simulate more physiological conditions than cell cultures . In one In one study of post-mortem patients, immunohistochemistry and immunofluorescence revealed viral antigen (spike protein) in pneumocytes and hyperplastic cells around the bronchioles, mucosal epithelia, submucosal glands, gland ducts of the trachea, glands of the small intestine, distal tubules and collecting ducts of the kidneys, islets of Langerhans, glands and intra-islet ducts of the pancreas, and vascular tissues of the brain and heart . Few virSingle-cell RNA sequencing (scRNA-seq) demonstrated ACE2 receptor expression was primarily restricted to lung pneumocytes, gut absorptive enterocytes, and nasal mucosa goblet secretory cells . In geneHuman autopsy studies  have shoThese studies suggest that the virus has a varying range of expression within each organ, which may be influenced by levels of the ACE2-receptor and related entry factors  within each organ-type . This fuThe physiological status of the individual significantly affects COVID-19 morbidity and mortality , 26. NotThe cellular surface receptor ACE2, a key regulator of the Renin-Angiotensin Aldosterone System (RAAS). It is speculated to be the primary SARS-CoV-2 viral target for entry. SARS-CoV-2 is thought to infect multiple organs in part due to the widespread distribution, expression, and polymorphisms of ACE2 , 29.2+ concentrations. ACE2 also plays an important role in inactivating Des-Arg9-Bradykinin (DABK), a bradykinin involved in inflammation. This inactivation promotes C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIPS2), keratinocytes-derived chemokine (KC), and tumor necrosis factor-\u2329 (TNF-\u03b1) activity, drawing leukocytes into the affected tissues [ACE2\u2019s molecular function in the human RAAS pathway is to cleave Angiotensin I to produce Angiotensin 1\u20139, and break down Angiotensin II into Angiotensin 1\u20137. RAAS moderates blood pressure and osmolarity by means of hormonal feedback control. In response to binding of ACE2 to the ACE2 receptor (ACE2R), blood vessels vasoconstrict. This process is mediated by G-protein-signaling, activating phospholipase C and increasing cytosolic Ca tissues .Decreased ACE2 receptor expression can have detrimental effects. Computational models of COVID-19 suggest the role of a bradykinin storm in the pathophysiology of the disease. In this model, the Kallikrein-Kinin and Renin-Angiotensin-Aldosterone Systems are integrated, with cross-talk mediated by the degradation of bradykinin by ACE and prolylcarboxypeptidase . This beThe ~600-kDa trimeric S proteins can bind to ACE2 through the RBD required for membrane fusion  is essential for many aspects of cellular physiology and viral replication. Experimental data on the relation between Ca2+ signaling and SARS-CoV-2 infection and replication is sparse. However, studies of other coronaviruses  have reported that these viruses utilize Ca2+ for host fusion [2+ ion, while the SARS-CoV-2 spike (S) protein has two FP domains, FP1 and FP2, and binds to two Ca2+ ions for host cell entry [2+ homeostasis in ways that promote viral infection and reproduction  [2+ concentrations, resulting in virus-induced cell lysis [The calcium ion  , 37. Thill lysis , 37. Thell lysis .Fig. 3SA2+ by forming membrane channels, are a characteristic of a diversity of virus. SARS-CoV-1/2 each encode viroporins. SARS-CoV-1 encodes for three viroporin proteins ORF3a, E and ORF8b, which alter ion homeostasis within the cell, and have important roles in pathogenesis and promoting viral fitness. SARS-CoV-2 encodes two of these viroporin proteins, E and ORF3a; however, the ORF8 protein of SARS-CoV-2 is highly divergent from SARS-CoV-1 ORF8b and lacks the viroporin sequence of SARS-CoV-1 ORF8b.Viroporins, transmembrane pore-forming proteins that alter membrane permeability to ions including Ca2+ homeostasis in the host, by acting as calcium ion channels, enhancing the virion\u2019s entry and replication potential [2+ ions. The SARS-CoV-2-ORF3a protein is 274 aa in length, harbors three helical TMD, and is a Na+ or Ca2+ ion channel protein. The alteration of Ca2+ homeostasis by SARS-COV-1-E and SARS-COV-2-E proteins promotes SARS-CoV-1/2 fitness and elicits the production of chemokines and cytokines, contributing to pathogenesis. Ion channel activity modulation by the SARS-CoV-1-ORF3a protein also modulates viral release [2+ homeostasis may contribute to morbidity and mortality. COVID-19 patients have been noted to have low serum calcium levels overall [The E and ORF3a proteins of coronaviruses impact Caotential . The SAR release . Therefo overall .2+ dysregulation could lead to increased cellular oxidative stress and shifts in metabolic activity. Low Ca2+ may also be coupled with viral infection and internalization through the ACE2R, which synergizes with Ca2+ signaling pathways. Understanding these reverberations will increase our insight into the basic biology of the effects of SARS-CoV-2 infection on the various organ systems.We speculate that Ca2+ signaling. As infection proceeds, SARS-CoV-2 manipulates, or totally reprograms, the normal metabolism and signaling of the host cell, optimizing the molecular environment to enable the viral replication cycle. This involves interfering with signaling pathways that regulate processes of DNA repair and replication, immune response, transcription, metabolism, cell cycle, and apoptosis [Viral infection and hijacking of cell-surface receptors begin to trigger activation of multiple intracellular pathways in addition to Capoptosis .SARS-CoV-2 infection alters phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), Type I and III interferon, transforming growth factor-\u03b2 (TGF-\u03b2), Toll-like receptors (TLR), and nuclear factor kappa-light chain enhancer (NF-\u03baB) pathways. These pathways are dysregulated in the setting of SARS-CoV-2 to antagonize host antiviral responses and are vital for viral replication, entry, propagation, and/or apoptosis/viral release. For instance, severe COVID-19 is characterized by an inflammatory profile dominated by NF-\u03baB activity . The SARHost antiviral immunity requires an optimal and coordinated response to control viral infections; this immunity is mediated by several host sensors, notably pattern recognition receptors (PRR). PRRs identify damage- and pathogen-associated molecular patterns . SARS-CoV-2 infects the cell via the endosomal compartment, and may activate TLRs, such as TLR4, resulting in increased NF-\u03baB activity and expression , 43. TheInhibition of IFNs and ISGs is a tactic used by several viruses to evade host antiviral responses , and SARThe cytokine TGF-\u03b2 triggers the Janus kinases (JAKs) / signal transducer and activator of transcription (STAT) proteins (JAK/STAT) pathway in certain contexts, while suppressing it in others . It has The innate immune response is a first step to protecting against pathogens, which stimulate the interferon signaling pathway and expression of IFN-I, leading to an antiviral cellular response . CoronavViruses rely on host cell machinery to propagate, promoting anabolism for generation of macromolecules needed for virion replication and assembly see Fig.\u00a0. ConsequWe reported recently through study of COVID-19 patient samples  that heavy suppression occurs of mitochondrial functions in various organs . SpecifiSARS-CoV-2 infection of human monocytes  and humade novo purine synthesis intermediates are accumulated, intracellular glucose and folate are depleted, and lactate levels are elevated [Following host cell infection, the SARS-CoV-2 replication/transcription complex synthesizes ~30\u2009kb viral genomes as well as the subgenomic RNAs required to encode for viral structural and mechanistic proteins. Between 1\u20135\u2009h post-infection, the percentage of coronavirus-encoded protein per total cellular protein translation may increase by as much as 20,000 times, with the fraction of viral to cellular RNA ultimately reaching as high as 90% intracellularly . To accoelevated . This su2+ signaling and increased mitochondrial generation of ROS. How SARS-CoV-2 induces host cell nucleotide metabolism remains unanswered.Virus-infected cells also commonly exhibit the Warburg effect - increased glycolytic metabolism in the presence of inadequate oxygen for oxidative phosphorylation - to supply reducing equivalents and precursors for macromolecule biosynthesis, and to support generation of ATP needed for also increasing nucleotide and lipid biosynthesis. Metabolic shifts include dysregulated CaMitochondrial metabolism and function are highly impacted in multiple ways. With the shift towards glycolysis, there is a reduction in oxidative phosphorylation affecting the mitochondria and its function. SARS-CoV-2 may interact with the mitochondria to destabilize its oxidative phosphorylation capacity. Coronavirus replication requires the formation of double-membrane vesicles (DMVs) derived from endoplasmic reticulum (ER). These DMVs serve as a site for viral replication and help conceal the virus from host cellular defenses. Interestingly, mitochondrial stress is known to induce mitochondria-derived vesicles (MDVs) that communicate with the ER. It is conceivable that SARS-CoV-2 disruption of mitochondrial function results in the induction of (double-membrane) MDVs. SARS-CoV-2 RNA present in the mitochondria induces mitochondrial dysfunction. Increased DMVs can provide opportunity for viruses to hide and replicate .+/K+ Transporting Subunit Beta 1 (ATP1B1), ATPase H\u2009+\u2009Transporting V1 Subunit A (ATP6V1A), acyl-coenzyme A dehydrogenase (ACADM), Alpha-aminoadipic semialdehyde synthase (AASS), Peptidase, Mitochondrial Processing Subunit Beta (PMPCB), Pitrilysin Metallopeptidase 1 (PITRM1), Coenzyme Q8B (COQ8B), and Peptidase, Mitochondrial Processing Subunit Alpha (PMPCA); these proteins are each components of critical mitochondrial metabolic pathways. SARS-CoV-2-encoded ORF9b protein interacts and localizes with Translocase Of Outer Mitochondrial Membrane 70 (TOMM70) [SARS-CoV-2 and all subgenomic RNAs are enriched in the host mitochondria, and viral genome\u2019s 5\u02b9 - and 3\u02b9 -UTRs contain distinct mitochondrial localization signals , indicat(TOMM70) . These mAnother example of COVID-19\u2019s mitochondrial-related impacts is the over-production of cellular ROS . ROS andSARS-CoV-2 is thought to suppress the ROS-associated Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Nrf2 is a transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage. Dysregulation of the Nrf2 pathway will exacerbate the pro-oxidative stress caused by the virus . SARS-CoDue to the multiple effects of SARS-CoV-2 that alter cellular metabolic and oxidative states, there are multiple directions to deplete NAD+, loss of cellular ATP and reduced Poly-ADP-ribose polymerase (PARP) activity, each of which have cytotoxic effects of their own . In geneCellular metabolism adapts to the alterations induced by SARS-CoV-2 infection of the cell see Fig.\u00a0. These aATP production from oxidative phosphorylation, glycolysis, and other pathways is critical to support cellular physiology, but this molecule also has signaling properties, which can be particularly beneficial in epithelial cells . PerturbIn the endothelium, nitric oxide directly affects mitochondrial metabolism through interaction with cytochrome C, providing cytoprotection against free radicals. However, reduction of NO bioavailability, due to the increased oxidative stress state caused by SARS-CoV-2-elevated superoxides, results in the formation of peroxynitrites (ONOO-). The reduced NO diffusion to neighboring vascular smooth muscle may impair vascular function . PeroxynLike ATP, NO acts as a biological signaling molecule. This dissolved gas rapidly diffuses across cell membranes and regulates various functions across the body . The vasThe causative agent of the COVID-19 the pandemic, SARS-CoV-2, has caused loss of incomes, economic crises, morbidities, and loss of life worldwide. Here, we describe the virus and review state-of-the-art information about the processes it utilizes to enter and reprogram the human host machinery. We detail research on early infection using evidence from patient samples, organoids and cells, and non-human animal studies. Each of these has limitations but taken together provide unique observational and mechanistic insight on SARS-CoV-2 infection.COVID-19 is a pleiotropic condition. Viral insults and subsequent cellular metabolic adaptations differ in the context of cell-type, genotype and environmental influences. Thus, much of what we have presented applies to specific cell types and contexts, and we have attempted to cover these contexts.Key avenues of future research on SARS-CoV-2 infection and propagation include: 1) defining the mechanisms of how the virus enters cells, and the protein and receptor molecules that are critical to this process; 2) elucidating the dynamics of how protein machinery is captured and retrofitted for viral purposes in a cell-specific manner; 3) understanding how the host genetics and environment can affect the ability of the virus to infect; 4) understanding the impact of SARS-CoV-2 on glycolysis and oxidative phosphorylation; and 5) revealing how the mitochondria adapts to ultimately shift its physiology from steady-state.In the best-case scenario for the SARS-CoV-2 virus, infection leads to a cascade of intracellular adaptations in which multiple networks are remodeled, from transcription to metabolism to signal transduction, shifting the invaded host cell from its original physiology into a SARS-CoV-2 replication system, and causing the emission of new viral particles and signaling molecules. The subsequent disease events will reverberate across the body\u2019s cells and organs. This will be the subject of our Part 2 review (in preparation).Supplemental Table 1"}
+{"text": "Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is highly contagious and remains a major public health challenge despite the availability of effective vaccines. SARS-CoV-2 enters cells through the binding of its spike receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 (ACE2) receptor in concert with accessory receptors/molecules that facilitate viral attachment, internalization, and fusion. Although ACE2 plays a critical role in SARS-CoV-2 replication, its expression profiles are not completely associated with infection patterns, immune responses, and clinical manifestations. Additionally, SARS-CoV-2 infects cells that lack ACE2, and the infection is resistant to monoclonal antibodies against spike RBD in vitro, indicating that some human cells possess ACE2-independent alternative receptors, which can mediate SARS-CoV-2 entry. Here, we discuss these alternative receptors and their interactions with SARS-CoV-2 components for ACE2-independent viral entry. These receptors include CD147, AXL, CD209L/L-SIGN/CLEC4M, CD209/DC-SIGN/CLEC4L, CLEC4G/LSECtin, ASGR1/CLEC4H1, LDLRAD3, TMEM30A, and KREMEN1. Most of these receptors are known to be involved in the entry of other viruses and to modulate cellular functions and immune responses. The SARS-CoV-2 omicron variant exhibits altered cell tropism and an associated change in the cell entry pathway, indicating that emerging variants may use alternative receptors to escape the immune pressure against ACE2-dependent viral entry provided by vaccination against RBD. Understanding the role of ACE2-independent alternative receptors in SARS-CoV-2 viral entry and pathogenesis may provide avenues for the prevention of infection by SARS-CoV-2 variants and for the treatment of COVID-19. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), has remained a major challenge for public health since the first case was reported in December 2019. Although SARS-CoV-2 vaccines are widely available, fully vaccinated people are susceptible to variants ,4. The mbasigin or extracellular matrix metalloproteinase inducer (EMMPRIN), acts as an alternative receptor for SARS-CoV-2 entry into cells with low or undetectable ACE2 expression [CD147, known as pression ,39. CD14pression ,46. CD14pression ,51,52,53pression , which mPlasmodium falciparum [scid IL2Rgammanull) [CD147 is a functional receptor for various pathogens, including measles, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), SARS-CoV, Kaposi\u2019s sarcoma-associated herpesvirus (KSHV), and lciparum ,61,62,90lciparum . The datlciparum ,37,39, pmmanull) . In contmmanull) . We havemmanull) . Anti-CDmmanull) , which immanull) . CD147 hmmanull) . Thus, tanexelekto or uncontrolled) is a tyrosine-protein kinase receptor, initially named UFO for its unidentified function as a protein with oncogenic potential [AXL , zika virus, Ebola, Lassa virus, Marburg, Hantaan virus (HTNV), and Andes virus (ANDV) ,69,70,71Bohan et al. showed that co-transfection of ACE2 and AXL or other PS receptors (Tim1 and Tim4) promotes ACE2-dependent SARS-CoV-2 infection . E-64, aCD209L/L-SIGN/CLEC4M, CD209/DC-SIGN/CLEC4L, and CLEC4G/LESCtin are C-type lectins. These receptors function as cell adhesion molecules and pathogen receptors; they play an important role in immune responses ,106,107.Two independent groups identified CLEC4G/LSECtin as SARS-CoV-2 receptors using cell-based library screening approaches ,44. Zhu Hoffman et al. used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric spike and the RBD of SARS-CoV-2 . AnnotatLDLRAD3 and TMEM30A were also identified as ACE2-independent SARS-CoV-2 receptors through the genome-wide CRISPR activation screen . LDLRAD3Zhu et al. showed that, similar to CLEC4G, both LDLRAD3 and TMEM30A bind to the NTD of SARS-CoV-2 spike proteins . The funGu et al. identified several SARS-CoV-2 binding proteins using a high-throughput receptor profiling system. After screening 5054 human membrane proteins for interaction with the SARS-CoV-2 extracellular spike and Fc fusion protein, KREMEN1 and ASGR1 were found to serve as ACE2-independent SARS-CoV-2 receptors .KREMEN1 is expressed in various tissues, including the brain, esophagus, endocrine and reproductive tissues, and skin and is a negative regulator of Wnt signaling ,118. KREAsialoglycoprotein receptor-1  is a calcium-dependent C-type lectin receptor and is expressed primarily in hepatocytes . ASGR1 iKREMEN1 and ASGR1 interact with SARS-CoV-2 spike proteins but not with SARS-CoV spike proteins . Both reACE2 is an important and well-studied receptor for SARS-CoV-2 infection, but its distribution cannot explain SARS-CoV-2-mediated pathology. A number of alternative ACE2-independent receptors with broader distribution patterns may contribute to SARS-CoV-2 infection and pathogenesis. Indeed, the effects of alternative receptors on lung pathology have been demonstrated in SARS-CoV-2-infected mice. The molecular mechanisms of SARS-CoV-2 entry via alternative receptors are not always consistent and require further investigation . Most st"}
+{"text": "The constantly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) fuel the worldwide coronavirus disease (COVID-19) pandemic. The spike protein is essential for the SARS-CoV-2 viral entry and thus has been extensively targeted by therapeutic antibodies. However, mutations along the spike in SARS-CoV-2 VOC and Omicron subvariants have caused more rapid spread and strong antigenic drifts, rendering most of the current antibodies ineffective. Hence, understanding and targeting the molecular mechanism of spike activation is of great interest in curbing the spread and development of new therapeutic approaches. In this review, we summarize the conserved features of spike-mediated viral entry in various SARS-CoV-2 VOC and highlight the converging proteolytic processes involved in priming and activating the spike. We also summarize the roles of innate immune factors in preventing spike-driven membrane fusion and provide outlines for the identification of novel therapeutics against coronavirus infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. The initial case of SARS-CoV-2 was reported in Wuhan 2019, and reported to be a zoonotic spill-over event, possibly from bats to the human population , followeSARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins including the spike (S), envelope (E), nucleocapsid (N), and membrane (M) A. When aUpon contacting the target cell, spike proteins on the SARS-CoV-2 virions attach to the angiotensin-converting enzyme 2 (ACE2) to enter the cell . ACE2 isHaving once entered the target cells, the viral RNA genome is rapidly translated into effector molecules that play vital roles in supporting the viral replication cycle. Structural proteins, such as spikes, M, and E, undergo complex trafficking events for their maturation before being displayed onto the plasma membrane or assembled onto virions ,17 FiguB. Non-stViral entry is the initial and essential step in the infection cycle. Hence, it is important to understand the mechanism of SARS-CoV-2 entry, either by the ancestral strain or the ongoing Omicron subvariants. Molecular events targeting the spike not only limit the initial SARS-CoV-2 viral entry, but also inhibit downstream viral replication to reduce potential cellular damage and host pathology.Among all SARS-CoV-2 encoded proteins, spikes are responsible for the initial host receptor recognition, membrane fusion, and virion assembly. Similar to the influenza A virus (IAV) hemagglutinin (HA), human immunodeficiency virus (HIV) envelope, the SARS-CoV-2 spike glycoprotein is a class I fusion protein that requires host receptor binding to initiate its activation . Upon biThe ancestral strain SARS-CoV-2 spike protomer has 1273 amino acids and is expressed and inserted into the endoplasmic reticulum (ER)membrane, before being trafficked through the Golgi network for a complex series of post-translational modifications, including adding 22 N-linked glycosylation and C-terminal palmitoylation ,31,32. OThe physical number of spikes residing on virions may directly influence the strength of viral attachment, infectivity, and immune antigenicity. In situ cryo-electron tomography of the ancestral SARS-CoV-2 virions revealed that each spherical viral particle has a diameter of 85 \u00b1 10 nanometers and is estimated to carry 30\u201340 spike homotrimers ,36,37,38The SARS-CoV-2 S1 subunit contains a receptor-binding domain (RBD) A. The RBThe S2 subunit contains highly conserved regions that catalyze the membrane fusion. The internal fusion peptide is a highly hydrophobic membrane-interacting region ,59 that Over the period of the COVID-19 pandemic, the spike has accumulated a variety of mutations along the S1 and S2 subunits, causing SARS-CoV-2 VOC to partially evade approved vaccines and population immunity. However, the steps involved in the priming and activation of the SARS-CoV-2 spikes remain relatively conserved. The host-mediated proteolysis of spikes could be a promising antiviral target. Yet few protease inhibitors or neutralization antibodies are available to block the activation of the spikes. Here, we emphasize the two proteolytic steps involved in spike priming and activation. Initial amino acid sequence alignments of the original SARS-CoV and SARS-CoV-2 spike show the addition of a polybasic cleavage site at the S1/S2 junction ,33. IndeOne of the functional outcomes of the S1/S2 cleaved SARS-CoV-2 spikes has been described in terms of syncytium formation ,68,72,73Although S1/S2 cleaved spikes are primed for membrane fusion, this cleavage also penalizes the overall stability of spike trimer. Spontaneous shedding of the S1 subunit has been reported in the absence of host receptor binding , and canPrevalence of the spike H665Y substitution within the S1 subunit has been ranked highest among all SARS-CoV-2 VOC and subsequent Omicron subvariants ,94,95, sActivation of the spike-mediated membrane fusion involves the proteolytic processing at the spike S2\u2032 cleavage site . Unlike TMPRSS2 belongs to the family of type 2 transmembrane serine proteases (TTSPs) and is involved in the entry of many pathogenic viruses ,108,109.However, newly emerged spike Omicron subvariants became less dependent on TMPRSS2-mediated spike activation at the plasma membrane and are insensitive to original inhibitors such as camostat mesylate ,99,118. Other members of the TTSP family also promote SARS-CoV-2 spike-mediated entry in a context-dependent manner . TMPRSS1Cathepsin L has been implicated in the activation of the spikes of SARS-CoV and SARS-CoV-2 in the target cell endosomes ,128. PhaSecreted and soluble protease may also play a role in the activation of the spike protein and exacerbating host pathogenesis. For instance, matrix metalloproteases (MMPs), such as ADAM10 and ADAM17, cooperatively cleave SARS-CoV-2 spikes into S2\u2032 at the cell surface in TMPRSS2-deficient cells ,140,141,Spike substitutions and deletions not only profoundly impact viral transmission and immune evasion, but also critically determine host receptor and protease tropism. It is still unclear what protease is primarily involved in the activation of Omicron subvariants; nor inhibitors that selectively and efficiently block the endocytic route of SARS-CoV-2 entry. More systemic, physiological and context-dependent approaches on spike-activating proteases are warranted, in order to fully understand the mechanism of SARS-CoV-2 spike-mediated entry. Viral entry is the first and crucial step during the viral life cycle. Targeting viral entry could block the subsequent steps of virus infection and inhibit downstream viral replication and host cell pathology. The host innate immune response serves as the initial defense against a broad range of pathogens and shapes an essential adaptive immune response to clear viral pathogens . At the Interferon-stimulated genes (ISGs) are the cellular intrinsic defense against viral infection. Interferon-induced transmembrane proteins (IFITMs) are members of the membrane-bound ISGs , comprisSome independent evidence supports that the spike of SARS-CoV-2 interacts with human IFITMs, especially with IFITM2. IFITM2 interacts with SARS-CoV-2 spike proteins on the cell surface, mainly promoting subsequent virus\u2013cell fusion in the early endosome . It has Cholesterol-25-hydroxylase (CH25H) is one of the intracellular ISGs, which synthesize 25-hydroxycholesterol (25HC) from an intracellular pool of cholesterol . 25HC haThe lymphocyte antigen 6 complex member E (LY6E) gene is a member of the lymph stromal cell membrane Ly6 superfamily. It is a glycosylphosphatidyl-inositol (GPI)-anchored cell surface protein, rich in lipid raft microdomains, which regulate cytoskeletal rearrangement and the membrane-dependent pathways, such as the endocytosis and signaling pathways. LY6E can effectively reduce cell infection caused by multiple coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) , but proCells express endogenous antiviral factors to inhibit proteolytic activation of spikes. For instance, activation of the class II major histocompatibility complex transactivator (CIITA) leads to the upregulation of CD74 p41. P41 is a unique short isoform of the CD74 that carries a functional thyroglobulin type-1 domain, which inhibits cysteine proteinases, such as cathepsins . InteresBefore the involvement of adaptive immune responses, timely innate immune responses are paramount for sensing and achieving the antiviral states in SARS-CoV-2 infected tissues. In target cells such as the lung epithelia, pattern recognition receptors (PRRs) are responsible for the initial recognition of SARS-CoV-2 components either extracellularly or intracellularly . These iIn addition to ISGs and soluble factors, intracellular inflammasomes are intracellular innate immune sensors constitutively expressed in the respiratory epithelia and circulating immune cells. For instance, NLRP1 senses the SARS-CoV-2 dsRNA and cleavage by 3CL protease in human lung epithelial cells ,172; in Mycobacterium tuberculosis. Interestingly, it provides significant protection against the SARS-CoV-2 infection in a mouse model of authentic infection [Lastly, antigen-independent immunity can be raised against SARS-CoV-2 spike-mediated entry. The Bacillus Calmette\u2013Gu\u00e9rin (BCG) vaccine is a non-viral, live-attenuated vaccine for the bacteria nfection . Moreovenfection . Injectinfection . Althougnfection ,183, furThe SARS-CoV-2 virus will continue to evolve, and it is currently crucial to establish the fundaments of its biochemical modes of entry. From host receptor recognition, protease-mediated proteolytic processing, structural rearrangement, and fusion pore formation, each of these steps requires further investigation in physiologically relevant and context-dependent models. Regardless of the virion or cellular spikes, molecular mechanisms underlying the activation mode and membrane fusion are still elusive. Understanding the converging proteolytic processing of spikes ensures rapid identification, screening, and application of novel protease and fusion inhibitors. There is also a continued need for the identification of new host factors that regulate SARS-CoV-2 infection, especially for emerging new VOC. Host-derived innate immune factors are likely to act as broad-spectrum, endogenous inhibitors of spike-mediated viral entry. While there have been many successful works in this field, we should still notice that some factors exhibit contradictory mechanisms and phenotypes in different models. Overcoming this problem may require the utilization of 3D and physiologically relevant models such as the lung organoid and air\u2013liquid interface culture of the human airway epithelia. Further, we should also pay attention to the differences in the ISGs\u2019 antiviral effects against ancestral strains and newly emerged variants. Overall, these findings further our understanding of the host\u2013virus arms race during viral entry and provide wider clues for novel antiviral therapeutics against emerging and re-emerging infectious diseases."}
+{"text": "Measurements wereperformed on polymeric NPs, as these represent one of the major classesof NPs under development for drug-delivery aims. Determinations forpolystyrene NPs  and for NPs made of PEGylatedpoly-d,l-lactide-co-glycolide  were accomplished within 108\u20131012 particles mL\u20131 range,depending on the NPs size and composition. NPs size and concentrationwere maintained during analysis, as verified for NPs eluted from theLOV by particle tracking analysis (PTA). Moreover, concentration measurementsfor PEG\u2013PLGA NPs loaded with an anti-inflammatory drug, methotrexate(MTX), after their incubation in simulated gastric and intestinalfluids were successfully achieved , showing the suitability of the proposed methodto support the development of polymeric NPs targeting intestinal delivery.Nanoparticles (NPs)concentration directly impacts the dose deliveredto target tissues by nanocarriers. The evaluation of this parameteris required during NPs developmental and quality control stages, forsetting dose\u2013response correlations and for evaluating the reproducibilityof the manufacturing process. Still, faster and simpler procedures,dismissing skilled operators and post-analysis conversions are neededto quantify NPs for research and quality control operations, and tosupport result validation. Herein, a miniaturized automated ensemblemethod to measure NPs concentration was established under the lab-on-valve(LOV) mesofluidic platform. Automatic NPs sampling and delivery tothe LOV detection unit were set by flow programming. NPs concentrationmeasurements were based on the decrease in the light transmitted tothe detector due to the light scattered by NPs when passing throughthe optical path. Each analysis was accomplished in 2 min, renderinga determination throughput of 30 h Still,the regulatory approval of drug delivery nanoparticles (NPs) requiresa thorough and robust characterization of their main physicochemicalproperties, along with evidence of reproducible manufacture.5 and drug encapsulationefficiency 6 are among the critical parameters to determinethe dose delivered to target tissues, and consequently, for the therapeuticresponse.7 Accurate assessment of NPs concentrationis required to set dose\u2013response correlations during formulationdesign and is a requisite at industrial level to control the reproducibilityof NPs manufacturing batches and to comply with current8 and future needs at quality control and regulatory levels.9NPs concentration (defined as the number of NPs per volumeof formulation)9 namely, fororganic NPs, whose mass cannot be quantified by inductively coupledplasma mass spectrometry,10 as occurs forinorganic NPs.11 Therefore, for organic NPs, concentrationmeasurements are mainly performed by single-particle analysis techniques,such as particle tracking analysis (PTA) and tunable resistive pulsesensing (TRPS).12 PTA measurements result fromlight-scattering-based tracking of individual NPs in a given illuminationvolume. However, PTA analysis requires extensive sample dilution,which can induce NPs aggregation, causing inaccurate measurements.13 Also, results depend on the parameters set for video acquisitionand processing, which are operator-dependent, thus requiring trainedpersonnel and often suffering from variability if undertaken by differentoperators.13 TRPS is a more robust technique, in whichNPs concentration is assessed while they pass through a porous membrane,driven by voltage application.12 Nevertheless, TRPSrequires NPs dilution in electrolytes, analysis at different pressurepoints, calibration with particle standards prior to each sample analysisand for each pressure point, and experienced operators12 capable of optimizing measurement parameters, which renders thetechnique not straightforward for routine applications at industrialor medical facilities. Considering these limitations, other techniques,such as nano flow cytometry (nFC), multiangle light scattering (MALS),and differential centrifugal sedimentation (DCS), have been also usedfor measuring NPs concentration, and/or to provide a means for resultsvalidation. In nFC, single-particle counting is performed by lightscattering (label free) and/or fluorescence measurements after NPsirradiation by a laser beam.14 Still, light-scatteringmeasurements for NPs with <300 nm are challenging and were onlyperformed so far resorting to custom-made, not broadly available nFCsetups,15 while fluorescence detection requires NPslabeling and purification before analysis. Regarding concentrationmeasurements by ensemble techniques, such as MALS and DCS, both requirepost-analysis conversions of the obtained NPs size distributions,which entails NPs properties, such as size, density, refractive index,and media viscosity to be well-known for accurate estimations.16Still, measuring NPs concentration resortingto the currently availabletechniques poses several challenges,18 Nevertheless, label free, simpler, and fit for purpose methods suitableto characterize the number of organic NPs in formulation batches forroutine quality control operations are yet required. In this context,an automated, miniaturized and simple method to quantify NPs resortingto the sequential injection lab-on-valve (SI-LOV) system20 is proposed. LOV is a mesofluidic platform that provides precisehandling of small volumes (\u03bcL) of samples/reagents in rigidminiaturized channels resorting to flow programming and to a multipositionselection valve,21 thus affording robust and reproducibleoperations with downscaled consumption of samples/reagents.21 LOV accommodates sampling, online dilution andmixing by flow reversal, and online detection as it integrates anoptical detection unit, enabling several operations in a single setup.This system has successfully been employed to set simpler, fasterand less-laborious quantification of small molecules,22 sorbent-based sample pretreatment,24 and molecular recognition procedures.25 Furthermore,considering the need of ensuring minimal stress and dilution duringthe analysis of organic NPs, the LOV platform emerges as a valuablealternative for NPs characterization as it offers (i) inert bore conduits(ca. 1.0 mm), larger than conventional size-exclusioncolumns and ultrafiltration/dialysis membranes, (ii) low operationflow rates , and (iii) short distance (10 mm) between the sampling port andthe detection unit.Considering the above-mentioned challenges, improved, alternative,and orthogonal procedures to the currently available strategies forNPs concentration measurements have been pursued.d,l-lactide-co-glycolide (PEG\u2013PLGA)NPs as PEG\u2013PLGA NPs are among the most produced due to thebiocompatibility, biodegradability, and \u201cstealth\u201d propertiesconferred by the PEG\u2013PLGA polymer.26 Moreover, the application of the established method was furtherextended to assess NPs stability when exposed to surrogate biologicalmedia, to investigate NPs suitability for oral intake.Given the previous context, in this work,a miniaturized and nondestructivemethodology was set under the LOV to evaluate the concentration ofpolystyrene NPs with different diameters . Thedeveloped framework was used to determine the concentration of empty and methotrexate (MTX)-loaded PEGylated poly-Supporting Information, along with preparation details.Details on the reagents andsalts used for preparing the PEG\u2013PLGA NPs, the phosphate-bufferedsaline solution (PBS) used as carrier in the SI-LOV system and fordiluting NPs suspensions, and the simulated gastric and intestinalfluids are provided in 7 of 5.5, 34, and 0.29 \u00d7 1012 particlesmL\u20131 were acquired from Sigma-Aldrich . These were designated as NPs A, B, and C for simplicity.Different concentration levels were prepared from each standard byadequate gravimetric dilution in PBS buffer.Polystyrene NP standards withmonodisperse distributions, with 188 \u00b1 4, 102 \u00b1 3, and 502\u00b1 13 nm of diameter, and with estimated particle concentrations27 (details on Supporting Information). Their hydrodynamic diameter and polydispersityindex (PDI) were characterized by dynamic light scattering after preparation and after NPs purification (details on Supporting Information). Moreover, the size andconcentration of PEG\u2013PLGA NPs final suspension were characterizedresorting to particle tracking analysis . For this, NPs were diluted 16200\u00d7 in ultrapurewater prior to analysis. A 60 s video (n \u22653) was recorded for the suspensions of empty and loaded NPs (datawill be available upon request). Videos were processed using the NTA2.3 software .PEG\u2013PLGANPs were prepared weekly by the single emulsion-solvent evaporationtechniqueFigure S1) is describedin Supporting Information. Optical detectionand quantification of nanoparticles was performed in the LOV integrateddetection unit (further details on Supporting Information)28 by measuring the light attenuation  when nanoparticles were present in the opticalpath.The configuration of the SI-LOV  system used for nanoparticlesquantification (Table S1): first, the syringe pump was filledwith carrier solution (PBS). Subsequently, 20 \u03bcL of carrierwas dispensed into the flow cell for performing a reference scan.Then, the multiposition valve was switched to the sample port, andthe sample was aspirated into the holding coil at 3 \u03bcL s\u20131. After flow reversal, the NPs present in the holdingcoil were directed at 2 \u03bcL s\u20131 to the detectionunit, where the light attenuation values were monitored at 280, 302,320, and 480 nm. These last two steps can be repeated  for replicate analysis of the same sample. Finally,the carrier remaining in the syringe pump was discarded to waste toclean the holding coil and to prepare the system for the measurementof the following sample.The analytical routine comprised five steps  and quantification (LOQ), precision, andaccuracy)29 are provided at Supporting Information.Different concentrations of polystyreneNPs  and ofPEG\u2013PLGA and PEG\u2013PLGA\u2013MTX NPs were analyzed followingthis analytical routine. For this, working solutions were preparedby submitting the NPs stock suspensions to vortex (3000 rpm) for 30s, followed by adequate dilution in PBS. Each working solution wassubmitted to vortex immediately before its introduction into the LOVchannel, which was just performed prior the start of each analyticalcycle. Likewise, PEG\u2013PLGA-MTX NPs were also analyzed afterincubation for 2 h with simulated gastric fluid (containing or notpepsin), and for 4 h with simulated intestinal fluid. Further experimentaldetails on analysis conditions, control experiments and on the dataanalysis performed for calculating peak height, peak area, peak width,and method analytical features . A brief overviewon the fundamentals of NPs light scattering can be found in Supporting Information. Herein, as incident wavelengths(\u03bbinc) from 280\u2013500 nm and NPs with 100\u2013500nm of diameter were targeted, scattering events were putatively consideredto obeyed to Mie theory.31 Thus, the total lightscattered is a function of (i) NPs size, (ii) NPs concentration, (iii)NPs extinction efficiency (Qext), and(iv) the optical path as described by equation S1, with Qext being dependent onthe \u03bbinc, and on the differences between NPs\u2019material and surrounding medium refractive indexes.31NPs concentration measurements werebased on the decrease of light reaching the detector when part ofthe incident light was scattered by NPs passing through the LOV flowcell, for negligible absorbance eventsinc for all the NPs under study  since the magnitude of scattering intensity isinversely proportional to \u03bbinc.31In this work, although data acquisition was performed at 4wavelengths, light attenuation measurements were taken at 280 nm,as this wavelength provided increased sensitivity in relation to higher\u03bb32 and the sample volume,33 theseparameters were further studied to implement a reliable analyticalroutine for NPs quantification.Moreover, the measurements were performed under sequentialinjection(SI) conditions, as these offer reproducible features regarding themaintenance of NPs in suspension as they pass through the detector,beyond miniaturization. Since several variables will affect the dispersionof NPs in the carrier fluid, namely, the flow rateFigure S3 depicts the signal profiles (lightattenuation vs. time) when afixed volume (30 \u03bcL) of polystyrene NPs A (188 \u00b1 4 nm)was aspirated from the sampling port and sent to the detection unitat different flow rates . Good repeatability concerning signal height (RSD < 2%) andarea (RSD < 5%) was observed independently of the flow rate tested.Nevertheless, experiments at the lowest flow rate (1 \u03bcL s\u20131) resulted in the broadest peak profile and in thelowest signal height (0.304 \u00b1 0.004), which was caused by theincreased NPs dispersion in the carrier when the time of sample displacementfrom the holding coil to the detector increases.32 Experiments at 4 \u03bcL s\u20131 resultedin the narrowest peak, with a peak height (0.35 \u00b1 0.1) similarto the obtained for experiments at 2 \u03bcL s\u20131 (0.355 \u00b1 0.002), although with RSD values at least twice largerfor peak height and area. This fact is likely attributed to the fasterpassage of NPs through the detector, causing some irreproducibilityprobably due to inadequacy in data frequency acquisition.34 Thus, the flow rate of 2 \u03bcL s\u20131 was selected for further assays, providing fast analysis withoutcompromising detection performance.peak area = 0.181 (\u00b10.003)\u00d7 sample volume \u2013 0.24 (\u00b10.11),, suggesting that NPs did not agglomeratein the carrier plug when passing through the LOV detection unit forthe range of tested volumes. A similar effect was described by Ruzickaet al. when small molecules (<3 nm) were analyzed in the LOV flowsystem:33 when no increase in peak heightwas observed, the central segment of the sample was not diluted inthe carrier (Ruzicka\u2019s dispersion coefficient = 1).Different volumes (10\u201360\u03bcL) of polystyrene NPs A were sent to the detection unit toselect the best conditions for quantitative measurements. A markedincrease (\u224825%) in peak height (light attenuation vs. time)was observed with increasing sample volume up to 30 \u03bcL 1. For voThus, the same principles were verified herein for NPs analyzedin a flow system. Considering the results obtained, 30 \u03bcL wasselected as the sample volume for further assays, providing a goodcompromise among sensitivity, sample spending, and time of analysis(120 s per determination).\u201311 [NPs A] \u2013 0.002 (\u00b10.001), R2 \u2265 0.9996 where [NPs A] is given in number of particles mL\u20131, Table S2). Similarly,a linear increase in peak area was also observed (Table S2), while peak width was constant along all the concentrationstested (Table S3), confirming the reproducibledispersion of NPs using the LOV platform.The flowconditions set above were applied to evaluate analytical readout forincreasing NPs concentrations. A linear increase in peak height wasobserved for increasing concentrations of polystyrene NPs A (lightattenuation 280 nm= 1.70 (\u00b10.01) \u00d7 10n = 10) were aligned with the required bybioanalytical method guidelines,29 provingthe method suitable for quantification purposes.Intermediate precisionvalues for peak height (RSD \u2264 3.6%) and peak area (RSD \u226411.3%)  and 502 \u00b1 13 nm (NPs C), a linear increasein peak height and area for increasing concentrations of NPs B andC was also observed, demonstrating the method feasibility to distinguishbetween NPs concentrations for NPs within 100\u2013500 nm size range(Table S2). Indeed, whenthe NP size was \u22482\u00d7 smaller to that of NPs A, a decreaseof \u224815\u00d7 in the sensitivity was obtained for peak heightand area. The different sensitivity attained for NPs of the same material,density and concentration but with different sizes is a result ofthe increase in the light scattered when particle size increases.36 Indeed, the ratio (light attenuation/particle concentration) isdirectly proportional to the extinction efficiency (Qext), and to the square of particle diameter, as describedby Mie theory (equation S1) when multiplescattering is not verified (as occurs in the concentration rangesused for NPs A and B).37 For NPs B, thesquared diameter and the Qext are \u22483\u00d7and \u22485\u00d736 lower in relationto that of NPs A, thus corresponding to the \u224815\u00d7 sensitivitydecrease observed experimentally.However, the working range and sensitivity for these NPs was differentthan the obtained for NPs A . However, it has been described that when NPsize increases, deviations from the expected Mie theory Qext values occur.39 Indeed, deviations of \u22482\u00d7 have been described for NPswith a size higher than that of the incident wavelength. These havebeen attributed to the increase of particle\u2019s geometrical crosssection, that affects Qext values,39 which could possibly justify the increased sensitivity attained.On the contrary, when NPssize was \u22482.7\u00d7 higher inrelation to that of NPs A (500 nm), an increase of \u224815\u00d7in the sensitivity has also been observed. In this case, the sensitivitywas 2\u00d7 higher than the theoretically expected considering the Supporting Information, Section 2). Indeed, althoughNPs A, B, and C have the same overall density (1.05 g cm\u20133), when the same number of NPs is sampled from each formulation anddispersed in 1 mL, a different volume will be occupied by the particlesbecause of their differences in size,7 resultingin different scattering intensities tested , and thus, this parameterwas not influenced by NPs size. Hence, the gathered results confirmthat transport within the flow conduits was not affected by NP sizeand concentration, and that the analytical signal (decrease in thelight received by the detector due to scattered light) was proportionalto NPs concentration for NPs of the same material and size and withmonodisperse distributions , beingaligned with the NPs light scattering fundamentals described  for systemassessment is recommended, which is a usual practice in flow analysissystems, considering its three principles for reliable, nonequilibriumdeterminations: (i) repeatable insertion of sample into a flowingstream, (ii) controlled dispersion of sample in the carrier phase,and (iii) reproducible timing of events, including sampling and detection.Thus, the present method was responsiveto NPs concentration, enablingto distinguish concentrations between batches of particles of thesame material and with similar particle sizes. Still, independentparticle size measurements must be performed prior to analysis foradequate result interpretation.26 For organic NPs holding a core and surface withdifferent compositions (and densities) as these, the expression ofNPs dose as NPs number mL\u20131 in detriment of massof polymer mL\u20131 is highly recommended for a correctinterpretation of cytotoxicity/efficacy results, especially when comparisonswith other formulations are envisioned during NPs development stages.7PEG\u2013PLGA NPs areamong the most produced particles for drug-delivery aims, as PEG\u2013PLGAis an FDA approved polymer, with biodegradable, biocompatible andstealth properties presenting a safe and long-circulation profile.Figure S4).A linear increase of peak height and area was observed for increasingconcentrations (0.139\u20131.39 \u00d7 1012 particlesmL\u20131) of PEG\u2013PLGA NPs . Intraday RSD values were <6% and <10% forpeak height and peak area, respectively. Likewise, interday RSD valueswere \u226413%.As observed for polystyrene NPs, differences <6% in peak widthwere obtained along all the tested concentrations (Table S2). This is consistent with light scattering fundamentals, consideringthe lower refractive index of PEG and PLGA (1.46\u20131.47)41 in relation to that of polystyrene (1.59\u20131.62).42 Indeed, the intensity of the scattered light will be lower whensmaller differences exist between the refractive index of the NPsand that of the medium (\u22481.34\u20131.35).30 This is further amplified by the size differences betweenthe PEG\u2013PLGA NPs and the polystyrene NPs A under study (NPsA size is \u224830% higher than that of PEG\u2013PLGA NPs), justifyingthe different scattering intensities attained.However, the sensitivity of these determinationswas lower (\u224850\u00d7 lower slope) in relation to the achieved for polystyreneNPs with a similar size , the first time a NPs formulation is analyzedin the LOV system, since each NPs formulation has its own physical/lightscattering properties. Such would allow to establish a LOV signal/particleconcentration reference for subsequent concentration measurementsof NPs of the same size and composition that could be performed solelyon LOV afterward, due to method reproducible features and using aparticle calibrant for daily system assessment as recommended above.Figure S6). Different concentrations of PEG\u2013PLGA-MTX NPs with 151 \u00b13 nm, polydispersity values \u2264 0.07, and with monodisperse distribution were analyzed rendering a linearcorrelation between NPs concentration and peak height/area , which absorbslight within 200\u2013450 nm S6. Diffeght/area 1.tcalc| > 3.70, ttab = 2.0, \u03bd >48, p = 0.05, concerning the slopes for analyticalsignal vs.NPs concentration, inc, despite the smaller size (ca. 20 nm) of empty NPs, whereas for peak area higher sensitivity wasobtained for loaded NPs (as expected considering the higher NPs size).This trend for peak height at 280 nm was not consistent with the analyticalsignal obtained at 480 nm (Table S4), awavelength at which the loaded compound (MTX) does not absorb . At 480 nm, the slope for both peakheight/area vs. NPs concentration was significantly higher for loadedNPs in relation to empty ones . These resultssuggest that, at 280 nm, part of the light illuminating the particlesis absorbed by the loaded compound, with less light available to bescattered by the particles while these pass through the flow cell.Therefore, particle concentration estimations and comparisons betweenbatches that contain loaded compounds must be performed using a \u03bbinc at which the loaded compounds do not absorb light or resortingto peak area values.However, slight differences in peak height andarea were observedbetween empty and loaded NPs at 280 nm  andsizes (\u2248100\u2013500 nm).9 to 1011, 109 to 1010, and108 to 109 particles mL\u20131 werefeasible for polystyrene NPs with ca. 102, 188, and502 nm, respectively (Table S2). LOD andLOQ values within 107 and 109 were determinedfor these NPs . Likewise, working ranges for PEG\u2013PLGA andPEG\u2013PLGA\u2013MTX NPs were from 1011 to 1012 particles mL\u20131, with LOD/LOQ values inthe 1010 particles mL\u20131 range (Working ranges from 10ectively S2. LOD a7\u2013109 particles mL\u20131),13 the most usedtechniques for NPs counting, thus enabling measurements resortingto less diluted samples . This is advantageousdue to the instability commonly caused in the nanoformulations byextensive dilutions (such as those required for PTA).5The workingconcentration ranges were higher than the commonlyapplied for PTA and TRPS analysis  and size   as confirmedby PTA. This suggests that NPs integrity was unaffected during analysis,and that there was no detectable NP loss in the flow system. Thisis aligned with LOV system features, namely, the inert and relativelylarge bore (ca. 1.6 mm) conduits when compared tomicrofluidic systems. Furthermore, analysis was accomplished usingflow rates close to 2 \u03bcL s\u20131 and without submittingthe particles to extensive dilutions in the system , thus maintainingNPs properties, in opposition to the commonly verified in column-basedprocedures .The analysis of a control sample before and afterLOV analysis(by collection of the fluid leaving the flow cell) revealed no significantdifferences in NPs concentration (|n = 5), making the analysis of 6 samples h\u20131 feasible,which is also advantageous considering other NPs concentration measurementstrategies .12Finally, each sample was analyzed within 10 min  for regular concentration measurementsof NPs from distinct materials/composition, expanding the availabletoolset with a fast, user-friendly, easily implementable, and reproducibleprocedure for NPs concentration control. In addition, the proposedmethod is not destructive, allowing NPs reuse for further characterizationexperiments upon their elution from the LOV, as it does not causealterations in NPs size .Besidesthe above-mentioned advantages, NPs measurements underLOV dismiss experienced operators, optimization of analysis parametersbetween samples, or laborious calibration procedures .43 with consequent overestimation of NPs number. Therefore,the feasibility of the developed method to determine PEG\u2013PLGA\u2013MTXNPs concentration upon their exposure to gastric and intestinal simulatedfluids was exploited and results compared to those from PTA measurements.Recoveries within 102\u2013115% were determined by direct analysisof PEG\u2013PLGA\u2013MTX NPs after their incubation with simulatedgastric fluid  at 37 \u00b0C  in biological media, such as gastricfluid is required, for instance, when administration by the oral routeis aimed. Still, concentration measurements of organic NPs in biologicalfluids pose some challenges, as counting of protein aggregates asNPs have been reported for TRPS and PTA measurements,at 37 \u00b0C 3, suggesLikewise, NPs concentrationmeasurements after NPs exposure to pepsin-free gastric fluid by LOVwere consistent  with thevalues determined by PTA. Thus, NPs stability upon exposure was confirmedby the developed method and validated by PTA measurements, suggestingthe stability of the studied PEG\u2013PLGA\u2013MTX NPs undergastric pH.43 as strongly suggested consideringthe results attained with the LOV method in NPs concentration values in relation to the same NPs concentrationin protein-free gastric fluid. This increase in NP concentration islikely attributed to PTA bias when measuring NPs dispersed in mediacontaining protein,V method 3c, for wV method 3b.Figure S8), suggesting the absenceof NPs disruption under these conditions. Similar findings were verifiedby PTA, with no marked changes in NPs concentration values  after 4 h of incubationin this fluid. These results were further supported by the maintenanceof NPs size upon incubation (163 \u00b1 2 vs. 169 \u00b1 3 nm).NPs stability upon incubation in enzyme-free simulated intestinalfluid (pH 6.8) for 4 h at 37 \u00b0C resulted in recoveries of 103\u00b1 1%  were feasible, with no changes in particle size andconcentration being found when eluted fractions were analyzed by PTA.In this work, a miniaturized, simple, fast,and nondestructiveprocedure for NPs quantification based on optical measurements wasdeveloped under the SI-LOV platform. The set method allowed reproduciblemeasurements of NPs concentration, for NPs made of polystyrene (100\u2013500nm), and for PEG\u2013PLGA (\u2248130 nm) and PEG\u2013PLGANPs loaded with a therapeutic agent (\u2248150 nm). Low sample consumption(30 \u03bcL) and fast analysis ,providing a simple tool to evaluate NPs suitability for gastric passageduring formulation development stages.The simplicity of theproposed procedure makes this method advantageousin relation to other strategies, such as PTA, TRPS, and MALS, forroutine quality control operations. This, combined with the portablefeatures of LOV along with its multiple sampling ports (6), rendersthe method promising for implementation in routine evaluations ofNPs concentration at development laboratories. Furthermore, the setmethod provides a new analytical tool for measuring nanoparticle concentration,affording a means for result validation, as required due to the variabilityof results reported among techniques.The gathered results suggestthe feasibility of the proposed methodfor measurements of NPs of different sizes, made of different materials(refractive index \u2265 1.46), only requiring their previous characterizationby an alternative method the first time these are analyzed by theLOV method to establish a reference concentration. Additionally, asusual in flow injection systems, daily calibration is recommended.e.g., serum, saliva, or urine). Likewise, the insertion of size exclusionmaterials upstream the LOV optical path is currently under considerationtoward extending method applicability to formulations containing nonhomogeneousnanoparticle populations.Future studies on NPs presenting refractive indexes and/or diametersdissimilar from those tested herein can further extend method applicabilityfor NPs characterization at industrial level, for instance, to evaluatethe reproducibility of NPs batches, to screen for alterations throughoutNPs storage, lyophilization or sterilization, and even to evaluateNPs stability when present in biological fluids ("}
+{"text": "Cutaneous melanoma is one of the most malignant types of skin cancer, with an extremely poor prognosis. Immune cells infiltrated in the tumor microenvironment (TME) affects melanoma initiation, progression, prognosis and immunotherapy strategies in melanoma. The potential utility of TME-related genes as a prognostic model for melanoma and as a predictor of immunotherapeutic response merits further exploration. In this study, we determined that an immune-related gene, protein tyrosine phosphatase receptor type C (PTPRC), was positively correlated with the positive prognosis of melanoma patients. Integration of this gene with TNM classification created a predictive model that showed better performance in determining overall survival than others. PTPRC expression was positively correlated with the levels of immune checkpoint molecules, and PTPRC knockdown significantly enhanced the migration, invasion, and proliferation of melanoma cells. Finally, immunohistochemical results from HPA and Real-time quantitative PCR of clinical tissues confirmed that PTPRC expression was higher in melanoma than in normal skin. In conclusion, PTPRC served as a potential predictor of survival and response to immunotherapy in melanoma patients. The risk model combining the PTPRC and TNM classifications holds the potential to be a promising tool for prognostic prediction of cutaneous melanoma. This will help in the effective clinical management of melanoma patients. The etiology of malignant melanoma has not been fully understood. It is generally considered to be multifaceted, with race and genetics, trauma and irritation, sunlight, immunity, and other factors all likely to be involved2. Targeted therapies and immunotherapy have emerged over the past decade or so, improving overall survival (OS) for melanoma, particularly in immunotherapy. Immune checkpoint inhibitors such as anti-CTLA-4 monoclonal antibodies (Ipilimumab), anti-PD-1 monoclonal antibodies (Navulizumab and Pabrolizumab), and LAG-3 blocking antibodies (Relizumab) inhibit the reactivity of the corresponding immune checkpoint proteins and have proven to be clinically efficacious6. However, a subset of patients remained resistant to this immunotherapy, and some even experience autoimmune adverse effects8. Therefore, it is clinically meaningful to explore potential biomarkers to evaluate prognosis and assess whether patients would benefit from immunotherapy.Melanoma is a highly aggressive skin cancer originating from melanocytes, leads to approximately 55,500 deaths per year9. The two most important members in the TME infiltrating are mesenchymal cells and immune cells, playing an important role in tumor biology10. There is growing evidence that cancer cells can activate different immune pathways that lead to immunosuppressive functions and determine the immune microenvironment of tumors11. The association between robust lymphocytic infiltration and better patient survival has been well documented inovarian, head and neck, breast, uroepithelial, colorectal, lung, hepatocellular carcinoma, esophageal cancer, and melanoma12. Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) is a tool that uses gene expression data to predict tumor purity, and the presence of infiltrating stromal/immune cells in tumor tissue. The ESTIMATE algorithm is based on a single-sample genomic enrichment analysis and produces three scores: Stromal score (captures the presence of stroma in the tumor tissue); Immune score (represents the presence of immune cells in the tumor tissue), and Estimate score (infers tumor purity). Several studies have established that these scores correspond with clinicopathological aspects and chemotherapeutic treatment resistance in diverse tumor types and are thought to be valuable as prognostic indicators for patients15. In addition, TNM staging is valuable for cancer staging, prognosis determination, and treatment option selection, but it does not allow for accurate patient assessment from an immunological perspective16. Therefore, combining TNM classification with tumor microenvironment-related markers may be more useful for predicting the heterogeneous clinical behavior and prognosis of melanoma patients.The tumor microenvironment (TME) is the soil on which tumorigenesis and development dependHerein, we explored the differentially expressed immune score-based genes related to the prognosis of melanoma, which resulted in the identification of one immune-related genetic biomarker that can be used to guide the prediction of prognosis and response to immunotherapy. Then, the function enrichment and immune landscape related to the prediction model were investigated. Next, we explored at how suppressing biomarker\u2019 mRNA expression effected melanoma cell migration, invasion, and proliferation. Finally, we validated the biomarker protein levels and mRNA expression levels in the Human Protein Atlas (HPA) database and clinical specimens. In conclusion, these data suggest that the biomarker identified in this study, combined with the TNM staging of tumors, may be practical predictors of heterogeneous clinical behavior and prognosis in melanoma patients. It may provide a basis for future clinical treatment, especially immunotherapy.P\u2009<\u20090.001; Fig.\u00a0P\u2009<\u20090.001; Fig.\u00a0P\u2009=\u20090.076; Fig.\u00a0A total of 470 tumor samples in the TCGA database and 332 tumor samples in the GEO datasets were acquired. The ESTIMATE algorithm was used to calculate ImmuneScore, StromalScore, and ESTIMATEScore for each sample. Based on the median of each score, the samples were divided into high or low-score groups, and Kaplan\u2013Meier survival analysis was performed for each of the three scores. The results revealed that melanoma patients with higher ESTIMATEScore P\u2009<\u20090.00; Fig.\u00a01aSince the immune microenvironment plays an important role in melanoma behavior and disease prognosis, and the previous results indicate a strong relationship between ImmuneScore and clinical OS. To further explore the role of immune-related genes in melanoma, we divided the patients to high- and low- score groups according to their ImmuneScore and then identified DEGs. The analysis found 1684 significantly upregulated genes and 154 significantly downregulated genes in TCGA, and 61 significantly upregulated genes in GEO Fig.\u00a0a,b.FigurWeighted gene co-expression network analysis (WGCNA) was conducted to identify the critical gene modules closely associated with the high-immunity status of melanoma Fig.\u00a0c\u2013f. In TP\u2009<\u20090.05) , emphasizing the critical role of PTPRC in prognosis  GO terms, cellular component (CC) GO terms, molecular function (MF) GO terms Fig.\u00a0a, and thP\u2009=\u20090.01). Consistent with the trend of ImmuneScore, the expression of PTPRC significantly declined in T4 . Of the 466 melanoma patients in TCGA, 46 (10%) carried somatic mutations in PTPRC, and patients with PTPRC mutations had a significantly higher TMB than wild-type samples , and the high-PTPRC group had a higher TMB . Kaplan\u2013Meier analysis showed that PTPRC mutation was associated with a positive prognosis .Next, RNA-Seq data from 556 normal skin samples from the GTEx database and 470 melanoma samples from the TCGA were combined, and normal as well as disease cases from the GEO database were also analyzed, we observed that PTPRC was significantly overexpressed in melanoma Fig.\u00a0f,g, and As previously mentioned, PTPRC overexpression associated with better prognosis of melanoma patients. A prognostic model based on the expression of PTPRC was constructed. The RiskScore of each patient was calculated, based on the median RiskScore, we categorized the samples into low- and high-risk clusters. Survival analysis showed that the high-risk cluster exhibited a significantly poorer OS rate than the low-risk cluster in both the training and testing groups Fig.\u00a0a,b. The As previously mentioned, PTPRC is an immune-related gene and is associated with prognosis of melanoma patients. GSEA analysis was conducted to further investigate the differences in enrichment pathways between the high- and low-PTPRC groups. We discovered that pathways enriched in the high-PTPRC group were mainly related to immune-related pathways, including B cell receptor signaling pathways, cell adhesion molecules, cytokine signaling pathways, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, and T cell receptor signaling pathways. In contrast, pathways enriched the low-PTPRC group was associated with metabolisms, such as glycosylphosphatidylinositol anchor biosynthesis, RNA polymerase, oxidative phosphorylation, glyoxylate, and dicarboxylate metabolism Fig.\u00a0a. To expP\u2009=\u20090.049)  Fig.\u00a0d.Figure Next, to determine whether PTPRC affects migration, invasion and proliferation of melanoma cells, siRNA targeted PTPRC was applied to knockdown PTPRC expression in A375 and MEL-28 cell lines  in TME that has good predictive power for both prognosis and response to immunotherapy in melanoma patients through a comprehensive bioinformatics analysis.Melanoma is an aggressive skin cancer with increasing morbidity and mortality. Although current immunotherapy has made significant progress, there are still many melanoma patients who do not respond to immunotherapy or who experience adverse reactions20. It controls immune function by regulating lymphocyte survival, cytokine responses, and T cell activation signaling. Altering PTPRC can lead to severe combined immunodeficiency21. In addition, CD45 activity is essential for an efficient immune response. In particular, in NK cells, CD45 expression is highly correlated with NK cell maturation23. As a gene closely related to immune function, PTPRC has consistently been reported to be related to autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis24. It was also reported that PTPRC was associated with favorable disease-specific survival in lung cancer27. However, the function and mechanism of PTPRC in melanoma have not been elucidated.PTPRC, also known as CD45, is expressed in almost all hemopoietic cells except mature red blood cells and is an essential regulator of T and B cell antigen receptor-mediated activation. It is also an important protein on the cell surface of the blood and immune systemIn line with other reports, we found that the immune component of TME plays a critical role in the clinical outcome of melanoma patients, including survival and TNM stage classification. Then, a comprehensive and systematic analysis of the data using various databases showed that high expression of PTPRC was significantly associated with a better prognosis, while a RiskScore calculated from PTPRC expression revealed a worse prognosis in the high-risk group, suggesting that PTPRC expression was expressed by infiltrating immune cells rather than cancer cells present in the tissue, which is associated with an immune response. To demonstrate the possible protective role of PTPRC in melanoma development, we showed that low expression of PTPRC enhanced the migration, invasion, and proliferation of tumor cells by knocking down the expression level of PTPRC in the melanoma cell lines A375 and MEL-28. Next, consideration of PTPRC expression based on TNM classification effectively improved the reliability of predicting the prognosis of melanoma patients, and thus the model may be able to provide a prediction of the prognosis of melanoma patients in the clinic.28. These results all suggest that PTPRC may play an important role in predicting and improving the response of melanoma to immunotherapy. Finally, we confirmed by immunohistochemistry results in HPA and qPCR that PTPRC expression is higher in melanoma than in normal skin.Tumor-infiltrating lymphocytes determine tumor progression and aggressiveness and are a source of important prognostic information for patients. Analysis by GSEA and CIBERSORT showed that the PTPRC high expression group was significantly associated with immune-related biological processes and activated immune infiltrating cells, which is consistent with previous studies. Furthermore, PTPRC was co-expressed with a variety of immune checkpoint genes, including PDCD1, PD-L1/CD274, CTLA4, LAG3, and HAVCR2. PTPRC expression levels increased significantly after anti-CTLA4 and anti PTPRC expression levels increased significantly after anti-CTLA4 and anti-PD1 treatment, probably because inhibition of these checkpoints resulted in activation of the PTPRC-expressing immune cell population. Also, PTPRC expression is positively correlated with TMB, and studies have shown that tumors with high TMB are thought to be more susceptible to immune checkpoint inhibitors (ICI) as they express more neoantigens and are therefore more likely to be recognized and targeted by T cells30. Recently, high LDH has been linked to a poor response to anti-PD1 therapy, implying that it could be used as a predictive biomarker31. However, this remains to be proven. Numerous studies have shown that elevated serum S100B levels are associated with increased aggressiveness and decreased survival in melanoma, suggesting that S100B can be used as a prognostic marker35, S100B is also valuable for monitoring patients during treatment, with increased levels associated with disease progression and decreased levels associated with disease regression36. However, given the low incidence of elevated serum levels of S100B in patients with early-stage disease, S100B was not considered useful for screening or early detection. Nevertheless, the two predominant serum markers are elevated in most non-neoplastic diseases and are susceptible to a variety of confounding factors when making judgments. With the boom in immunotherapy and its revolutionary improvements in the treatment of melanoma, there is an urgent need for appropriate prognostic biomarkers to estimate risk and an even more urgent need for predictive biomarkers to determine which patients are likely to respond to immunotherapy. Studies have shown that TMB38, IFN \u03b3-related gene expression profile40, infiltration of CD8\u2009+\u2009TILs42 T, and HLA heterozygosity44 all have a possible role in predicting response to ICI, but none have been found to correlate with prognosis. As for PTPRC, which was studied in this paper, it also predicts prognosis and responsiveness to immunotherapy. In conclusion, it makes sense that PTPRC has been proposed as a biomarker for melanoma.In recent years, significant developments in molecular analysis, genomics, and cancer biology have led to the discovery of many new cancer biomarkers. Cancer biomarkers can be classified into three categories: diagnostic, prognostic, or predictive. Lactate dehydrogenase (LDH) and S100B have been found in research to be the most useful serum prognostic indicators. In metastatic melanoma, serum LDH is a potent independent prognostic indicator, and increased LDH is linked to a lower OS in patients with advanced disease. To date, LDH is the only widely used prognostic serum marker for melanomaOverall, we identified the value of PTPRC as an indicator of TME status remodeling in melanoma and also as a potential predictor of response to immunotherapy. In addition, the model created in combination with TNM classification allows for more precise stratification of patient survival. It is important to note that while these results suggest a clear clinical link between PTPRC expression and melanoma, it has the limitation that only data from various databases the validation of mRNA levels and some simple in vitro experiments. Therefore, further additional in vivo and in vitro studies are necessary to seek results that logically support these clinical associations.This study identifies a new immune-related prognostic indicator for use in melanoma that may also serve as a potential predictor of response to immunotherapy. Targeting PTPRC may be a treatment approach for melanoma based on the hallmark immune infiltration landscape observed in our study.https://portal.gdc.cancer.gov/) as a training set. The standard skin samples were retrieved from Genotype-Tissue Expression Project (GTEx) (https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project). Another 332 melanoma patients\u2019 information obtained Gene Expression Omnibus (GEO) datasets (https://www.ncbi.nlm.nih.gov/geo/)  was used as a testing set. Somatic mutation information for melanoma patients was obtained from TCGA and International Cancer Genome Consortium (ICGC) (https://dcc.icgc.org/). The results of melanoma single-cell sequencing were also obtained from GEO datasets. Batch corrections were applied when different cohorts were combined. All of the above datasets are accessible to the public.RNA-Seq data and clinical information of 470 melanoma patients were obtained from The Cancer Genome Atlas (TCGA) (45.The immune infiltration (ImmuneScore), overall stromal content , and combined (ESTIMATEScore) of each melanoma sample were calculated using ESTIMATE algorithmsSamples were divided into high- and low- immune score groups, and DEGs were identified based on the conditions: |log2 fold change (log2 FC) |>\u20091.0, false discovery rate (FDR)\u2009<\u20090.05 using the \u201climma\u201d R package. The \u201cpheatmap\u201d R package was used to generate heatmaps.The power value was filtered out in the module construction through weighted gene co-expression network analysis (WGCNA). The independence and average connectivity of different modules were tested using the gradient method . When the degree of independence arrived at 0.8, the appropriate power value was determined, and then the module construction was carried out through the WGCNA. In addition, the corresponding genetic information for each module was extracted. The minimum number of genes was set to 50. A Venn diagram was used to intersect the key gene module closely associated with the high-immunity in melanoma and the genes differentially expressed between the high-immunity and low-immunity groups.Survival analysis was performed through the \u201csurvminer\u201d and \u201csurvival\u201d R packages. Kaplan\u2013Meier (K\u2013M) survival curves were drawn to analyze the relationship between DEG expression and the overall survival (OS) of melanoma patients.The enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were carried out by \u201cclusterProfiler,\u201d \u201cenrichplot,\u201d and \u201cggplot2\u201d packages in R. GO results included molecular function (MF), biological process (BP), and cellular component (CC). FDR\u2009<\u20090.05 was statistically significant.https://cn.string-db.org/), the interaction between cross genes was obtained. Core genes were identified using the CytoHubba plug-in of Cytoscape (http://www.cytoscape.org) (version 3.9.1) with the highest confidence (0.70) as a threshold.Through PPI network analysis of the STRING website  between the PTPRC wild-type and mutant groups, the nonparametric Wilcoxon rank-sum test was used. A two-sided Univariate and multivariate Cox regression analyses were performed to confirm whether this gene could serve as an independent clinical prognostic predictor. Then the Risk Model was constructed. The area under curve (AUC) values were calculated for each model, and the corresponding 1 -, 3 -, and 5-year ROC curves were plotted. The following formula was used to calculate the riskScore from the risk model for all clinical cases: RiskScore\u2009=\u2009\u2211ki\u2009=\u20091\u03b2iSi.GSEA was performed to explore the potential mechanisms of genes affecting prognosis, and obtain the signaling pathways of up-regulation and down-regulation. FDR\u2009<\u20090.05 was considered statistically significant. The infiltration of 22 immune cell types in tumor tissues between the low- and high-risk groups were estimated by CIBERSORT.To study the relationship between the PTPRC and the expression level of immunosuppressive molecules related to ICIs, we used \u201climma\u201d package to perform the co-expression correlation analysis of PTPRC with ICIs. Gene expression data with immunotherapy was downloaded from the GEO database (GSE91061) and analyzed to determine the expression level between responders and non-responders. The R package Seurat was used for most preprocessing steps for the scRNA-seq datasets downloaded from the GEO database (GSE120575). The batch effect was removed at first, and then t-SNE for data visualization was calculated with fast interpolation-based t-SNE.https://www.proteinatlas.org/).The immunohistochemistry expression graph of related genes was obtained from the HPA database (2 in a humidified incubator. Cells from generations 3\u20134 were utilized for subsequent experiments.The human tumor cell line A375 (CL-0014) and MEL-28 (CL-0717) were purchased from Procell. Cells were cultured in DMEM medium  supplemented with 10% FBS  and 1% 10\u2009kU/ml penicillin/10\u00a0mg/ml streptomycin  at 37\u00a0\u00b0C with 5% COThe transfection reagent mixture was prepared as follows: 5\u00a0\u03bcL of LipofectamineTM2000 , 250\u00a0\u03bcL of serum-free medium, 5\u00a0\u03bcL of negative control siRNA or PTPRC siRNA (2\u00a0\u03bcg), and 250\u00a0\u03bcL of serum-free medium. A375 and MEL-28 cells transfected with PTPRC siRNA (5\u2032-GCTGCACATCAAGGAGTAATT-3\u2032) were named si-PTPRC group, and transfected with negative control (NC) siRNA were named si-NC group. The reagents were mixed well in two EP tubes, and left for 20\u00a0min. The complete culture medium in the corresponding wells was discarded, and the mixed transfection reagents were added, and the corresponding treatment was carried out 48\u00a0h later. The transfection efficiency was determined by the expression levels of protein  and mRNA.3 cells/well). Thereafter, the cell viability was assessed using CCK-8 (China Elabscience Biotechnology) at 0, 12, 24, 36, and 48 h according to the manufacturer\u2019s instructions.After transfection with si-NC or si-PTPRC in A375 cells and MEL-28 cells, the cells were seeded into 96-well plates  were implanted into the upper chamber, and 600\u00a0\u03bcL of DMEM containing 10% FBS was placed into the lower chamber. The Transwell plates were then incubated at 37\u00a0\u00b0C in a 5% CO2 incubator for 48 h, fixed with 4% formaldehyde for half an hour, and stained with 0.01% crystal violet. Unmigrated cells were carefully removed with a cotton swab, and then the cells that migrated into the lower chamber were counted under a microscope.After transfection of A375 cells and MEL-28 cells with si-NC or si-PTPRC, cell migration assays were performed using 24-well Transwell plates . Cancer cells . Reverse transcriptase reactions were performed using the PrimeScript\u2122 RT kit (Takara). \u03b2-Actin was used to normalize the expression levels of mRNAs for genes. Normalized CT values were used to calculate ploidy differences between groups. Primer sequences are shown in Supplementary material Table P\u2009<\u20090.05 was considered statistically significant.All bioinformatic statistical analyses were performed using the R software package version 4.2.3. Kaplan\u2013Meier survival analysis and the log-rank test were used to derive prognostic values and evaluate patient survival in different subgroups in each dataset. Or continuous data, the non-parametric Wilcoxon rank sum test was employed to examine the connection between the two groups. The Kruskal\u2013Wallis test was used to compare more than two groups. Using univariate and multivariate Cox regression , clinical characteristics from the high and low-risk groups were evaluated for prognostic factors. Spearman correlation analysis was used to assess correlation coefficients. Experimental data were analyzed using GraphPad Prism 9, and This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The Third Xiangya Hospital of Central South University. And informed consent has been obtained from all subjects and/or their legal guardians.Supplementary Information 1."}
+{"text": "The last 20 years have seen considerable research on the nature and biopsychosocial impacts of compassion training on self and others. This training is usually focused on calming and slowing the mind and body and on individual imagery practices and mantras. This study explored the effects of three variations: 1. The impact of using energizing music to generate activation and \u201cdrive\u201d for compassion; 2. To focus on imagining \u201cbreathing in and breathing out a white light or mist of compassion\u201d to bring compassion to the world; and 3. While listening to energizing music, participants were guided to imagining connecting to the compassion (Sangha) community, imagining oneself as linking with others as part of communities seeking to help the world.From approximately 1,600 members of the Compassionate Mind discussion list, participants were invited to take part in a new energizing focused self-practice study. The study involved listening to recorded guidance on the evolutionary model of compassion and the need to address the potentially harmful side of our nature. This was followed by a 4 1/2-min tonglen-informed guided practice of breathing in and breathing out compassion accompanied by energizing music. Forty-three participants completed several self-report scales measuring compassion orientation, wellbeing, social safeness, and positive affect before and following 2 weeks of practice. Participant experiences were recorded from 6 open explorative questions.d = \u22120.76). In addition, qualitative data revealed that the participants had experienced the practice as energizing, inspiring, and felt socially connected and that it had significant impacts on other aspects of their lives. Some participants noted that engaging with suffering also stimulated sadness.Self-report measures taken before and following 2 weeks of practice revealed significant increases in self-compassion, compassion to others, openness to compassion from others, activated positive affect, safe positive affect, social safeness, and wellbeing, with the largest effect size relating to compassion for the self (This study found that pairing energizing music with breathing practices and specific compassion visualizations, focusing on the desire to bring compassion to the world and be part of a compassionate community, was well-accepted and had a range of significant positive impacts. This study indicates the potential value of exploring energizing in comparison to the more standard soothing and settling practices as ways of stimulating the biopsychosocial processes of compassion. These are first to explore the processes that facilitate people's detection and preparedness to move toward and engage with suffering, and second, the processes that influence people's efforts to work out what to do and actually do them , they suggested that training in compassion needs to teach abilities to be sensitive and have the courage and wisdom for skilful engagement but also ways to be helpful. Hence, the second element of the compassion algorithm is the action and response function. When guiding people in compassion, it is important how and what people learn about compassion  and to envision a golden light extending from their heart to the heart of the other person (p. 4).The rapid movement from awareness of suffering to positive responses with the wish to be free of suffering stimulates different physiological systems . There is good evidence that music can have a variety of impacts on emotional states as exemplified by how it is used in film scenes. There is also good evidence that music can have major therapeutic benefits  was introduced to several different practices of tonglen by a Buddhist monk called Choden and his colleagues during training in Samye Ling   showed interest, only 43 participants completed measures both before and after using the practice for 2 weeks. The final group consisted of 35 female and 8 male participants aged 25\u201368 years .Initially, a study invitation was sent via email to the Compassionate Mind Foundation Google discussion list of ~1,600 members , inviting them to take part in the study. The only exclusion criterion was the inability to understand spoken and written English. Although many participants , a musician interested in the role of music to create emotional textures, had been practicing compassion exercises (such as the flow of life and tonglen practices), using different types of music. He identified one piece of music by Thomas Bergersen that, for him, generates energy for compassion. Out of curiosity, he offered to share his experience with participants at an online workshop to explore their experience. Participants were very enthusiastic and fed back that combining the music with this guided meditation generated feelings of being energized, connected, and joyful. With this anecdotal evidence, the authors decided to explore these experiences in a more standard scientific way.Final Frontier from the album Sun (https://www.youtube.com/watch?v=BAzCf0ascW8), for permission to use the music with a guided meditation and it was granted for a small fee.The authors contacted Thomas Bergersen, composer of the music called that have been built for us, not by us, to pursue survival and reproductive biopsychosocial goals \u2014this is the cultivation of compassion. Although given an evolutionary orientation  then developed a video that provided a brief overview of the CFT evolutionary approach to compassion which was followed by guided meditation. This included the following information: 1. We, like all living things, have bodies, brains, and minds Gilbert, . A lion Gilbert, , 2019a, Gilbert, . Hence, Rather than breathing in suffering and breathing out compassion, participants were guided to imagine breathing in a compassion-based white light or a mist that fills the body, then breathing out white light and mist, whilst imagining it reaching out into the world to address the suffering of others. Additionally, participants were guided to focus on feeling that this was something they really wanted to do, and how wonderful that would be if they could do it and help the world move toward a more compassionate orientation. This is linked to what is called a Bodhicitta wish . They provided written consent in accordance with the Declaration of Helsinki's ethical principles for medical research involving human subjects . Participants were also invited to complete a number of open-ended questions about their experience and how the meditation made them feel.Participants were asked to complete the following self-report measures both before and after 2 weeks of practice:Gilbert et al.  developeThe Compassionate Engagement and Action Scales . The participants were asked to complete several single-item questions derived from common reflections people had made using the meditation. They were not derived in any specific order but simply developed to understand how the meditation was experienced. Participants were asked to rate the extent to which the practice made them feel more, for example, energized, joyful, and socially connected, on a 10-point Likert scale .What were your standout experiences?Can you describe how the practice made you feel?Could you describe any impact the practice may have had on you?Did you notice any change in your experience and understanding of compassion?How do you think the practice might change the way you act in the future?Any other feedback?Participants were asked the following six open-ended questions exploring their experiences following 2 weeks of practice:In addition, participants were invited to reflect on their experiences and share their observations from weeks 1 and 2. We analyzed this data separately from the six open-ended questions.n = 72/115) only completed the pre-measures. This left 37.4% (n = 43) of participants who completed all of the measures. This formed the basis of the analysis. Data were checked for normality and outliers; skewness and kurtosis values ranged within acceptable levels and no statistically significant violations were found  was used to explore the data. This provided an opportunity to explore theoretical issues, enhance understanding of the data  was based initially on word frequency. Responses containing words with the highest frequency were then coded and grouped into categories. We then returned to all responses for each question to ensure themes had not been missed. The responses for each category were extracted and compiled into documents that covered each of the open-ended questions separately. This analysis sought to explore and identify critical processes  and mindfulness practices (33/43 participants) as shown in Participants were able to practice the meditation without music if they wished. The majority of participants reported that they practiced with the music three or more times in week 1 (30/41) and week 2 (23/41), see .t-tests (two-tailed) which were conducted to compare the pre- and post- questionnaire responses. After 2 weeks, there were significant increases in self-compassion, compassion to others, openness to compassion from others, activated positive affect, safe positive affect, social safeness, and wellbeing, with small to medium effect sizes. Differences in relaxed positive affect approached significance (p = 0.55).Interestingly, those who practiced without the music three or more times had a change in self-compassion score of 6.64 in the first week whereas those who always practiced with the music had a change in score of 9.48.Results of the qualitative data analysis are reported here under the headings of each open-ended question with the following themes. All names are pseudonyms.1. What were your standout experiences?Energy, energizing, exhilarating: Participants reported an increase in energy and how this increase influenced their thinking after the experience.I noticed that on a couple of occasions I was surprised at the energy that I had which usually I wouldn't have. I even noticed that I had become more flexible and more aware of and not wanting to set into routines from which I would be reluctant to change. I realized I was more encouraging of myself to try different things. HettyListening the first time - exhilarating, emotional, uplifting. Feeling energised after each practice and that I have more to offer than I give myself credit for. HilaryConnection to self: The experience encouraged participants to connect with themselves and find the motivation to \u201cre-experience\u201d positive and negative events. It also encouraged more appreciation of what they had to offer.This was a moving experience. I connect with my life and trying to re-experience the positive and negative events connected. I feel with more energy and I started planning workshops. I would like to learn more and to use these exercises with myself and others. VeronikaConnection to others: Feeling a connection to others was reported in relation to \u201cnameless/faceless\u201d others, other participants in the study, colleagues, and \u201ca sense of goodness in the world.\u201dThe emotional power of actually visualising others in the world doing this exercise and expending compassion to a nameless/faceless other. Because of the study, I felt a connection with the other participants of the study and was able to visualise others doing the same exercise as I and that helped me to accept compassion from others. There was one occasion where I had a difficult interaction with a colleague and utilised the exercise to extend compassion to them and the emotional connectedness and universal human experience I felt with them was very powerful to the point I became tearful. It helped me to see an alternative view of the disagreement and fix the situation. CleoA rousing sense of connectedness, a stirring of energy in my chest, feeling of being powerful with compassionate connection (rather than power in regards to others). A sense of goodness in the world. PatA global compassionate community: Feeling connected to a global movement of compassionate others was described as \u201camazing,\u201d \u201cemotional\u201d, and \u201cbeautiful.\u201d Some participants reported feeling a transhistorical connection to \u201cgenerations of human beings\u201d with a sense of their contributions to \u201cmaking life a little better for all of us\u201d.Imagining being joined in a global circle of empowering and compassionate white light at various times throughout the day was an amazing experience. DanielleI really enjoyed imagining being part of a whole movement of people all breathing out compassion into the world. LisaThe meditation made me appreciate the effort and the contribution that humans have made, over the years. The music made me see generations of human beings, swarming like bees, all busy trying to make life a little better for all of us. And this is beautiful. Kirsty2. Can you describe how the practice made you feel?Part of a compassionate community: The experience of feeling \u201cunited\u201d and \u201cbelonging\u201d was reported as powerful for many participants. Furthermore, participants emphasised how this made them feel more connected and hopeful.A sense of belonging, strong, and the unity of my whole light joining others'. KellyLike I am part of a compassionate world, I mostly felt that in my chest. I noticed my chest would actually expand and take up more space. It made me feel more hopeful for a compassionate world and positive about the world. It made me feel safer. PatConnected to compassionate motivation and part of a compassionate community. RitaUnited, like a compassionate power ranger or another team of superheroes. SaraIt made me feel connected with others, both those doing the study and those others in the world who work daily to spread compassion. It made me feel more prepared for the rest of the day and able to take on anything that came my way. CleoUplifted, joyful, hopeful, inspired by others, a strong sense of belonging. HilaryPhysical experiences: Physical experiences as a result of, and during, the practice included \u201ccalm,\u201d \u201cwarm,\u201d \u201cpowerful,\u201d \u201cstrengthened,\u201d and \u201cALIVE.\u201d One participant described how their experiences ranged from feeling \u201canxious and activated\u201d to \u201ccalm and soothed.\u201dCalm but energised, optimistic, ready. DanielleIt made me feel warm, as though my heart were expanding. GracePowerful, connected, courageous. IngridIt made me feel ALIVE and invigorated. LisaSense of being physically and emotionally strengthened, grounded, nourished, determined. TinaEnergized. Connected with my body and centred. I enjoyed the music... VeronikaA range from anxious and activated to calm and soothed. WilmaI felt that the music and energy can linger in the body especially when [I] think about it, certain rhythm and image comes up from the body, interconnected within self and the outside world. MariaChanges in emotional experience over time: Some participants reported feeling \u201coverstimulated\u201d, \u201coverwhelmed\u201d and \u201coverloaded\u201d with one participant describing a range of difficult emotions that they worked through (Carl). However, participants also noted over time that these challenges were reduced and became easier with practice.A whole range of emotions came up, fear, sadness, the feelings got better over the two weeks. CarlA bit euphoric, on the edge of overstimulated. I have a pretty good acquired positive affect tolerance but I found it not quite but almost demanding to take in the intensity of the musical track. FreireAt first a little distressed and overwhelmed even when I adjusted the volume. After the first week I started enjoying the music. Una3. Could you describe any impact the practice may have had on you?Reminders and re-connection: Perhaps unsurprisingly, given the global environment that participants found themselves in, many reported feelings of re-connection and being reminded that there are compassionate others in the world and they are not alone.I think that I've been struggling for the past 2 years with the pandemic, restrictions, isolation, war, callousness in the world, etc, and this practice helped me re-connect to a feeling that there are other things to be aware of \u2013 joy, collaboration, overcoming dark forces\u2026we all have a dark side (or sides) but we can work together to make the world better. DanielleA consolidated reminder that I am not alone! KellyI feel more connected to the world as a whole and the people within it. More hopeful. CarlI liked the visualisation of white coming in and out as I was breathing, it made the practice more tangible for me. I live with the impact of trauma and it helped me to feel safer in that there are a lot of compassionate people in the world when I can be quite threat focused. PatI feel motivated to plan and practice this exercise. This exercise made me think about what is happening in other countries and the war and how other peoples are suffering right now. I feel the necessity of help and support and do something to make a better life. VeronikaIt has lifted my energy levels and confidence to engage with others. I also have more space and energy to be compassionate to others again, not being debilitated by my own stress, anxiety and depression. ElizabethA lovely reminder that I am not alone - something to call upon and use to connect. Hilary4. Did you notice any change in your experience and understanding of compassion?Expanded understanding and appreciation for the dimensions of compassion: Participants reported how the experience had expanded their understanding of, and appreciation for, experiencing a different dimension of compassion. Some participants noted the significance of experiencing compassion as a drive, and as activating, as opposed to previous experiences of soothing.Yes, compassion is being connected. It is also transcending the immediate reality. AndrewA lot of the time I practice calming and soothing compassionate skills and this helped me to activate compassion focused drive rather than soothing. This is really very useful. PatCompassion is action... I want to do.. I would like to help. VeronikaI think the music directed me toward specific aspects of compassion that are not usually at the core of my awareness and practice; rather than empathy, connecting with suffering, loving kindness and being with the difficulty, trauma and suffering of humanity that is normally where my practice rests, I felt a much larger, expansive, joyful, fierce, transpersonal and cosmic level of compassion. I'm certain that reflects Gilbert's take on compassion and was refreshing and uplifting for me. AnnaIt made me think that compassionate practice doesn't have to be slow. Excitement and enthusiasm within the music can still have compassionate qualities. I have used the white smoke visualisation while running for example. So the practice could contribute to exercise routines to calm anxiety and breathing. GretaEmbodied experience of compassion: The embodied experience of compassion and its impact was highlighted by participants. Participants spoke of how the theoretical concepts of compassion became felt, and in turn, this aided in motivation and commitment to being compassionate.Yes, I have come to have a more energised, joyful, lighter sense of compassion - something I had been working on for a long time. The music paired with the CS practice helped me feel these things (as opposed to thinking about or wanting them). DanielleGrounding and a \u2018felt\u2019 or embodied sense of compassion. RitaYes. We are always telling our clients it's not their fault. For the first time, this did not just come across as a conceptual idea. I actually felt this for the first time this past week. I was able to be far more observant of my own process with an interested and curious attitude. I was able to provide myself with reassurance in a difficult client situation and....I ACTUALLY FELT REASSURED. I have had that happen before, but not like this. The reassurance felt....believable. EddieIt helps to strengthen my understanding and embodiment of compassion, inhaling the sensitivity as awareness is also based on energy and exhaling to help the others which in return helps self too. Maria5. How do you think the practice might change the way you act in the future?Use exercise to develop own compassion practices: Participants described how they hoped the exercise would help them to develop their own personal practices with others.Be more energetic with compassionate endeavours, not just calm and soothing. TinaI would be well served to integrate these components into how I experience and practice compassion. it makes it much bigger than what I am able to generate and give, but tapping into a larger stream. AnnaWell, if things progress as they have, I suspect that doing this practice each day  can only strengthen my own sense of compassion, and my compassionate self, and motivate me to continue working to bring compassion to others. It has helped me slow down when I needed to address my own suffering. I think the biggest change is really feeling from the inside out reassured. EddieIf the impact would remain each time, i believe with time would make me more calm, attentive, compassionate. JudeHopefully making me to do small compassionate things with more care and beauty. KirstyThis is a practice of building a compassion mind and its neurological pathway. It will be reactivated whenever and where ever is needed. MariaCall upon exercise for personal use: The exercise was highlighted as something participants would return to when they were distressed.I think when I'm feeling stuck, afraid, beaten down, or just crappy, I can listen to it and re-connect to my inner compassionate warrior, or even imagine it lifting me. DanielleI think it will protect me from feeling so low when distressed - less isolated. KellyI'll use these techniques to help myself cope with stressful situations and to moderate my responses to difficulty in future. ElizabethI think it will enhance my stamina, my sense of myself against challenges. HilaryUse exercise to engage with others more: Connecting with compassion being expressed by others was an area that participants thought the exercise would help them with.I would hope that it would help me grown in kindness and empathy toward others and myself. It would nice to release myself from the distress of being judgmental of myself and others and more loving. HettyReminding me to draw on the compassion of others, even though I might not know, are putting compassion out into the world. IngridPossibly connecting more easily, authentically and openly with others who are showing compassionate motivation and behaviours. Rita6. Any other feedback?Positive feedback: Positive feedback reported changing views about compassionate motivation in participants themselves and others around the world.I am 100% glad that I had the opportunity to take part in this study. It has really changed how I feel about myself and the world around me. I am full of energy and enthusiasm which is a very welcome experience for me. I haven't felt like this for a number of years. OliviaHelped with own and others' fears, blocks, and resistances (FBRs)This practice has made me realize my struggle with myself where I couldn't imagine breathing out compassion towards others, because I couldn't accept that I could be a storehouse of compassion. And then I realized that even as I thought this about myself, I could feel compassion towards myself for feeling so badly about myself and that was a wonderful feeling! HettyReally helpful practice for strengthening determination and ability to keep bringing compassion into the world, especially when this is very challenging. The musical component in particular felt as if it helped me to replenish my energy and the sense of belonging to compassionate community strengthened my commitment and determination to keep going when compassion and connection feels very hard (is being unconsciously rejected/resisted). TinaThe following section reports the participants' reflections and observations from weeks 1 and 2.Participants engaged with the practice and communicated openly about their experiences. Some reported feeling energized by the music whilst others felt the music was too intense.The music felt inspirational. I imagined breathing compassion (in and out). I imagined others around the world being compassionate and imagined being connected to them. The music, however, didn't match my sense of compassion which is more tranquil. HarryThe music was music therapy - very powerful and evocative and made me imagine how your research team/Paul Gilbert imagines the properties of compassion - if I had to choose a piece of music to evoke compassion, it would not have chosen that one. very interesting to lean toward the dramatic, energizing, dynamic, expansive qualities within compassion as evoked in the music. my compassion mode is much more quiet, soft and tender, so it was an interesting stretch to enter the practice with the music. I appreciated what was evoked in me. FreireI like the music better, the more I do it, and it is never the same meditation, there were always new images every time I do the meditation. KirstyParticipants continued to practice the exercise, with many reporting that they had engaged more with the music, were feeling more energetic, confident, and connected, and were adapting the exercise where they felt it was needed. There were also powerful reports of the influence of the practice on FBRs. Some participants described how they had used the practice over time, adjusted to the practice, or adjusted their thinking and understanding of the experiences they were having .I made more sense of the imagery this week! I think if I can't imagine it then it won't happen, that's the energising for me. I got an image of white light and could use it, that felt incredible. I enjoyed the music practise a lot more in the second week. I still wanted more of a choice of music , the cut off crescendo was less distracting. I used the music on HiFi speakers without the verbal guiding, I'd memorised that, that was the most impactful experience. CarlI noticed that doing the practice with the music was much more effective if I took additional time to practice beforehand; doing the 5 min practice (with music) alone was not the most powerful access to compassion, despite the music being evocative. it feels like 5 min of compassion practice of any kind is too short; more time allows me to really find a deeper connection with compassion and then the 5 min practice at that point is very accelerating. AnnaAfter the challenges of week 1 with accepting compassion from others I attempted to visualise this in conversations where I felt the other people expressing positive emotions to me. In these conversations I would visualise the stream of light and compassion coming from them to me. I found that this actually helped me to find the parts of the conversation and their behaviour that would indicate compassion and care that I would usually miss. CleoYes, following up from the last box, I really have begun to feel far more compassionate toward myself as much as I am toward others. I have taken far more time to address my own suffering as it has arisen. Going into client sessions after having completed the practice, I have noticed being more engaged and open - far less tired. I really cannot overstate how much I have enjoyed and benefited from this practice of energizing compassion. To go from low motivation to address my own suffering to feeling that deep sense of belonging and connectedness and wanting to help myself...there is no better feeling. EddieIt is important to keep in mind that compassion is about connecting to suffering and the first movement to suffering can be stressful and distressing  did so.The self-report measures showed significant changes pre-to-post in the study variables. The effects included feeling energized, joyful, socially connected, hopeful, courageous, and wise see . In addiexhilarating, emotional, uplifting. Feeling energised after each practice and that I have more to offer than I give myself credit for.\u201d There were also experiences of feeling strengthened and energized. For example, Danielle noted \u201cI think when I'm feeling stuck, afraid, beaten down, or just crappy, I can listen to it and re-connect to my inner compassionate warrior, or even imagine it lifting me.\u201d Participants reported that this \u201cnew\u201d practice had enabled them to expand their understanding and appreciation for the dimensions of compassion. It gave them a more embodied experience of compassion. They also reported how they intended to continue using the practice in both a personal and professional capacity. Participants also noted an increased sense of social connectedness, belonging, and being part of a group of others, rather than pursuing compassion alone. The qualitative analysis indicated that many participants enjoyed using the music, however, nine participants did not like it. Consequently, we are exploring variations where individuals can choose their own music that will give them a sense of activation and enthusiasm in follow-up studies.In regard to the experiential and qualitative findings, stand-out experiences included increased energy and connection to self and others. As noted in our report on the physical sensations, many experienced feelings of warmth, calmness, and strength. These themes arose in other parts of the interview too. For example, Hilary noted that listening for the first time felt \u201cAs often noted, the first movement to compassion is to address suffering. This can be distressing and stressful. Although we did not set out to explore this, some clients did note that they experienced distressing emotions when they connected to the realities of the human dark side and suffering in the world. Given the global environment that participants  experienced at the time of the study\u2014war in Ukraine, the continuing COVID threat, and climate change\u2014this distress is important to anticipate but equally not to be overwhelming.n = 43) who were already familiar with the basic evolution-based compassion model. Indeed, only 4.7% of participants reported that they did not use compassion-based practices very much (for mindfulness and other meditation practices this was 9.3%), suggesting that the majority of participants in this group were regularly engaging in related practices. Subsequent research will therefore need to work with na\u00efve participants and explore if the practice can have the same powerful effects. The small numbers also made it difficult to investigate specific effects like practicing with and without music. This will need to be addressed in subsequent studies. In addition, subsequent studies could invite clients to choose their own energizing music.This study recruited a small number of members from a compassion discussion list (Another limitation inherent to studies incorporating self-report measures is the risk of demand characteristics biasing results. However, as this was a small proof-of-concept trial, it is hoped that the rich experiences reported as part of the qualitative analysis may help to support the quantitative responses. Subsequent studies may incorporate a single or double-blind design with a control group to mitigate against possible demand characteristics. There was little data collected from male participants. It is unclear whether this was because male participants showed less interest in the exercise than female participants, or whether this was a natural variation resulting from the sampling methods used. Future research should therefore aim to address this, so that we gain a better understanding of how male participants, in particular, experience the exercises.In summary, as a proof of concept, this study has shown the potential value of integrating energizing music with an energizing compassion focus, which had a positive impact on participants. Clearly, subsequent research will wish to identify and explore aspects of specific components. For example, to what degree did the energizing music or the sense of being part of a community impact results, and which aspects carried the most powerful impact? This study was not designed to explore that but rather whether this combination of energizing and engaging in a sense of community was acceptable to participants and of value. This is worthy of subsequent research including physiological and long-term effects.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Available by contacting the corresponding author.The studies involving human participants were reviewed and approved by University of Derby College of Health, Psychology and Social Care Research Ethics Committee. The participants provided their written informed consent to participate in this study.PG and JB were involved in all aspects of the study. PP, HG, and JB analyzed data. All authors contributed to the article and approved the submitted version."}
+{"text": "Bradypus and Choloepus sloths over periods of days to weeks. We investigate how fluctuations in the environmental conditions affect the activity of sloths inhabiting a lowland rainforest on the Caribbean coast of Costa Rica and examined how this might relate to their low power lifestyle. Both Bradypus and Choloepus sloths were found to be cathemeral in their activity, with high levels of between-individual and within-individual variation in the amounts of time spent active, and in the temporal distribution of activity over the 24-hour cycle. Daily temperature did not affect activity, although Bradypus sloths were found to show increased nocturnal activity on colder nights, and on nights following colder days. Our results demonstrate a distinct lack of synchronicity within the same population, and we suggest that this pattern provides sloths with the flexibility to exploit favourable environmental conditions whilst reducing the threat of predation.It is usually beneficial for species to restrict activity to a particular phase of the 24-hour cycle as this enables the development of morphological and behavioural adaptations to enhance survival under specific biotic and abiotic conditions. Sloth activity patterns are thought to be strongly related to the environmental conditions due to the metabolic consequences of having a low and highly variable core body temperature. Understanding the drivers of sloth activity and their ability to withstand environmental fluctuations is of growing importance for the development of effective conservation measures, particularly when we consider the vulnerability of tropical ecosystems to climate change and the escalating impacts of anthropogenic activities in South and Central America. Unfortunately, the cryptic nature of sloths makes long term observational research difficult and so there is very little existing literature examining the behavioural ecology of wild sloths. Here, we used micro data loggers to continuously record, for the first time, the behaviour of both  For the majority of animals, time is clearly divided between periods of activity and periods of inactivity . During Activity patterns vary greatly across and within taxonomic groups, with different taxa being primarily diurnal, nocturnal, crepuscular or, occasionally, a combination of all these . SpeciesBradypus and Choloepus) are cryptic canopy mammals that are considered to be cathemeral and nocturnal in their activity patterns, respectively  (Panthera onca) and ocelots , harpy eagles (Harpia harpyja)  and tayras (Eira Barbara) , cathemerality and asynchronous activity of individuals within the same population may also function as an effective predator avoidance strategy   ), big caability) .Understanding the temporal activity patterns of sloths and their ability to withstand environmental fluctuations is of growing importance when faced with a dramatically changing world. Climate change alongside with various anthropogenic pressures  are threatening sloth populations throughout South and Central America, with both species of sloth in Costa Rica recognised as conservation concerns in the country . In ordeBradypus and Choloepus sloths inhabiting a lowland rainforest on the Caribbean coast of Costa Rica to record, for the first time, their behaviour continuously over periods of days to weeks. We examined how environmental conditions impact their activity budgets and how all of these factors might relate to their uniquely low power lifestyle.Here, we used micro data loggers attached to wild Considering the known link between sloth metabolism, digestion, and ambient temperature , we hypoThis research was approved by the Swansea University Animal Welfare & Ethical Review Process Group (AWERP), and the Costa Rican government and associated departments  permit number; R-033-2015.Bradypus variegatus sloths  and four adult Choloepus hoffmanni sloths   . The exa female) . However female) .Alouatta caraya), and predators including tayras (Eira barbara), spectacled owls (Pulsatrix perspicillata), boas (Boa constrictor), and ocelots . Notably, the sloth\u2019s primary diurnal predator, the harpy eagle (Harpia harpyja) is extinct in the region.All sloths were from the same region of secondary forest surrounding the Sloth Sanctuary of Costa Rica . This forest is protected from development and has a high level of canopy connectivity, however, there are some anthropogenic stressors present in the area including human settlements, domestic dogs, and a major highway. There is a variety of other species present in the area including other arboreal folivores such as howler monkeys  without anaesthesia. Choloepus sloths were anaesthetised during capture using 1 mg/kg of ketamine  and 0.008 mg/kg of dexmedetomidine  administered intramuscularly. Anaesthesia was reversed prior to release using 0.008 mg/kg of anti-sedante . Body mass measurements were taken for each individual at capture ).All via elastic, backpack-style harnesses, positioning the devices firmly on the upper back  data loggers , programper back   was braided into the harness of these animals which allowed the backpack to fall off when exposed to heavy rain.After being equipped with a backpack, all sloths were released in the same location they were originally found. Backpacks were retrieved manually when the sloth was in an appropriate position for recapture close to the ground following daily checks. Due to the difficulty of recapturing Bradypus sloth data and 176\u00a0h of Choloepus sloth data to coincide with tag deployment periods. Ambient temperature (\u00b0C), humidity (%), wind speed (m/s), maximum gust speed (m/s) and rainfall (mm/15 min) were recorded every 15\u00a0min by a weather station (Davis Vantage Vue) mounted 17\u00a0m above the ground on the edge of the study site. Light intensity was measured using a HOBO Pendant\u00ae UA-002-64-Temperature/Light Data Logger (64K) mounted adjacent to the weather station.Corresponding weather data was recorded for 997\u00a0h of http://www.wildbytetechnologies.com/software.html). Standard metrics for looking at animal behaviour , specifically designed to enhance manual identification of Daily Diary datasets. Data from all sensors within a single tag and derivatives (VeDBA and rate of change metrics) were displayed as time-based parallel traces that were inspected over both time- and sensor resolution scales.To determine overall activity budgets, the total percent time allocated to different behaviours was calculated for each dataset.Bradypus and Choloepus sloths differed in their overall levels of activity, the mean hourly percent time spent active was calculated and compared using a Wilcoxon rank-sum test. The significance of between-individual variation in the mean hourly percent time spent active was calculated using Kruskal-Wallis tests.To ascertain whether vs climbing downwards for all sloths combined was determined using a Welch\u2019s two-sample t-test. The total time spent climbing downwards  was compared across different times of the day using an analysis of variance (ANOVA) followed by a Tukey Honestly Significant Differences (Tukey\u2019s HSD) test. This was repeated for the total time spent climbing upwards. For these tests, the 24-hour day was broken down into 4 bins: morning (06:00\u201312:00), afternoon (12:00\u201318:00), evening (18:00\u201300:00) and night (00:00\u201306:00). The significance of the relationship between climbing upwards and light intensity was tested by using a point bi-serial correlation case of the Pearson\u2019s product-moment coefficient (as \u201cclimbing up\u201d is a Boolean variable).The significance of the difference in the total amount of time spent climbing upwards Bradypus sloths and four individuals totalling 11 days for Choloepus sloths). Corresponding daytime and night-time ambient temperatures were also calculated.To determine the effect of the environmental conditions, the mean hourly percent times spent active, mean hourly ambient temperatures, and mean hourly maximum wind speeds were calculated. Using this data, and assuming diurnal hours to be between 05:00 and 17:00, and nocturnal hours to be between 17:00 and 05:00, the daily percent times spent active diurnally and nocturnally were calculated for each sloth for each day that had corresponding weather data  using normal data that was centred and box-cox transformed (power transformation of 0.3608). The significance of the relationships between maximum wind speed and the mean percent time spent active for the day and night were tested using Spearman\u2019s Rank correlations.The significance of the relationships between the percent time spent diurnally active and the mean diurnal temperature for both \u03b1\u00a0=\u00a00.05, followed by a Dunn\u2019s post hoc test to identify which periods of the day differed from each other. Based on the significance level, sloths are identified as either nocturnal (significantly higher activity during the night compared to the day), diurnal (significantly higher activity during the day compared to the night) or cathemeral .In order to determine the significance of the difference in percent time spent active between different periods of the day for each sloth, the 24-hour day was again broken down into 4 bins: dawn (04:00\u201307:00), day (07:00\u201316:00), dusk (16:00\u201319:00) and night (19:00\u201304:00). The percent times spent active during each bin were compared using a Kruskal-Wallis test with a significance threshold of Bradypus sloths and 206\u00a0h for Choloepus sloths. Bradypus sloths were inactive for a total of 85.5% of time, comprising 62.7% and 22.8% of time spent sleeping and resting, respectively. There was significant between-individual variation in their mean hourly percent time spent active , with total time spent in inactive behaviours ranging from 49.3\u201397.1% , however Choloepus sloths also demonstrated significant between-individual variation , with total time spent in inactive behaviours ranging from 37.8\u201395.2%  compared to climbing downwards, where the equivalent values were  . Sloths spent more time making vertical movements within the canopy during nocturnal hours. This included significantly more time spent climbing downwards during the night compared either to the morning or afternoon . Similarly, sloths spent a significantly higher proportion of total active time climbing upwards at night compared to the morning, afternoon, or evening . There was no significant correlation between the occurrence of upwards climbs and light intensity for all sloths .Both Bradypus or Choloepus sloths . However, Bradypus did spend significantly more time active nocturnally (17:00\u201305:00) on days which had cooler ambient daytime temperatures (05:00\u201317:00) , with individuals differing significantly in their response to this. Overall, there was also a strong significant negative relationship between the percent time Bradypus sloths spent nocturnally active and the ambient night-time temperature (17:00\u201305:00) , with increased activity on colder nights (R squared = 0.731).The mean and standard deviation in ambient temperature during the study period was of 26.3 \u00b1 3.0 \u00b0C, ranging from an overall recorded minimum of 20.5 \u00b0C to an overall recorded maximum of 32.8 \u00b0C. There was no significant difference in the amount of time spent diurnally active and the mean ambient temperature during daytime hours (05:00\u201317:00) for Choloepus or Bradypus during either the day or the night.Maximum wind speed did not significantly affect the amount of time spent active for either Bradypus sloths were significantly more active during both the daytime  and at night  compared to at dawn, and they were significantly more active during the day than they were at dusk . However, there was significant variation in the periodicity of activity between individuals  (Phascolarctos cinereus)   and koalinereus) . While oinereus)  and inculic rate .All sloths were found to spend more time climbing upwards than downwards, suggesting that it may take a longer \u201cperiod\u201d to cover the same distance when climbing upwards due to the added effort of moving against gravity. This is particularly pertinent in sloths, with their low power usage, because the biomechanical power invested for climbing upwards is given by the rate of change of potential energy  so that power usage is directly proportional to speed. Sloths were also found to spend more time making vertical movements within the canopy during the night than they did during the day, although overall levels of activity did not differ within nychthemeral cycle. This may represent an increased tendency to descend to the ground during the night, although this would seem surprising considering the increased threat of predation from nocturnally active large cats . AlternaCholoepus sloths were previously considered to be strictly nocturnal , their physiology, and the environmental conditions. Inter-individual differences in activity have historically been ignored or considered to be of little scientific value, however such variances likely form an adaptive flexibility of substantial biological importance .Bradypus sloths are known to engage in territorial disputes over access to females  based on the ambient conditions . This, cMyrmecophaga tridactyla) (Dasypus novemcinctus) (Phascolarctos cinereus)   and the cinctus) , as wellinereus) . Howeverlic rate . It has lic rate , and so lic rate .Bradypus sloths to reduce activity at dawn and dusk? Predatory animals including medium and large cats often display crepuscular patterns of activity in order to exploit the vulnerability of early-starting nocturnal  prey species that are operating at their visual limits in terms of light intensity  and Very High Frequency radio transmitter (VHF). Transmitter antenna is wrapped around the chest strap of the harness to avoid catching on branches.Click here for additional data file.10.7717/peerj.15430/supp-2Figure S2g and periods between 5\u201320 s. Downwards climbing includes all climbing activity occurring for more than 15 s with a corresponding increase in pressure typically greater than 0.02 mbar per 5 s. (b) Climbing upwards. Climbing activity is identified by characteristic wave forms in the sway signal, with mean values around 0, amplitudes of 0.5 g and periods between 5\u201320 s. Upwards climbing includes all climbing activity occurring for more than 15 s with a corresponding decrease in pressure typically greater than \u22120.02 mbar per 5 s. (c) Climbing. Climbing  identified by characteristic wave forms in the sway signal with mean values around 0 and periods between 5\u201320 s. In this case, surge acceleration always reads close to 1 and is relatively stable while the heave signal typically reads \u22121. Horizontal climbing is identified by stable pressure values. (d) Grooming. Behaviour identified by stability in pressure (not shown), apparently variable body position manifest in baseline levels of the acceleration and much more dynamism in the movement than shown for locomotion. Wave forms are not particularly obvious, but peak-to-peak periods are typically less than 5 s.(a) Climbing downwards. Climbing activity is identified by characteristic wave forms in the sway signal, with mean values around 0, amplitudes of 0.5 Click here for additional data file.10.7717/peerj.15430/supp-3Figure S3g and periods of 30 s or greater.(a) Sleeping: Identified by static signals across all channels for duration longer than 10 min. (b) Resting. Static accelerometer and magnetometer signals lasting less than 10 min, or typically slow changes in surge and heave with wave forms (if visible) with amplitudes less than 0.5 Click here for additional data file.10.7717/peerj.15430/supp-4Figure S4Each actogram represents a different dataset (#1 \u20134) and each row represents 24 h of data. Percent activity calculated every 15 min and dark phases shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-5Figure S5Each actogram represents a different dataset (#5 \u20136) and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-6Figure S6Actogram represents dataset #7 and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-7Figure S7Actogram represents dataset #8 and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-8Figure S8Actogram represents dataset #9 and each row represents 24 h of data. Percent activity calculated every 15 min and dark phases shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-9Figure S9Each actogram represents a different dataset (#10 \u201315) and each row represents 24 h of data. Percent activity calculated every 15 min and dark phases shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-10Figure S10Actogram represents dataset #16 and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-11Figure S11Each actogram represents a different dataset (#17 \u201319) and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-12Figure S12Each actogram represents a different dataset (#20 \u201321) and each row represents 24 h of data. Percent activity calculated every 15 min. Dark phases are shown by grey shadingClick here for additional data file.10.7717/peerj.15430/supp-13Figure S13Actogram represents dataset #22 and each row represents 24 h, however only 12 h of data were collected for this individual. Percent activity calculated every 15 min. Dark phases are shown by grey shadingClick here for additional data file.10.7717/peerj.15430/supp-14Figure S14Actogram represents dataset #23 and each row represents 24 h of data. Percent activity calculated every 15 min and dark phases shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-15Figure S15Actogram represents dataset #24 and each row represents 24 h of data. Percent activity calculated every 15 min and dark phases shown by grey shading.Click here for additional data file.10.7717/peerj.15430/supp-16Table S1Click here for additional data file.10.7717/peerj.15430/supp-17Table S2Click here for additional data file.10.7717/peerj.15430/supp-18Table S3Click here for additional data file.10.7717/peerj.15430/supp-19Table S4Periods of the day separated into: dawn (04:00\u201307:00), day (07:00\u201316:00), dusk (16:00\u201319:00) and night (19:00\u201304:00). Based on the significance level, sloths are identified as either nocturnal (significantly higher activity during the night compared to the day), diurnal (significantly higher activity during the day compared to the night) or cathemeral .Click here for additional data file.10.7717/peerj.15430/supp-20Supplemental Information 1Click here for additional data file."}
+{"text": "Sensor-based human activity recognition is becoming ever more prevalent. The increasing importance of distinguishing human movements, particularly in healthcare, coincides with the advent of increasingly compact sensors. A complex sequence of individual steps currently characterizes the activity recognition pipeline. It involves separate data collection, preparation, and processing steps, resulting in a heterogeneous and fragmented process. To address these challenges, we present a comprehensive framework, HARE, which seamlessly integrates all necessary steps. HARE offers synchronized data collection and labeling, integrated pose estimation for data anonymization, a multimodal classification approach, and a novel method for determining optimal sensor placement to enhance classification results. Additionally, our framework incorporates real-time activity recognition with on-device model adaptation capabilities. To validate the effectiveness of our framework, we conducted extensive evaluations using diverse datasets, including our own collected dataset focusing on nursing activities. Our results show that HARE\u2019s multimodal and on-device trained model outperforms conventional single-modal and offline variants. Furthermore, our vision-based approach for optimal sensor placement yields comparable results to the trained model. Our work advances the field of sensor-based human activity recognition by introducing a comprehensive framework that streamlines data collection and classification while offering a novel method for determining optimal sensor placement. As part of pervasive computing, sensor-based human activity recognition (HAR) involves the classification of time series data produced by inertial measurement units (IMUs). IMUs primarily capture and measure quantities, such as acceleration and angular velocity, generating discrete temporal data. These data can be segmented into windows and classified, typically utilizing machine learning (ML) models. By leveraging these models, the activity recognition system can classify various activities based on the analyzed patterns within the time series data. The range of movements encompasses both low-level activities, such as walking and standing, as well as high-level activities that involve combinations of multiple low-level activities. HAR demonstrates promising applications across diverse domains, including healthcare, sports, and smart environments ,2,3. SimTo bridge this gap in human activity recognition, we introduce HARE (human activity recognition engineering), a comprehensive framework that revolutionizes the traditional HAR process as illustrated in To assess the effectiveness of HARE, we employed a two-fold evaluation approach. Firstly, we utilized publicly available datasets commonly used in sensor-based HAR. Secondly, we conducted a controlled laboratory study involving ten subjects who performed nursing activities. The combination of existing datasets and a proof-of-concept study allows us to evaluate the performance and applicability of the HARE framework comprehensively. The evaluation allowed us to assess HARE\u2019s performance in data collection, labeling, sensor placement, and real-time feedback, as well as its ability to preserve privacy during these processes. Through our evaluation, we found that multimodality increased the F1 score by up to 4.4% and on-device training by up to 58.7%. Moreover, HARE\u2019s sensor placement recommendations demonstrated similarity to the optimal placements suggested by the best-performing deep learning model, specifically a CNN-LSTM architecture. Our findings demonstrate the effectiveness of HARE in addressing the current challenges in the field of HAR.Combining synchronized data collection from wearable devices, a mobile phone camera, and 2D keypoints from pose estimation. This coordination enhances robust data acquisition and supports a multimodal classification approach to HAR from IMUs and 2D keypoints.Presenting an on-device working lightweight method using 2D pose estimation for determining optimal sensor placement, requiring only 500 data points, even from publicly available video footage of target activities.Accommodating diverse classification tasks using wearable, video, or skeleton inputs, with options to modify underlying classification and landmark detection models for specific cases, like face, eye, or hand recognition.Employing transfer learning for on-device training, which preserves data privacy and enhances model performance. The integration of active learning, demonstrated through a returned confusion matrix of each activity\u2019s performance, supports continual data recollection and model refinement.Providing instant activity recognition and detailed statistics on person-specific and activity-based metrics. These insights can enhance business processes and support further research.Our research makes the following contributions to the field of HAR:In the following sections of this paper, we provide a detailed roadmap of our study. Sensor-based HAR utilizing wearable sensors boasts diverse applications, extending from healthcare to surveillance ,7,8. HARData collection and labeling in sensor-based HAR are significant challenges . ExistinHARE offers a comprehensive and promising solution that addresses the challenges of data collection and labeling. It leverages real-time pose estimation from video recordings to facilitate data anonymization and relabeling, which mitigates privacy concerns associated with video data. Beyond this, HARE integrates capabilities for synchronized data collection from multiple standalone IMU devices and video recording. This enables a single or multimodal real-time classification approach not confined to a specific area of interest.The issue of sensor placement arises as a crucial aspect when conducting studies in HAR. The classification accuracy heavily relies on the data received, which varies based on the location and number of sensors employed for different body parts . PreviouBuilding upon the approach introduced in , we exteReal-time activity recognition  has emerOur proposed approach provides several additional features that make it particularly well-suited for use in real-world scenarios, unlike the aforementioned approaches, which have focused primarily on accuracy and computational complexity. For example, our model is both adaptable and uses less computing power, which allows it to be trained on-device and run efficiently on resource-constrained devices. Furthermore, we provide accuracy on all activities performed, which gives the opportunity to recollect data on poorly recognized activities. In addition, we include metadata statistics on the performed activities and persons, which provides the possibility for further analysis and process mining. These features are an improvement upon previous work and highlight its potential for practical use cases.Privacy is a major concern in the field of HAR, where the collection and processing of personal data are an integral part of the process . SeveralHARE stands out in this facet by offering on-device training aided by pre-trained models, which ensures that data are not leaving the device and thus maintains data privacy. Additionally, HARE provides a confusion matrix that encourages the collection of new data on poorly recognized movements, fostering active learning and improving performance.Building upon the work introduced in , we delvIn contrast to these multimodal approaches, HARE takes a unique approach by utilizing a single device to collect data from IMUs and videos. These data are then transformed in real-time into 2D human pose estimations, resulting in an inherently given multimodal data stream that facilitates recording and classification processes. This distinctive methodology distinguishes HARE from existing multimodal approaches in sensor-based HAR.HARE represents a significant advancement for the HAR community. Several studies have investigated some features independently to a limited extent. However, such studies remain narrow in focus as they cover just one aspect. We highlight the need for the unification of the features. HARE combines all necessary features for sensor-based HAR in one application and enriches it with newly developed functionalities. It offers a more comprehensive set of features, including support for a wide range of sensors, synchronized multimodal recording and classification, real-time classification, on-device training, and active learning. The framework is highly adaptable and, therefore, a versatile tool for a wide range of use cases. A comprehensive comparison of HARE with other tools and research methods in the HAR field, showcasing the unique and enriched functionalities, is shown in As depicted in HARE\u2019s architecture revolves around a single MainActivity, containing four primary screens, as depicted in Xsens\u2122 DOT : GuidesWebDAV Client : EnableCharts Library : FaciliTree View Library : AssistThe following libraries and modules integrated into the app are instrumental in its functionality:Due to its compact size and lightweight design, we opted to utilize the Xsens\u2122 DOT sensor as a standalone device for unobtrusive data recording. We developed our framework using the Xsens DOT Android software development kit (SDK version v2020.4). This SDK enabled functionalities such as scanning, connecting, and receiving data seamlessly. The Xsens DOT sensor communicates with the host device via Bluetooth for data transmission. While there is no specific connection limit in the Xsens DOT SDK services, the maximum number of sensors that can be connected simultaneously depends on the central devices\u2019 hardware and operating system constraints. In the case of Android, up to seven sensors can be connected. The output rate for real-time streaming can be adjusted, ranging from 1 Hz to 60 Hz, while the recording mode supports rates up to 120 Hz. All sensors are time-synchronized after the initial synchronization process. Sensor connection and synchronization are achieved within 3 to 5 s. The transmitted data from the Xsens DOT sensor includes calibrated orientation data (quaternion), inertial data, and magnetic field data. This comprehensive set of information allows for accurate and precise analysis of human movements.Connecting the IMUs to the app enables data recording with various sensor data outputs, including quaternions, (free) acceleration, angular velocity, and magnetic field normalized to the earth\u2019s field strength. The raw data from these sensors, including the quaternion data representing sensor orientation, are used directly without extensive preprocessing. The app provides functionality to adjust the output rates of these sensors based on the requirements of the activity recognition task.For video data, frames are captured at a rate dependent on the device\u2019s hardware capacity, and the data are used to generate real-time pose estimations. The pose estimation data and the raw inertial sensor data form a comprehensive dataset for activity recognition. This multimodal dataset enhances the robustness of the model by incorporating both positional and movement data. If not required, video data can be excluded, retaining only pose estimation and/or inertial sensor data for analysis.All recorded data, including metadata, inertial sensor data, and pose estimation data, are synchronized and formatted into a consistent dataset. The deep learning model is adept at handling this multivariate time series data, applying neural network architectures to extract relevant features directly from the raw sensor inputs. This methodology leverages the strength of deep learning to analyze complex patterns in raw data, facilitating accurate activity classification without the need for traditional feature engineering or extensive preprocessing steps.Pose estimation is a computer vision technique for detecting human figures and their corresponding poses from image and video data (OpenPose ). In HARTo facilitate real-time performance, video frames undergo preprocessing to meet the model\u2019s input specifications. This includes normalizing pixel values, resizing frames to the required resolution, and maintaining a consistent frame rate, which is essential for the subsequent time series analysis. The deep learning algorithm employed by the MoveNet model identifies and tracks keypoints, applying a confidence threshold to ensure landmark detection accuracy. Any occlusions or instances of partial visibility are managed through post-processing techniques designed to smooth the temporal trajectories of the keypoints.HARE is designed to enhance pose estimation capabilities by enabling the integration of additional pre-trained models suitable for various use cases. For instance, MediaPipe offers a suite of pre-trained models that include face detection, face mesh, iris landmarking, and hair segmentation, which can be leveraged to extend the functionality of our application . As demoIntegrating pose estimation sequences and IMU data is pivotal in HARE. These data sequences differ in their informational content. Pose sequences, derived from video data captured by the same smartphone\u2019s camera used for IMU data collection, store relative positions, detailing spatial relationships of specific body points. In contrast, IMU data comprise position-independent sensor readings, providing a complementary perspective. Fusing these data types results in an enriched informational set.Both data types are treated as time series, allowing their integration to be treated as a multivariate time series. This is suitable as input for a deep learning model designed to classify activities from sensor data alone. To achieve this integration, the pose estimation data undergo specific preparation. Pose sequences are recorded at a frequency of 10 Hz and are synchronized with the higher-frequency IMU data, typically collected at 60 Hz. An interpolated data series of pose estimation for each sensor datum\u2019s timestamp is created for synchronization. More significant gaps in the data are addressed using value imputation , a methoAdditionally, keypoints in all pose data series are centralized and reduced to 12 keypoints, focusing on the most informative points to eliminate redundancy. This reduction, combining stationary keypoints such as those from the head and hips, streamlines the dataset for efficient processing and analysis.The final step involves concatenating the resultant pose sequences along the time axis with the IMU sequences to create a comprehensive multimodal input. This integration is crucial for the HARE system\u2019s effectiveness, ensuring that time-aligned multimodal data enhances the activity recognition\u2019s accuracy and reliability. Through this process, HARE leverages the full spectrum of data modalities, providing a robust foundation for the application\u2019s deep learning models.The outcome of the pose estimations yields 2D keypoints for each timestamp. Since the position data exhibit a causal relationship with acceleration, we consider each keypoint an accelerometer. Consequently, we interpret each keypoint as a potential location for sensor placement .To calculate the optimal sensor placement, we employ a three-phase algorithmic procedure. In the first phase, we pre-process the selected pose estimations by consolidating specific keypoints. Head-related and hip keypoints are replaced with a single average keypoint, while the remaining 12 keypoints are centralized around a center of mass point  within each data series. To align with real-life scenarios, we reduce the number of keypoints to five, selecting the wrists, ankles, and pelvis. This subset of keypoints has been shown to provide rich information for full-body pose estimation . The heak refers to the k-th keypoint, which is a potential position for sensor placement.i represents the i-th activity out of the total number of different activities present in the dataset.k-th keypoint.Each vector In the second step, we introduce a cross-validated feature metric, denoted as Each activity requires a minimum sequence length of 500 data points (corresponding to a 50-s recording at 10 Hz), making it possible to run efficiently on a mobile device. The i and j at the same keypoint k.The term The denominator A higher In this formula:In the concluding phase of our sensor placement optimization, we prioritize sensor locations based on the For scenarios that require the deployment of multiple sensors, we facilitate the evaluation of sensor pairings through vector concatenation. This process involves the sequential joining of activity-specific vectors from each potential sensor location. For instance, to assess the combination of a right wrist and pelvis sensor, we concatenate the activity vectors from the right wrist with those from the pelvis, creating a new, extended vector representing the activity data for this dual sensor setup. Such composite vectors enable direct comparison across various two-sensor combinations, helping to determine the most effective pairings for capturing distinct activity patterns.By utilizing a vision-based virtual sensor approach, we can determine the optimal sensor placement more efficiently than physical IMUs and subsequent model training and evaluation. Thus, having physical sensors or collecting IMU data is not required. Instead, existing or self-recorded videos of targeted activities suffice to receive sensor placement recommendations.Ensuring data quantity and quality is crucial for the training process of an ML model. Therefore, HARE provides a dashboard for data exploration and analysis. As shown in We integrated a real-time activity recognition TFLite model, a lightweight, fast, cross-platform, open-source ML framework specifically designed for mobile and IoT . There iOffline training refers to the option to train a model outside the app . Using tThe second option includes the possibility of training a model on-device . This enWe provide pre-trained classification models for different use cases and allow users to fine-tune the model using transfer learning after collecting and labeling small amounts of new data. In that case, the deep learning model\u2019s layers will be frozen, excluding the last, and trained for a defined number of epochs. After completing a training run on a device, the model updates the set of weights in storage that can be saved in a checkpoint file for later use. As an interactive outcome, we display a performance list for each activity in a confusion matrix before and after on-device training. In doing so, we facilitate a human-in-the-loop  process,To evaluate the functionality and advantages offered by HARE, we collected data on nursing activities under the guidance of a professional nurse. To showcase its versatility and effectiveness, we conducted an experiment specifically focusing on nursing activities. Nursing activities were chosen due to their diverse and complex nature, demanding precise and efficient data collection and classification. By deploying HARE in this real-world scenario, we effectively demonstrate its capability to address the challenges associated with data collection, classification, and privacy preservation. Moreover, this experiment is an illustrative example of how our framework can be adapted to different domains and applications, highlighting its potential to contribute to the HAR field.For the purpose of this study, we engaged in data collection employing a set of five Xsens DOT sensors positioned at key anatomical locations: specifically, the left wrist, right wrist, pelvis, left ankle, and right ankle. These sensors were configured to deliver data at an output rate of 60 Hz, yielding an expansive dataset. Within each sensor, we obtained a rich stream of information, consisting of 14 sensor streams per sensor . These streams encompassed a spectrum of data, including four-dimensional quaternion values, four-dimensional angular velocity data derived from the quaternion values, three-dimensional acceleration values, and three-dimensional magnetic field values. In our experiments, the video recording for pose estimation was conducted in a lab environment measuring approximately 4 m \u00d7 7 m. The mobile device used for recording was positioned at a distance of approximately 2 to 3 m from the subject. This distance was found to be optimal for capturing the subject\u2019s full range of motion while maintaining the quality of the recorded video. It is important to note that the pose estimation model employed in our app normalizes the subject to the screen center. Therefore, variations in the distance between the mobile device and the subject do not significantly impact the pose estimation results. This normalization ensures consistent pose estimation accuracy regardless of minor changes in the recording setup. Our dataset comprises 13 different activities by ten subjects. The result was a compilation of 51 recordings, resulting in nearly 8 h of documented data. The distribution of subjects across various activities is illustrated in \u22124 and an input size set at Offline training involved the utilization of three deep learning models. To enhance the performance, we employed hyperparameter optimization through grid search , resultin method . For ourC represents the set of classes, s denotes the vector of predictions, and Here, The architecture of the three models is defined below.1.The CNN-LSTM model is composed of six layers. The input layer is followed by two convolutional layers, two LSTM  layers, 2.The ResNet model is composed of 11 layers. Next to the input and output layers, it has a repeated sequence of three convolution layers followed by a batch normalization layer and an activation layer .3.The DeepConvLSTM model is composed of eight layers. After the input layer, it is followed by four consecutive convolution layers and two LSTM layers before the softmax classifier .\u223c46 s and \u223c1249 s, and we used an 80:20 train-test ratio. One recording can contain only one specific activity or multiple activities performed multiple times. This validation technique reflects the model\u2019s performance when used within HARE; (3) lastly, we evaluate leave-one-subject-out cross-validation.For evaluation, we also used three different cross-validation techniques, namely, (1) k-fold cross-validation on time windows of length 600 with As shown in Making the bed, e.g., has a low accuracy and is confused with assist in getting dressed. Similarly, skin care is confused with assist in getting dressed or push the wheelchair.From the data in We employed the CNN-LSTM model architecture described in The findings presented in To evaluate the performance of our on-device training, we employed the leave-one-subject-out cross-validation method, in which we trained the model on data from all subjects except one, using this withheld subject\u2019s data for validation. The model trained using the leave-one-subject-out cross-validation served as a reference model, which we compared with the model trained on the left-out subject\u2019s data to evaluate the effectiveness of our on-device training approach. We employed a two-step evaluation process to assess the effectiveness of our on-device training approach. First, we used the leave-one-subject-out cross-validation method, where we trained the model on data from all subjects except one and reserved this left-out subject\u2019s data for evaluation. The prediction accuracy obtained for the left-out subject served as a reference value. Next, we further evaluated the model\u2019s performance on the left-out subject\u2019s data using the leave-recordings-out cross-validation method. We split the left-out subject\u2019s recordings into an 80:20 ratio, with 80% for training and 20% for testing. This allowed us to provide an unbiased estimate of the model\u2019s performance and compare it with the reference value obtained from the leave-one-subject-out cross-validation. Since only four subjects performed all activities and had at least five recordings for validation, we only included them for comparison. We used 10 epochs to retrain the given model on all four subjects. On-device training, utilizing TFLite for classification, varies in duration. Given our setup with transfer learning and a batch size of 7, the training time was between 7 and 9 min for a small dataset. Given five sensors, we trained a total of 31 models for all possible sensor combinations with the CNN-LSTM model. The We also used the deep inertial poser (DIP) dataset, which provides valuable insights for evaluating our optimal sensor placement approach . The DIPV. This mapping is represented by the function The SMPL model is a foundation for representing human body pose and shape variations. It is defined by a set of pose parameters Our proposed approach leverages both IMU readings and the corresponding 3D pose estimations of arbitrary body points. Due to the limited availability of datasets that include synchronized IMU data and 3D pose estimations, the applicability of our approach is currently constrained to a few datasets, like the DIP dataset. The dataset consists of approximately 90 min of real IMU data collected from 10 subjects wearing 17 Xsens sensors. The dataset encompasses 64 sequences with 330,000 time instants. Participants were instructed to perform various motions falling into five different categories. These categories include controlled motion of the extremities  in (1) locomotion, (2) upper body, (3) lower body, (4) freestyle, and (5) interaction. The dataset comprises 13 activities, providing a diverse range of motions for validation and analysis. The dataset\u2019s modalities and division into categories make it particularly interesting and suitable for our approach. We chose the same five positions  to track the movements in 3D. As illustrated in Further, we conducted a small-scale experiment to demonstrate HARE\u2019s sensor placement capabilities. The experiment involved five participants engaging in six different activities of daily living (ADL): sitting, walking, ascending stairs, descending stairs, standing, and lying down. Each activity was performed for ten minutes. Three sensors were placed at the right wrist, pelvis, and right ankle. This setup aimed to capture a comprehensive range of motion data for each activity. The results from our sensor placement analysis indicated that the right wrist is the optimal location for sensor placement in both approaches.This paper addresses the development of an application combining various HAR functionalities in a single application. Special attention is given to real-time classification, a multimodal approach, on-device training, and optimal sensor placement.making the bed as assist in getting dressed and vice versa, are likely to be related to similar movement patterns. Differences between activities skin care and assist in getting dressed are also related to similar movements. On the other hand, the activity full body washing in bed is always predicted correctly. This result may be explained by the fact that this activity was the most recorded.The results from the single modality analysis show that k-fold cross-validation provides the best results. This is related to the training process of the network being able to see a portion of the data from all subjects. Theoretically, leave-recordings-out can comprise the same data as leave-one-subject-out or k-fold. This depends on how the data were recorded. Therefore, it is not possible to conclude about the model\u2019s performance, depending on the recording\u2019s composition. The given result for leave-recordings-out may be explained by the fact that the model has not seen recorded data on all subjects or activities per subject, which can be assumed from the lower accuracy compared to k-fold cross-validation. This is also supported by the performance on each activity, presented in Overall, the ResNet model performed best on k-fold cross-validation. The shallower model, CNN-LSTM, has more trainable parameters and performs best on the other two evaluation metrics. The discrepant results for leave-one-subject-out cross-validation in While multimodal classification approaches exist, the unique advantage of HARE is its integrated data collection. Unlike existing approaches that require multiple devices and subsequent synchronization, HARE simultaneously collects pose estimation and IMU data. This approach simplifies the process while ensuring seamless data integration for improved classification accuracy. However, the observed performance boost is relatively modest. Two potential factors may contribute to this outcome. Firstly, the variations in camera angles during data acquisition could impact the results. When recordings are captured from a side view, the keypoints tend to be closer together in the 2D space, posing a challenge for accurate classification. The viewing angle of the camera, therefore, plays a crucial role in the overall performance. Secondly, the absence of pose estimations under certain conditions could affect the results. When the camera fails to capture the entire body or significant portions of it, obtaining accurate pose estimations becomes infeasible. Out of the 51 recordings examined, ten recordings lack pose estimation data due to these limitations. These factors highlight the complexities and limitations inherent in combining IMUs and pose estimation data for improved classification. Understanding the influence of camera angles and addressing challenges related to pose estimation under various conditions are essential considerations for further enhancing the performance of our multimodal approach.The cross-validated feature metric Our approach comes with some limitations. Currently, we only offer the connection with the Xsens DOT sensors, and HARE is only available for the Android operating system. When forming pose estimations, distortions in the image can occur quickly if there are objects in front of the person or if the focus is shifted. This leads to low confidence values and, thus, gaps in data collection. Consequently, this would corrupt both a multimodal approach and the optimal determination of sensor positions. Furthermore, our method employs a 2D pose estimation technique that does not consider depth mapping. Consequently, when the person being observed turns or the recording angle changes, there may be inaccuracies in estimating the person\u2019s distance. While our optimization method for sensor placement has demonstrated its effectiveness, ease of implementation, and speed, it is essential to acknowledge that our study did not include a direct comparison with other methods for optimization. The main reason for this omission is the limited availability of readily implementable alternatives for direct evaluation.We designed an on-body sensor-based HAR application system in this research study and evaluated its features. In conclusion, our work introduces several innovations and contributions to HAR. First, we streamline the data collection process and integrate a novel approach for multimodal classification using a single device. This approach represents a significant advancement over traditional methods that rely on multiple devices and complex synchronization. Second, the pose estimation technique can effectively anonymize test subjects. Third, we demonstrate the possibility of determining the optimal sensor placement without the necessity of actual IMUs. To determine the optimal placement, videos from targeting activities are sufficient. Lastly, the possibility to infer a personalized, privacy-preserving on-device trained model and a multimodal real-time classification approach on different use cases.Our vision-based approach has demonstrated how integrating several HAR tasks into a single workflow can significantly enhance the efficiency and accuracy of HAR. We aim to further these innovations by exploring advanced pose estimation techniques, such as 3D pose estimation models, video recordings, and diverse sensor types. Future work will also address implementing and comparing other sensor optimization methods."
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