Dietary selenium intake was directly associated with female sex  and weight , and inversely associated with the percentage of total body fat  and HbA1c . Considering the noticeable young age of the individuals evaluated and the high frequency of disturbances in HbA1c concentrations in those with selenium consumption below the recommendation, it is suggested that adequate dietary intake or supplementation of this micronutrient should be guaranteed to prevent future possible complications associated with glycemic control disturbances.Numerous endogenous functions related to antioxidant processes, reproduction, and thyroid metabolism, as well as actions related to glycemic control, have been attributed to selenium. This study aimed to evaluate whether dietary selenium consumption is associated with variables of glycemic control in a sample of young Brazilian adults with Normal-Weight Obesity (NWO) syndrome. This was a cross-sectional study that evaluated 270 individuals with adequate body weight and excess body fat, who had their body composition assessed by dual-energy X-ray absorptiometry. Socioeconomic, health, and lifestyle questionnaires and three 24-h food records were applied. Glycemic control markers were also evaluated. The prevalence of inadequate selenium intake was analyzed by the Estimated Average Requirement (EAR) cut-point method. The prevalence of disturbances in glycemic control markers according to selenium consumption was compared by either the chi-square or the Fisher's exact test, with individuals classified according to the EAR values for selenium. The associations were evaluated by multiple linear regressions, using the backward strategy. The mean ± standard deviation (SD) age was 23.7 ± 3.3 years, and the mean ± SD daily selenium intake was 59.2 ± 26.4 μg. The overall prevalence of inadequate selenium intake was 59.2%. Individuals with selenium intakes below the EAR (≤45 μg/day) showed higher concentrations of glycated hemoglobin (HbA Selenium is a trace element that fulfills key roles related to human body homeostasis, thyroid gland function, and optimal functioning of the immune system , 2. MostObesity in the classic sense, defined by the Body Mass Index (BMI), has been extensively studied in several aspects, including selenium metabolism. However, more recently, emphasis has been placed on the substantial role of excess body fat regardless of BMI classification. In 2006, the Normal-Weight Obesity (NWO) syndrome, a metabolic condition of excess body fat in individuals with normal BMI, was defined . It is kAlthough the most selenium-rich food is widely available in Brazil, there are few studies on the consumption of selenium in the Brazilian population and none in individuals with NWO. Analysis of the dietary intake of 34,003 Brazilians older than 10 years revealed a mean selenium intake of 107.6 μg/day, with no difference between income classes or urban and rural areas. Higher selenium intakes were found in women and residents of the northern region of Brazil. Among the elderly, mean selenium intake was lower than that in other age groups but still above the RDA .INSR and ADIPOR1) and others that encode pyruvate metabolism enzymes   but converge concerning serum insulin and the Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) index and suggest that its action occurs through some mechanisms, including regulation of insulin signaling and glycolysis, pyruvate, and chromium metabolism , 12–15, A, PDHB) , 17.PKM, the gene encoding the glycolytic enzyme pyruvate kinase , and adiponectin concentrations  and increase the Quantitative Insulin Sensitivity Check Index (QUICKI); however, it had no effect on blood glucose, HOMA-IR, glycated hemoglobin , Brazil . The stu2)   and high2)  were inc2) . The staThe first data collection was performed from May to September 2015, and the second one, from January to June 2019. Data collection took place in two meetings after participant recruitment. During the first appointment, individuals received all information about the research, and the informed consent form was presented. Individuals who met the inclusion criteria and agreed to participate answered the socioeconomic, demographic, lifestyle, and health questionnaires and were referred for anthropometric assessment, body composition, and food consumption analysis. The second appointment was previously scheduled, and the individuals were instructed to fast for 12 h to collect blood samples for biochemical tests.All researchers on the team in both studies were trained and underwent standardization regarding the nutritional care protocol. In addition to this training, the researchers received training to measure anthropometric measures, according to the technique of anthropometry standardization recommended by Habicht .The application of a questionnaire referring to socioeconomic, demographic, and lifestyle data took place in an average of 30–40 min. During this stage, blood pressure was measured at three non-consecutive moments, and the mean values of systolic and diastolic pressures were used in the analyses .Body mass was measured on a digital platform scale , with a maximum load of 150 kg and an accuracy of 0.1 kg. Height was determined using a stadiometer  with a maximum reach of 220 cm and a precision of 0.1 cm, according to the procedures described by Lohman et al. .Individuals dressed in light clothing and without accessories were subjected to DEXA examination, using a Lunar DPX NT model DEXA scanner .1c).Blood was collected by a specialized technician, with disposable materials, in a strictly sanitized environment, from the median cubital vein for determination of the glycemic profile  −46.7]. Results ≥5.7% were considered altered  was calculated from results of insulinemia and fasting glycemia using the equation: [1/(log FPI + log FPG)]. This index assesses insulin sensitivity, and the value adopted as a reference for altered values was less than the 10th percentile of the sample . For thiDietary food intake was assessed using three 24-h food records (R24H), on alternate days and different weeks, including a weekend day . Aiming The information collected was transformed into standard home measures, and the data evaluation was carried out with a Brazilian software . This software calculates the results of 21 micronutrients, including selenium, in micrograms. Selenium intake was adjusted to the energy value, according to the residual method . In summA double-entry database was developed to check the consistency of the results. Descriptive analysis, including mean ± SD or median (interquartile range – IQR), was performed for all quantitative variables. Shapiro–Wilk's W test was applied to evaluate the significance (α = 0.05) of normality deviations observed in the residuals of mean comparisons tests and linear regression models.t-test or the Mann–Whitney test was applied to compare means. A comparison of the disturbances in glycemic control markers between individuals consuming selenium at levels below and above the EAR was performed using either the chi-square or Fisher's exact test, according to the number of individuals who presented disturbances. Associations between variables were assessed by multiple linear regression models, using the backward strategy. The variables that could associate with glycemic response and were added to the model included sex, weight, height, BMI, body fat percentage, and HbA1c, age, systolic and diastolic blood pressure, waist circumference, android and gynoid fat, the android/gynoid ratio, fasting blood glucose, insulinemia, and the HOMA-IR, HOMA-beta, and QUICKI indexes. Although selenium intake was the main focus of the analysis, other dietary variables that also could interfere with glycemic response were added to the model, including carbohydrate, total fat; saturated, monounsaturated and polyunsaturated fatty acids; and protein intakes.Student's P < 0.05 was considered statistically significant, and all analyses were performed in R software version 4.0.3  , with NWn = 58) of women and 41.2% (n = 33) of men showed selenium consumption below the EAR (P = 0.089).For selenium, individuals showed a mean intake of 59.2 ± 26.4 μg/day. The overall prevalence of inadequate intake was 59.2% (54.7% for men and 63.7% for women). To analyze glycemic control marker disturbances, individuals were separated into two groups according to selenium consumption: (1) below the EAR (≤45 μg/day)—91 individuals (33.7%); and (2) above the EAR (>45 μg/day)—179 individuals (66.3%) . When coThere were no differences in weight, BMI, waist circumference, body fat percentage (% BF), android fat, and gynoid fat between the two groups (below and above the EAR). Individuals in the group below the EAR showed higher values of the ratio between android/gynoid body fat compared to those in the group above the EAR .1c than those in the group with intake below the EAR (P = 0.002) (1c concentrations was found in the group with selenium consumption below the EAR compared with the one with intake above EAR (P = 0.001)  . In addi= 0.001) . No diff= 0.001) .n = 267). After all candidate variables were added into the multiple regression model using the backward strategy, the final model with the lowest Akaike criterion presented the variables sex, weight, height, BMI, body fat percentage, and HbA1c. Sex, weight, body fat percentage, and HbA1c were the variables most strongly associated with dietary selenium intake. The female sex was directly associated with a higher consumption of selenium , and a positive association was also observed for selenium consumption and weight . However, negative associations of body fat percentage and HbA1c with selenium consumption were observed . The final regression model is shown in 1c.Three individuals were excluded from the regression analysis due to missing data ; however, approximately one third of participants did not reach the expected minimum values. We also observed that dietary selenium intake was inversely associated with serum concentrations of HbA 90 days . The ass1c concentrations (P < 0.001); lower daily selenium consumption was observed among those with the highest HOMA-IR indexes (P < 0.001) and a negative correlation between selenium and insulin resistance was observed up to a consumption limit of 1.6 μg/kg/day , including 1,441 individuals with ages ranging from 10 to 85 years, verified that selenium supplementation reduced insulin secretion as assessed by the HOMA-beta index and increased the QUICKI index; however, significant results were not observed for disturbances in HOMA-IR and HbAg/kg/day . Althoug1c, even within different populations and with different doses of selenium , which acts on insulin resistance and increases lipid metabolism (+IV inhibited the activity of protein tyrosine phosphatases (PTP)  , responses (PTP) . In thisIpf1). A response to sodium selenite was also observed via increased mRNA levels of Ipf1 and Ins. The increased antioxidant activity of GPx culminates in beneficial effects on the level of pancreatic beta-cell mass and insulin synthesis; however, long-term increased insulin production and secretion can result in chronic hyperinsulinemia , observed a mean dietary selenium consumption of 108.10 μg/day, and decreasing consumption in μg/kg/day along the BMI ranges. The authors found a significant and dose-dependent association between tertiles of selenium consumption and weight, BMI, waist circumference, total body fat percentage, and gynoid and android fat percentages and demonstrated that dietary selenium can account, regardless of other factors, for 9–27% of the variation in body fat percentage , showed divergent results regarding the type of association between dietary selenium consumption and body weight but also observed an inverse association with body fat percentage, BMI, and body water (Thereafter, a study published by the same group of CODING (dy water . A simildy water .Possible explanations for the role of selenium in adipogenesis are also related to PPAR-γ, which is responsible for adipocyte differentiation and distribution of adipose tissue, and is thus related to increased tissue insulin sensitivity and weight control. In animals receiving sodium selenate, hepatic PPAR-γ expression was 2.5 times higher than in those with selenium deficiency or treated with sodium selenite . On the 1c concentrations. We were not able to measure blood selenium levels, which can also be considered a limitation. On the other hand, this is the first study to assess associations between dietary selenium intake and markers of glycemic control in individuals with NWO.Among the limitations of our study, the cross-sectional design can be considered, as it does not allow us to make inferences about cause and effect between dietary selenium consumption and the other markers. However, it suggests that there may be interactions between the consumption of this nutrient and glycemic control markers and, for this reason, may guide future investigations on dietary consumption and the effect of selenium on biochemical and metabolic pathways and the expression of genes related to variation in HbA1c concentrations, a long-term marker of disturbances in blood glucose, and body fat percentage. Considering that the individuals evaluated in our study had NWO, and the disturbances in HbA1c found in the group consuming selenium below the EAR, we can observe that despite their noticeably young age, these individuals showed cardiometabolic markers that are more commonly seen in older people. Therefore, further studies with individuals with NWO are strongly recommended, as one of the basic principles of nutrition is to reduce or mitigate the risk of non-communicable chronic diseases throughout life.In conclusion, dietary consumption of selenium above the minimum values recommended for groups by the dietary guidelines was associated with lower serum HbAThe raw data supporting the conclusions of this article will be made available by the authors, upon a reasonable request.The studies involving human participants were reviewed and approved by UFG Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.AS and AP: acquisition, interpretation, and analysis of data. LH: data interpretation and writing the manuscript. CC: conceptualization, experimental design, obtaining resources, interpretation of data, and writing the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Koala habitat is threatened by urbanisation, agricultural activities and by increased temperatures and droughts caused by climate change. Habitat loss may cause stress in koalas, which, in turn, may exacerbate disease occurrence. Stress is associated with an increase of glucocorticoids, of which cortisol is the main one in most mammals. Cortisol is heavily metabolized and excreted via the faeces, where its metabolites can be measured, without interfering with the animal. However, before a link between events causing stress in koalas can be established, baseline levels in stress-free koalas need to be established. Our study has established which diagnostic procedure is best suited to measure these metabolites and has evaluated the physiological baselines levels in male and female koalas and in koalas during the breeding and non-breeding season.Phascolarctos cinereus) is an endemic marsupial inhabiting four states of Australia. Urbanisation, declining habitat, drought and fires are threatening the survival of this flagship species. These threats may cause acute and chronic stress in koalas, which might also be associated with occurrence of infectious diseases in koala populations. Stress may induce an increase in cortisol reflected in increased faecal cortisol metabolite (FCM) values. To be able to use faecal cortisol metabolites to measure stress levels in this species, our aim was to determine baseline values for males and females during breeding and non-breeding season. A total of 351 defecations were collected fortnightly, twice a day, for 12 months from koalas at a wildlife facility in South East Queensland. Samples were analysed with three different enzyme immunoassays (EIAs): a cortisol, 5α-pregnane-3β,11β,21-triol-20-one (37e) and tetrahydrocorticosterone (50c) EIA. The latter, which also reacts with tetrahydrocortisol, the main metabolite in koala faeces, was found to have the highest biological sensitivity and, therefore, is the most suitable EIA to measure stress levels in koalas. Utilising this EIA, we found significant differences (p < 0.05) in FCM values between males and females, breeding and non-breeding season, and between morning and evening samples. Values of faecal cortisol metabolites established in stress-free koalas in this study can serve as a reference for future studies in koalas.The Koala ( Phascolarctos cinereus) is an endemic marsupial species, whose range spans throughout eastern and south eastern Australia. As many populations are declining, koalas have been listed as vulnerable in Queensland, the Australian Capital Territory and New South Wales under federal and state legislations, but are not listed in Victoria and South Australia [The Koala , such as cortisol and corticosterone, are produced by the adrenal cortex and regulate several metabolic and physiological processes ,22. Duriper se, a stressful event [Measuring GC values in plasma and saliva or GC metabolites in fur, urine and in faeces are various options to evaluate the stress response in wildlife and domestic animals ,27,28. Hul event , and samul event ,28.Furthermore, in stress-free animals, physiological GCs concentration in plasma shows variations due to a circadian rhythm, characterised by peaks in the morning for diurnal species and peaks in the evening for nocturnal species, as well as episodic fluctuations may also exist during the day ,26,29,30To determine whether increases in plasma GCs are well reflected in FCMs, ACTH challenges were performed for many species ,26. ThesThe ACTH challenge has also been used in many studies on mammals ,37 and mMost cited studies on marsupials have used cortisol enzyme immunoassays (EIAs) to detect FCMs. However, due to differences in the metabolism and lag-time of excreted CMs among species , not oneTo determine if FCM levels represent a response to a stressor, physiological baseline levels need to be established . It was,ad libitum. The health of the koalas was monitored according to procedures of Wildlife HQ.This study was conducted at the wildlife park Wildlife HQ located at the Sunshine Coast, South East Queensland, Australia. A total of 8 female and 5 male captive koalas housed in outdoor enclosures were included in this study. Of these, 5 females and 4 males were sampled throughout the year-long project, while 1 male and 3 females were not available for the duration of the whole project and were replaced by other koalas of the same sex and age category. Therefore, defecations of 9 koalas were collected each fortnight. The mean (min–max) age of the koalas at the start of the study was 4.5 years (1.5–13 years). Wildlife HQ koalas are provided daily with freshly cut branches of eucalypt fodder of the same species and quality obtained locally from areas where wild koalas also live and water was provided evening plasma cortisol values, and FCM values obtained from pellets collected between 15:00 and 18:00 represented morning plasma cortisol values.We placed rubber hollowed mats at the base of the tree stumps, where koalas were sitting, to separate the pellets from soil and to avoid contamination with urine. Fresh faecal pellets were collected from the animals immediately after each defecation. A total of 351 fresh defecations were collected fortnightly over a 12-month period (September 2019–August 2020). To ensure that pellets were collected fresh from each animal, researchers observed each koala until defecations were completed. Defecations were collected between 7:00 and 9:30 and between 15:00 and 18:00. Our previous study  showed tDue to disruptions caused by COVID-19, the number of fortnight samplings were reduced from 26 to 23 and one morning collection was missed for all koalas. A few collections were also missed for individual koalas. Fresh pellets were placed into sample tubes and stored at −20 °C in a portable freezer for transport and then stored at −80 °C until sample preparation and further analysis.We followed the extraction procedure described in Palme et al. . We usedDetails of the assays, including cross-reactions, have been published previously ,49,50. TIn addition, between 7 and 13 single pellets from separate defecations of 10 koalas were analysed using all three EIAs to establish whether concentrations of FCM were uniform across pellets of each defecation. Coefficient of variations (CVs) for each defecation and median CV for each EIA were calculated.For each of the 3  EIAs, descriptive statistics were produced for FCM values, which included histograms for all samplings, boxplots for individual koalas, boxplots by age, sex and breeding season and line graphs showing the observed FCM mean (with 95% percentiles) per month. Normality of FCM values was assessed by visually examining the histograms and by using the Shapiro–Wilk test, followed by a log-transformation of the FCM values for further statistical analysis.p-value. For categorical predictors with more than 2 levels, Wald-test p-values were calculated.Mixed effect linear regression  was usedp-value less than 0.05 indicates that the time series is stationary, signifying that the seasonal effect is minimal.To explore seasonality, we decomposed the FCM values as times series. As our data represented a short time series with only one observation per seasonal period, we used Fourier terms to approximate the seasonal patterns and decompose the time series . We thenThe descriptive and regression ana lysis was performed in STATA 16.1 , while the time series analysis was conducted in R version 4.0.2 . .The analyses of all pellets for 10 separate defecations showed a CV between 9.7% and 28.7% (median: 17.4%) with cortisol EIA, 8.4% and 17.7% (11.6%) with 37e EIA and 12.3% and 24.3% (17.6%) with 50c EIA.The box whisker plots indicating the variation of FCM values for each of the 13 koalas, measured with the cortisol, 37e, and 50c EIAs are shown in Histograms of the distribution of FCM values measured with the cortisol, 37e and 50c EIAs are shown in The measured FCM values were skewed for all three EIAs, with lowest median values for cortisol, followed by 50c and 37e. The widest interquartile range of values were detected with the 50c EIA, followed by 37e and cortisol EIAs.The median (IQR) FCM values measured by the cortisol, 37e and 50c EIAs by sex, age category and time of sampling are shown in The mean (with 95% percentiles) FCM values measured per month are shown in It is noticeable that there was a strong increase in FCM values detected by the three EIAs from September until January, coinciding with the breeding season of koalas . FCM valWe explored the seasonality of FCM values in more detail using time series analysis .p-value = 0.7924 for hypothesis of stationary time series) and 50c , but less so for cortisol .The decomposition of the time series dataset highlighted a strong seasonal trend for 37e , for 50c vs. cortisol was −0.0039 (p = 0.9423) and for 37e vs. 50c was 0.6776 (p < 0.0001). Thus, a strong, significant correlation was only observed for the relationship between FCM concentrations measured with the 37e and 50c EIAs.The Pearson Correlation coefficient for 37e vs. cortisol was 0.2426 (p = 0.022) than those of females. Sex differences were not detected with the cortisol or 37e EIAs. Age was not a factor affecting the values of log-transformed FCMs measured with any of the three EIAs. Log-transformed FCM values were significantly higher in the evening samples, when measured with the 50c EIA (p = 0.036). However, such differences were not detected with the other two EIAs. Log-transformed FCM values were significantly higher in the breeding season compared to the non-breeding season, when measured with the 50c EIA (p < 0.001). This association was also identified with the 37e EIA (p = 0.001), but not with the cortisol EIA. The univariate and multivariable analysis results are displayed in The impact of predictors on the log-transformed FCM values was then evaluated in mixed effect linear regression models. In the univariate analysis, sex of the koalas affected log-transformed FCM values when measured with the 50c EIA: values of males were significantly higher  where the broken down digested material is further metabolized by a large variety of bacteria . In thisOur study did not find any differences in FCM values between the three age categories with any of the EIAs. There are no comparable studies on koalas, nevertheless, research on other marsupials   showed nEstablishing average, minimum and maximum FCM baseline values was the main scope of this research as behavioural changes, during the breeding time as well as pregnancy itself , can leaKoalas’ breeding season may vary slightly across their northern–southern latitudinal range. Some authors have found that in South East Queensland the koala breeding season is between October and March and non-breeding is between April and September , while oWe also observed some variation in FCM values for the three EIAs between individual pellets of one defecation. These CV values were in the range or somewhat higher than those for the pool sample extracts analysed in the EIAs. Therefore, whenever possible, we would advise to collect and homogenise at least two pellets from one defection to lower the influence of any potential variation. Faecal decay  and stabWith this study, we found the tetrahydrocorticosterone (lab code: 50c) EIA best suited among three previously validated EIA for koalas. We have determined FCM baseline levels during the breeding and non-breeding season, differences between sexes and between morning and evening. Future studies on stress in koalas may now choose to compare their findings to our baseline values using this group-specific EIA, which can give consistent results.


Understanding the association between floods and bacillary dysentery (BD) incidence is necessary for us to assess the health risk of extreme weather events. This study aims at exploring the association between floods and daily bacillary dysentery cases in main urban areas of Chongqing between 2005 and 2016 as well as evaluating the attributable risk from floods.The association between floods and daily bacillary dysentery cases was evaluated by using distributed lag non-linear model, controlling for meteorological factors, long-term trend, seasonality, and day of week. The fraction and number of bacillary dysentery cases attributable to floods was calculated. Subgroup analyses were conducted to explore the association across age, gender, and occupation.After controlling the impact of temperature, precipitation, relative humidity, long-term trend, and seasonality, a significant lag effect of floods on bacillary dysentery cases was found at 0-day, 3-day, and 4-day lag, and the cumulative relative risk (CRR) over a 7-lag day period was 1.393 (95%CI 1.216–1.596). Male had higher risk than female. People under 5 years old and people aged 15–64 years old had significantly higher risk. Students, workers, and children had significantly higher risk. During the study period, based on 7-lag days, the attributable fraction of bacillary dysentery cases due to floods was 1.10% and the attributable number was 497 persons.This study confirms that floods can increase the risk of bacillary dysentery incidence in main urban areas of Chongqing within an accurate time scale, the risk of bacillary dysentery caused by floods is still serious. The key population includes male, people under 5 years old, students, workers, and children. Considering the lag effect of floods on bacillary dysentery, the government and public health emergency departments should advance to the emergency health response in order to minimize the potential risk of floods on public.The online version contains supplementary material available at 10.1186/s12199-021-00971-z. A series of extreme weather events caused by global climate change have posed a significant threat to public health. Extreme climate often leads to the prevalence of human diseases and results in the phenomenon that “a major epidemic occurs after a disaster” . Floods Bacillary dysentery is one of the intestinal infectious diseases caused by Shigella. According to the global disease burden report released by WHO in 2016, the disability-adjusted life expectancy of diarrheal diseases ranked fifth in the global disease burden . AccordiA growing body of epidemiological evidences supported that floods will increase the risk of bacillary dysentery incidence , 7. CaseChongqing is located in the west of China, in the middle and upper Yangtze River, which is one of the highest risk levels in floods disasters . Floods Chongqing is located in the west of China, in the middle and upper Yangtze River, which is one of municipalities under direct control of the central China’s government. And the terrain of Chongqing is mainly mountainous and hilly. The geographical scope of this study is the main urban area of Chongqing, which includes Yuzhong District, Jiangbei District, Shapingba District, Jiulongpo District, Nanan District, and Dadukou District, occupying 1.174 thousand square kilometers with a population of 5.0682 million by 2016, and the urbanization rate is 94.85%. The annual average temperature is between 16 °C and 18 °C, and the annual average precipitation ranges from 1000 to 1350 mm. Due to the impact of the subtropical monsoon climate, the floods could firstly happen in March, and end at November, mainly between June and September. Floods happened after September, which is extremely rare in the whole country [According to the Yearbooks of Meteorological Disasters in China, floods are defined as prolonged heavy rainfall or regional persistent rainfall (25.0–49.0 mm per day), rainstorm (daily precipitation ≥ 50.0 mm), and the local short time heavy rainfall, which cause river flooding and destroy buildings, thus causing geological disasters and results in economic losses and casualties. The floods events records were collected from the Yearbooks of Meteorological Disasters in China . When thBacillary dysentery is caused by Shigella, one of intestinal infectious diseases with main clinical manifestations like fever, diarrhea, and abdominal pain. Bacillary dysentery is infected mainly through fecal-oral transmission, and it’s the legal B class infectious disease in China. We obtained and verified daily disease surveillance data on bacillary dysentery between 2005 and 2016 from the National Notifiable Disease Surveillance System (NNDSS). In our study, all bacillary dysentery cases were defined based on the diagnostic criteria and principles of management for dysentery (WS 287-2008) launched by the National Health Commission of the People’s Republic of China . Only thIn this study, the annual bacillary dysentery morbidity between 2005 and 2016 was calculated, and its change was tracked during 12 years. Time sequence plots were used to depict the distribution of daily bacillary dysentery cases and the temporal change of floods. The descriptive analyses of daily bacillary dysentery cases and meteorological data were conducted by calculating the maximum, minimum, quartile, median, and mean value.Distributed lag non-linear regression model (DLNM) was applied to investigate the association between floods and bacillary dysentery. Considering the effects of temperature, relative humidity, and precipitation to the incidence of bacillary dysentery , 26, 27,Model:Yt represents daily bacillary dysentery cases, t represents time, l represents lag days. The maximum lag day is set at 7. “flood” is coded as categorical variable, with 1 refers to flood and 0 refers to non-flood. AT represents daily mean temperature, precipitation represents daily mean precipitation, ARH represents daily mean relative humidity, ns represents natural cubic spline, and dow is an indicator variable representing the day of week. The collinearity between lag days is reduced by fitting polynomial function [In the model, function , and natfunction , 26, 29.The attributable fraction (AF) is the proportion of cases that would not have occurred in the absence of exposure among the exposed population. The attributable number (AN) can be calculated by the total exposed population and AF. Traditional estimation of AF ignores the complexity of non-linear correlation and potential lag effects in time series analysis. In the distributed lag non-linear model, a lag dimension is added to the exposure-response relationship through the cross-basis function, thus obtaining the double dimension exposure-lag-response relationship function:  factors . The calt + l0,…,t + L) due to current exposure at time t [t attributed to a series of exposure events in the past  [There are two methods to estimate AF based on DLNM: forward and backward perspective. “Forward perspective” indicates risk in a future period time  , 33. We All statistical analyses were performed using R version 3.3.2 .5. During 2005–2010 and 2011–2016, the annual incidence showed distinctly different characteristics: between 2005 and 2010, the annual incidence fluctuated between higher levels of 90.80/105 and 104.56/105; while from 2011 until 2016, the annual incidence decreased steadily from 87.93/105 to 63.14/105  over a 7-day period was 1.393 95% CI 1.216–1.596). The CRR for female was 1.272 (95% CI 1.057–1.531) and 1.515 (95% CI 1.262–1.818) for male. The CRR for children under 5 years old and people aged in 15 ~ 64 years old were 1.266 (95% CI 1.040–1.541) and 1.534 (95%CI 1.252–1.878). The CRR for students, workers, and children were all statistically significant , and basic sanitation facilities . Floods The floods affect people’s health through influencing the transmission and spread of pathogen. The humid environment during the floods is conducive to the breeding and growth of Shigella and increases Shigella contamination opportunities for vegetable and fruits through water transmission . In ChonIn this study, we found that the annual incidence rate of bacillary dysentery in the main urban area of Chongqing fluctuated at high levels from 2005 to 2010, while after 2011, the annual incidence rate decreased year by year, indicating that the overall risk of bacillary dysentery in the population decreased. This may be related to the improved sanitary conditions and human interventions, such as timely diagnosis and treatment of bacillary dysentery patients, brought about by the rapid socioeconomic development in the last decade , 44. HowThe first limitation of this study is that the effect of social and economic conditions, medical and health facilities, access to health services, and other factors on the incidence of bacillary dysentery are not quantified. The second is that this study used data from the infectious disease network direct reporting system, and some people with bacillary dysentery but self-healing were not included in the scope of this study. As a result, the incidence of bacillary dysentery in the population may be underestimated.This study confirmed that floods would increase the risk of bacterial dysentery in the population within the fine time scale, and the risk of bacillary dysentery in the main urban area of Chongqing caused by floods is still serious. The key population includes male, people under 5 years old, students, workers, and children. Considering the lag effect of floods on bacillary dysentery, the government and public health emergency departments should advance the emergency health response in order to minimize the potential risk of floods to the public.Additional file 1: Supplementary Table 1 Annual incidence of bacillary dysentery between 2005-2016. Supplementary Table 2 Attributable fraction and number of bacillary dysentery incidence to floods between 2005-2010, 2011-2016.


Radix Puerariae (RP), a dry root of Pueraria lobata (Willd.) Ohwi, is used to treat a variety of diseases, including cancer. Several in vitro and in vivo studies have demonstrated the efficacy of RP in the treatment of colon cancer (CC). However, the biological mechanism of RP in the treatment of colon cancer remains unclear. In this study, the active component of RP and its potential molecular mechanism against CC were studied by network pharmacology and enrichment analysis. The methods adopted included screening active ingredients of Chinese medicine, predicting target genes of Chinese medicine and disease, constructing of a protein interaction network, and conducting GO and KEGG enrichment analysis. Finally, the results of network pharmacology were further validated by molecular docking experiments and cell experiments. Eight active constituents and 14 potential protein targets were screened from RP, including EGFR, JAK2 and SRC. The biological mechanism of RP against CC was analysed by studying the relationship between active components, targets, and enrichment pathways. These findings provide a basis for understanding the clinical application of RP in CC. The decline in the incidence and mortality of colon cancer in developed countries may be associated with increased cancer screening in developed countries. However, the rapid rise in mortality rates in developing countries and the fact that more than half of the patients were diagnosed as late-stage significantly increased the medical and economic burden in developing countries2. Despite progress in treatment and care in recent years, many treatments are still accompanied by severe adverse reactions and drug resistance, which can reduce the quality of life and increase patient suffering. Therefore, adjuvant therapy and alternative therapies with low toxicity are necessary to improve quality of life and prolong lifetime. Traditional Chinese medicine (TCM), as a critical adjuvant to tumour therapy, can improve the effectiveness of treatment, reduce drug resistance, and prolong survival time3. For example, adjuvant treatment with Chinese herbal medicine can reduce the hepatotoxicity of colon cancer patients4. Furthermore, alkaloids can inhibit the growth of colon cancer cells through the Wnt/β-catenin signalling pathway5. Previous studies have shown that the traditional Chinese medicine Angelica can increase the sensitivity of colon cancer cells to radiotherapy and chemotherapy. In addition, the traditional Chinese medicine ginsenoside Rh2 can reduce the resistance of colon cancer cells to oxaliplatin6.Colon cancer (CC) is the second most common cause of cancer death and the third most common cancer in the worldRadix Puerariae (RP) is a kind of Chinese medicine that was used for medicine in the Jin Dynasty. RP is a dried root of Pueraria lobata (Willd.) Ohwi, which has the effects of reducing heat, stopping diarrhoea, producing body fat, and benefiting7. Currently, RP is used in the treatment of many diseases, such as angina, hypertension, diabetes, optic atrophy, and retinitis8. RP is also commonly used in the treatment of cancer. In vitro experiments confirmed the anticancer activity of most components of RP, such as puerariae radix isoflavones, by inhibiting the growth of breast cancer cells9; puerarin, for example, inhibits metastasis and invasion of liver cancer through PTEN/AKT signalling10; and puerarin induces apoptosis of colon cancer cells by increasing the activation of caspase-311. In modern medicine, RP is also often used for the treatment of colon cancer, but the active components and targets of RP treatment for colon cancer are not known. Previous studies have demonstrated the clinical efficacy of traditional Chinese medicine prescriptions containing RP in diabetes through network pharmacology12. This study will verify the efficacy of RP on colon cancer through network pharmacology and cell experiments.13. The clinical efficacy and mechanism of TCM has been a major difficulty in the study of modern traditional Chinese medicine. Using network pharmacology method can realize the transformation of TCM research from experience medicine to evidence-based medicine, and accelerate the clinical research and application of TCM14. TCM Network pharmacology can show the complex relationship between drugs and disease in the form of a network. In the network, the drug target-disease target-pathways network and protein–protein interaction (PPI) network are commonly used to predict the efficacy of drugs to disease. The degree and clustering coefficient are common parameters for evaluating the network. The ADME system refers to the process of drug absorption, distribution, metabolism, and excretion in the body17. The ADME-based admetSAR method has been proven to be a successful method. We selected the parameters of HIA, Caco-2 and HOB in the admetSAR database to optimize the active ingredient screening strategy.Research on TCM and biological networks first appeared in 2007, before the term "network pharmacology" was proposed18. With the establishment of energy matching and geometric complementary models for ligands and receptors and the development of computer technology, the idea of simulating the interaction between ligands and receptors has been realized, namely, molecular docking analysis. Molecular docking refers to the prediction of binding patterns of ligand small molecules and receptor proteins by continuously optimizing their conformations, positions, amino acid residues and other binding modes when both ligand small molecules and receptor proteins are three-dimensional structures and the minimum energy of ligand small molecules19. The method involves screening suitable drugs by scoring affinity.In the nineteenth century, the receptor theory was proposed to consider the pairing of drug small molecules as ligands and protein macromolecules as receptors to form keys and locksIn this study, the active ingredient of RP and its possible anticancer mechanism were studied using network pharmacology. Key targets and pathways were experimentally verified by molecular docking and in vitro validation. The study flow is shown in Fig. http://lmmd.ecust.edu.cn/admetsar2) for further screening based on the results of human intestinal absorption (HIA), Caco-2, human oral bioavailability (HOB), and plasma protein binding (PPB) , cell component (CC) and molecular function (MF). In BP GO terms, peptidyl-tyrosine phosphorylation, regulation of cell adhesion, responses to drug, etc. may be associated with tumour regulation. CC terms were mainly enriched in the perinuclear region of the cytoplasm, membrane raft, focal adhesion, etc. In MF GO terms, protein kinase activity, kinase binding, and transmembrane receptor protein tyrosine kinase activity may be associated with tumours Fig. . In KEGGThe top 20 pathways with the largest number of genes were chosen to construct an RP component-target-pathway network with 183 nodes and 626 edges , JAK2 (3UGC), MAPK14 (2FST), NFKB1 (1SVC), ESR1 (3CBP), IL2 (4NEJ), and SRC (1O43). We conducted molecular docking between receptor proteins and ligand molecules through AutoDockTools 1.5.6. The result of AutoDockTools was output in the form of an affinity score, which is the core parameter of AutoDockTools Table .Table 2AThe anticancer effect of RP at different concentrations  on SW480 cells after 24, 48 and 72 h were verified by CCK-8 experiments. The proliferation of SW480 cells treated with different concentrations of RP decreased in a dose- and time-dependent manner  expression level in tumour tissues25. The genotoxic and cytotoxic effects of daidzein on human colon adenocarcinoma cells are dose-dependent26. These RP active ingredients were successfully screened by ADME parameters.RP is a Chinese herb with anticancer, anti-inflammatory, anti-oxidant and cardiovascular protective properties. RP is mainly used in the treatment of cancer, endocrine disease, cardiovascular disease, and neurodegeneration. The results of RP component screening showed that the activity of isoflavones was the strongest, followed by alkaloids and coumarins. Previous studies on the anticancer effects of RP have focused primarily on the role of puerarin, and few studies have examined other active components of RPPPI network analysis of common targets for RP and colon cancer showed that 14 targets had a high degree of SRC, LYN, JAK2, MAPK14, MAPK8, PTK2, PTK2B, EGFR, NFKB1, JAK1, PTPN6, SYK, FGR and ESR1. In molecular docking experiments, EGFR and 3'-methoxydaidzein showed high affinity, indicating that EGFR may be one of the key targets of RP in the treatment of CC.27. EGFR is a member of receptor tyrosine kinase (RTK), a family of proteins that needs to form heterodimers to function. The upregulation of EGFR activity is mediated by common truncated and mutated extracellular domains, and upregulated EGFR overactivates downstream procancer signalling pathways, including the AKT-PI3K-mTOR and RAS-RAF-MEK-ERK MAPK pathways28. These pathways then activate a variety of biological effects that are beneficial to the proliferation of cancer cells, causing cancer to occur and develop. This evidence suggests that RP may play a role in the treatment of colon cancer by downregulating EGFR protein expression.Epidermal growth factor receptor (EGFR) is a cancer-promoting tumour marker that regulates the homeostasis and growth of epithelial tissues and cells. EGFR is also considered to be associated with the development of tumour resistance due to the occurrence of point mutations and amplification following the use of anticancer drugs30. PI3K catalyses the formation of phosphatidylinositol 3,4,5-triphosphate (PIP3) on the cell membrane, and PIP3 acts as a second messenger to activate Akt. Finally, Akt regulates the key processes of cell growth and development by phosphorylating proteins involved in cell synthesis, metabolism, the cell cycle and apoptosis. RP may play an anti-CC role by regulating proteins enriched in the PI3K-Akt signalling pathway.In the GO functional analysis, we screened the first 20 terms out of the three parts BP, CC and MF based on a P value < 0.01. In BP GO terms, peptidyl-tyrosine phosphorylation, response to drug, CC terms membrane raft, and in MF GO terms, protein kinase activity, kinase binding, and transmembrane receptor protein tyrosine kinase activity may be associated with tumours. Then, we screened the first 20 pathways obtained from KEGG enrichment analysis according to the criteria of a P value < 0.01. The pathway with the most enriched genes was the PI3K-Akt signalling pathway, with 22 targets enriched in this pathway, including EGFR, HSP90AB1, IL2, JAK2, NFKB1, MAPK14, SRC, etc. Phosphatidylinositol 3′-kinase (PI3K)-Akt is a signalling pathway that regulates the basic functions of cells, such as transcriptional translation and growth and proliferation, and can be activated by toxic damage or cell stimulation31.The use of TCM has a long history, but its mechanism and target are not clear. Molecular docking experiments can further verify the accuracy of network pharmacology results by simulating the interaction between active ingredients and targets, and replace some exploratory experiments. Therefore, network pharmacology integration molecular docking experiment can improve the efficiency of TCM research. Both network pharmacology and molecular docking experiments are bioinformatics studies, which cannot directly reflect the dose–effect relationship, so the combination of the two has certain limitations32 (https://tcmspw.com/tcmsp.php), the Encyclopedia of Traditional Chinese Medicine33 (http://www.tcmip.cn/ETCM/index.php/Home/), Herb34 (http://herb.ac.cn/), and the literature. Then, canonical SMILES of these ingredients was obtained from NCBI PubChem35 (https://pubchem.ncbi.nlm.nih.gov). Finally, the SMILES descriptions were input into admetSAR (http://lmmd.ecust.edu.cn/admetsar2/) to screen active components of RP according to HIA, Caco-2 and HOB. AdmetSAR database can be divided into positive and negative results by various machine learning algorithms and molecular fingerprints36. The positive value indicates that the active ingredient has good ADME performance. We retained active ingredients with positive HIA, Caco-2 and HOB parameters. Ingredients that did not meet the screening requirements but were significantly bioactive in previous studies were retained.The components of RP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform37 (http://www.swisstargetprediction.ch/) to predict the target, the species was set as Homo sapiens, and the result of probability > 0.1 was selected. At the same time, colon cancer-associated target genes were obtained from GeneCards38 (https://www.genecards.org/) and Online Mendelian Inheritance in Man (https://omim.org/)39, and the results of colon cancer-target genes from both databases were combined. Then, we used Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) to obtain the gene target intersection of RP and colon cancer.The screened active ingredient smiles were uploaded to Swiss Target Prediction40 (https://string-db.org) to obtain the PPI network. The parameter was set to Homo sapiens, and the interactive score was set to the highest credibility. Then, the target intersection file was imported into Cytoscape 3.7.1 for visual analysis of the PPI network. We used the network analysis tools in Cytoscape to analyse the degree values of nodes and edges in the network. The higher the degree value is, the larger the node size and the brighter the edge colour in the network.Gene targets overlapping with RP and CC were input into the STRING database41 (http://metascape.org), selected “H. sapiens” as the input species, set a p value < 0.01, and then, we conducted Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The top 20 higher-score GO enrichment or KEGG pathways were analysed42.We submitted the intersecting target genes to Metascape43 to conduct visual analysis and construct an interactive network. First, we established a file in which the active ingredient and target correspond to each other, and the target and pathway correspond to each other. We then created an attribute file that named the active ingredient, target, and pathway as different types. Finally, we input the above two files into the Cytoscape 3.7.139 software. We used the network analysis tool in this software to analyse the network degree value so that the nodes in the network showed different sizes. A larger node represents a closer connection between that node and other nodes in the network.To understand the relationship between components, targets, and enrichment pathways, we used Cytoscape 3.7.144 (https://www.uniprot.org) database and filtered by reviewed and human organisms, and then, the higher resolution, single chain and X-ray pathway were selected according to the structural information. The selected crystal structures were downloaded from the Protein Data Bank database (https://www.rcsb.org/) in pdb format46. Then, the targets were deliganded and dewatered by PYMOL 2.3.4 software, hydrogenated and charge balanced by AutoDockTools 1.5.6 software, and treated by the Grid Option tool. Finally, targets and components were converted into pdbqt format. We used AutoDockTools to evaluate the affinity of components and target proteins by the same docking method. The docking complexes of ligand and receptor proteins and their binding residues were visualized by PYMOL software48.According to the interactive network results of RP components and targets, molecular docking experiments were carried out at the nodes to a larger degree. The 2D SDF structure files of components were obtained by NCBI PubChem, and the SDF files were input into Chem3D 20.0 software to minimize energy. The targets were entered into the UniProtThe human colon cancer cell line SW480 used in this study was obtained from the cell library of the Chinese Academy of Sciences . The cells were preserved in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin (Corning) and 10% foetal bovine serum (Gibco).4/mL. After overnight culture, the cells were treated with RP at 0, 5, 10, 15, and 20 μg/mL in DMEM for 24, 48, and 72 h. The cells were then incubated with Cell Counting Kit-8 at 37 °C for 2.5 h in a 5% carbon dioxide incubator, and their absorbance values at 450 nm were measured. The results were analysed and plotted with GraphPad Prism 8.0 software.The cell viability of CC cells was determined by Cell Counting Kit-8 (CCK-8) assay. SW480 cells were inoculated into 96-well plates at a density of 4 × 105 cells/well). When the cells reached a state of fusion into a monolayer, 3 uniform lines were drawn vertically along the plate with a pipette aspirator with a sterile diameter of 2 mm. The cells were then incubated for 0, 12, and 48 h with drug-free medium (serum-free medium) and drug-containing medium (serum-free medium containing 5 and 10 μg/mL RP). Each group was repeated three times. Photographs were taken with an inverted microscope  to observe the migration rate of cells in each group.SW480 cells in the exponential growth phase were inoculated in 6-well plates  one-way analysis of variance (ANOVA). p < 0.05 indicated statistically significant differences.Supplementary Tables.


Surgery was performed in 16/42 (40%) of children. A chronic cough and acute respiratory failure were correlated to the need for surgery. During follow-up, there was no difference in persistence of symptoms between conservative vs surgical treatment (p = 0.47). Conclusion: the management of tracheomalacia remains a challenge for pediatricians. Clinical manifestations, such as a barking cough and acute respiratory failure may suggest the need for surgery. Follow-up is crucial, especially in those patients affected by comorbidities, so as to be able to manage effectively the possible persistence of symptoms, including those that may continue after surgical treatment.Background: Few studies have been carried out with the aim of describing the clinical course and follow-up of patients with tracheomalacia. We aim to describe the symptoms at diagnosis and the post-treatment clinical course of patients affected by airway malacia. Methods: We retrospectively analyzed characteristics of pediatric patients with a diagnosis of airway malacia. Patients were classified into three groups: bronchomalacia (BM), tracheomalacia (TM) and tracheo-bronchomalacia (TBM). Demographic and clinical data, diagnostic work-up and surgical treatment were recorded. Results: 13/42 patients were affected by congenital syndromes (30.9%). Esophageal atresia with or without tracheal-esophageal fistula (EA/TEF) was detected in 7/42 patients (16.7%). Cardiovascular anomalies were found in 9/42 (21.4%) and idiopathic forms in 13/42 (30.9%). BM occurred in 7/42 (16.6%), TM in 23/42 (54.7%) and TBM in 12/42 (28.6%). At the diagnosis stage, a chronic cough was reported in 50% of cases with a higher prevalence in EA/TEF ( During infancy, the tracheal cartilaginous framework is a crucial factor for maintaining airway patency . TracheoPrimary or congenital airway malacia can be subdivided into either an isolated process or it can be associated with a condition or syndrome. The isolated primary tracheomalacia is usually benign because airway tone improves over time rather than progressively worsening. Secondary processes are related to extrinsic tracheal compression, such as from a vascular ring or mass, leading to progressive symptoms and morbidity. In addition, congenital abnormalities such as esophageal atresia with or without tracheal-esophageal fistula (EA/TEF) are often associated with tracheomalacia .When a diagnosis is suspected, based on clinical history and physical examination, as reported by the recent ERS (European Respiratory Society) statement, a flexible bronchoscopy represents the “gold standard” diagnostic test. The degree of TM/TBM can be assessed either bronchoscopically or radiologically . The patFew studies have been done with the aim of investigating the clinical features and follow-up of patients affected by this condition. Those few studies have a paucity of data on symptoms post-surgical treatment.This study aims to describe the symptoms at diagnosis and the clinical course in our cohort of patients affected by TM/TBM or BM, including both those conservatively and surgically managed.We retrospectively reviewed the records of pediatric patients (≤18 years) who were diagnosed with airway malacia and were subsequently followed at the Pediatric Pulmonology Unit of the Children’s Hospital V. Buzzi, Milano, Italy, from January 2010 to December 2019.-Subjects with bronchomalacia (BM).-Subjects with tracheomalacia (TM).-Subjects with tracheo-bronchomalacia (TBM).According to the level of the airway malacia, patients were classified into three groups: All data were collected from the medical records of patients diagnosed with airway malacia and these data included demographic characteristics, clinical history, imaging studies, bronchoscopy findings, surgical treatment, medications and outcomes.The current retrospective study protocol was approved by the Local Institutional Review Board. Informed consent was not obtained as this was a retrospective study; however, anonymity of the patients was preserved.Clinical data included: age, gender, presence of associated anomalies or pathologies and symptoms at the diagnosis and during the follow-up stages. In particular, we recorded the presence at the onset of the disease of a chronic cough, recurrent respiratory infections, recurrent pneumonia, dysphagia, reduced exercise tolerance and/or an exercise-induced cough, cyanosis, acute respiratory failure, stridor and/or neonatal respiratory distress. According to the ERS statement, there is no universally accepted classification. Considering the conditions associated with TM and TBM, the affected patients were classified as follows: congenital syndromes, significant and complex comorbidities, EA/TEF not associated with other congenital disorders and cardiovascular anomalies and idiopathic forms.The use of a CT thorax and/or a flexible bronchoscopy as a diagnostic evaluation was recorded. A multidetector CT was performed with an intravenous contrast agent, and, ideally, the examination was performed with breath holding at suspended inspiration. A bronchoscopy was performed under sedation, introducing the fiberoptic bronchoscope through a face mask. During spontaneous breathing, the larynx, trachea and bronchial systems were visualized in order to detect any anomalies. The presence and degree of tracheal narrowing, in inspiration and expiration, were evaluated from video recordings.According to the degree of the TM/TBM, malacia was described as mild (50–75% reduction), moderate (75–90%) or severe (>90%), as established by the recent ERS statement . The typp-value < 0.05 was considered statistically significant.All analyses were performed using Stata 16 . Data were described with the mean, standard deviation (SD), if continuous and as counts and percent if categorical. Comparisons between groups were made with a one-way analysis of variance. The association of categorical variables was assessed with chi square or the Fisher’s exact test. The association between the need for surgery and clinical features or symptoms was evaluated fitting logistic regression models. All tests were two-sided. A p = 0.9). The mean age at diagnosis was 2.1 ± 2.6 years (range 0–10.7 years). Of the 42 patients included in the study, 21 were female and 21 male (p = 0.6).According to the level of airway malacia, TM occurred in 23/42 patients (54.7%), BM in 7/42 (16.6%) and TBM in 12/42 (28.6%), with no significant difference between sexes (p = 0.8).In total, 13/42 patients were affected by congenital syndromes associated with malacia (30.9%), EA/TEF that was not associated with other congenital disorders was detected in 7/42 patients (16.7%), cardiovascular anomalies in 9/42 (21.4%) and idiopathic forms in 13/42 (30.9%), with no difference in males compared to females (Patient characteristics and onset symptoms are reported in p = 0.2) and airways malacia level (p = 0.26). A cough was more frequent in children with EA/TEF compared to other pathogenetic groups (p = 0.005).As reported in p = 0.6). A clinical presentation with recurrent respiratory infections was more prevalent in patients with congenital syndromes (71.4%) and in patients with EA/TEF (69.23%) compared to other groups , without, however, reaching any statistical significance (p > 0.05), as well as recurrent pneumonia (p = 0.123).The overall prevalence of recurrent respiratory infections was 57.4%, with equal distribution in BM, TM and TBM  compared to TM (0%) and TBM (16.6%), The prevalence of dysphagia was similar between BM, TM and TBM as well as according to the pathogenesis. Interestingly, stridor, cyanosis and neonatal respiratory distress were reported only in patients with TM or TBM, even if this was of no statistical significance. Furthermore, stridor was more frequent in patients with cardiovascular anomalies and idiopathic forms (46.7%).p = 0.3). A thorax CT scan was performed in 34/42 (80.9%) of patients and the result was diagnostic in 91% of cases, with no significant difference between BM, TM and TBM (In 36/42 (85.7%) of patients, a flexible bronchoscopy was performed and it was diagnostic in all cases. Among our patients, 33.3% of them presented with a severe form of malacia.p = 0.8). The need for surgery was no different between BM, TM and TBM (p = 0.9) and among different conditions associated with malacia (p = 0.22).Surgical treatment was performed in 16/42 (40%) of children, with no difference in males compared to females . No other significant associations between onset symptoms and need for surgery were detected. Particularly, the degree of severity of TM/TBM was not related to surgical treatment (p = 0.3).Clinical presentation with a chronic cough and acute respiratory failure was significantly related to the need for surgery  and reduced exercise tolerance (16.7%). Only one patient presented with chronic respiratory failure. p = 0.47). The overall prevalence of persistent symptoms during follow-up was 54.8%, with no difference in patients submitted to surgery compared to the others .In We reviewed a 10-year single-center experience of pediatric airway malacia and its management. BM, TM and TBM may be primary abnormalities of the large airways or they could be due to a secondary or acquired cause. As reported, we did not find any differences in the prevalence of these conditions between males and females . In agreThe mean age at diagnosis of more than 2 years in the patients we studied supports the data that, while severe malacia may be evident from birth, most patients typically exhibit symptoms later, resulting in a delay in diagnosis and in treatment ,10,11. In most of our patients, the diagnosis, as well as the measurement of the tracheal lumen reduction, were established by fibrobronchoscopy, the gold standard exam according to the ERS statement . A totalAccording to the data, in our study, a chronic cough and recurrent respiratory infections were the most common symptoms detected at the diagnosis, independent of the level of the airway malacia. In a recent Italian cohort of children affected by a chronic cough, in 32% of cases, tracheomalacia, due to an aberrant innominate artery, was found . BoogaarAmong our patients, other symptoms, such as reduced exercise tolerance and/or exercise-induced cough, seemed to be more prevalent in patients with BM compared to TM and TBM. We reported that the clinical presentation of a chronic barking cough and acute respiratory failure was significantly related to the need for surgery. Similar to our results, Okata et al. found that patients with episodes of acute life-threating events have a higher risk of surgical intervention .Despite the fact that the presence of a barking cough in the diagnosis of tracheomalacia has shown high specificity , our resEven though recent recurrent infections have been suspected as factors related to the need for surgery , we did In line with the study by Masters , our stuForty percent of our patients underwent a surgical intervention, and aortopexy was the most frequent type of procedure ,17. We dIn patients without comorbidities, an improvement of symptoms 1–2 years after post-surgical treatment has been reported ,20. In cThe expression of cytokines, IL-8, IL17A, and IL-1α, is higher in patients affected by tracheomalacia, and this has the potential to indicate the presence of chronic airway inflammation and also lung microbiota disequilibrium, with a prevalence of Pseudomonas at the BALF . These mIn contrast to what was reported in a small cohort by Zeng Hao Wong et al.  and in tWe recognize that this study has some limitations: first of all, the small number of patients, which makes it difficult to highlight any significant differences in clinical presentation, management and outcome between the different categories of patients; furthermore, the retrospective aspect of the study does not allow any definitive conclusions to be drawn on the best approach, surgical or conservative, for the patients. Moreover, a longer follow-up period could give further information on long-term outcomes in these patients. Additionally, further larger studies are key in order to confirm a correlation between a typical clinical symptom, such as a barking cough, not only with the diagnosis of TM, as already reported, but also with the need for surgery. On the other hand, our study highlights the importance of the follow-up, especially in those patients affected by genetic syndromes or with comorbidities, who may have persistent symptoms even after surgery, more than those who are not affected.The management of tracheomalacia remains a challenge for pediatricians. Clinical manifestations, such as a barking cough and acute respiratory failure, may suggest the need for surgery. Early diagnosis is important so as to allow for prompt surgical or medical intervention; follow-up is also crucial, especially in those patients affected by comorbidities, in order to be able to manage possible persistence of symptoms, after surgical treatment. As symptoms are varied and non-specific, early recognition and close monitoring of both surgically and conservatively managed patients are mandatory.


The Guzerá breed is one of the most relevant animal genetic resources for tropical and extreme environments, that is, low and high temperatures, and dry or humid environments. These animals were selected for beef, milk, or dual-purpose (beef and milk), and were extensively used to produce crossbred animals. Consequently, subjecting the breed to intense genetic bottlenecks in Brazil. The local scientific community and breeders have pursued a sustainable management and conservation program over the last 50 years. To evaluate the efficacy of these efforts, we characterized genetic diversity and structure in a Guzerá meta-population. DNA samples of 744 animals from one dairy, nine dual-purpose, and five beef herds were genotyped for 21 microsatellite loci. The genetic diversity estimates suggested a low fixation index, revealing a large genetic diversity in Guzerá herds. The dual-purpose herds/selection lines are the most uniform subpopulation, while the beef one preserved larger amounts of genetic diversity, representing a genetic diversity reservoir for the breed. In addition, the dairy herd showed to be genetically distant from other herds. Taken together, these results suggest that this Guzerá meta-population has higher genetic diversity, a lower degree of population subdivision, and a lower inbreeding level.is, Fit, and Fst estimates were obtained considering either farms or lineages as subpopulations. Mean Ho (0.73) and PIC (0.75) suggest that genetic diversity was efficiently conserved. Fit, Fis and Fst values (95% CI) pointed to a low fixation index, and large genetic diversity: Fit , Fis , and Fst . The dual-purpose herds/selection lines are the most uniform subpopulation, while the beef one preserved larger amounts of genetic diversity among herds. In addition, the dairy herd showed to be genetically distant from other herds. Taken together, these results suggest that this Guzerá meta-population has high genetic diversity, a low degree of population subdivision, and a low inbreeding level.The Brazilian Guzerá population originated from a few founders introduced from India. These animals adapted well to the harsh environments in Brazil, were selected for beef, milk, or dual-purpose (beef and milk), and were extensively used to produce crossbred animals. Here, the impact of these historical events with regard to the population structure and genetic diversity in a Guzerá meta-population was evaluated. DNA samples of 744 animals  were genotyped for 21 microsatellite loci. Ho, He, PIC, F Maximum conservation of genetic diversity is the best guarantee of long-time survival of a breed or a population under artificial selection. Significant loss of genetic variation due to Bulmer effect, inbreeding, and, especially in small populations, genetic drift in the populations under directional selection could imply in low response to genetic selection, decreasing genetic progress rate as well as adaptive performance ,2,3. On Bos indicus) cattle are originally from India, particularly from the dry lands of the Kankrej region. A relatively small population was brought from India at the end of the 19th century and expanded to Brazil, where it became adaptable to various, especially harsh, environmental conditions [Guzerá  [ [9] obtaIn 1993, FAO included the Guzerá in the World List for Domestic Animals Diversity. This list includes breeds of economic, scientific, cultural, and agricultural interest to be conserved through management. Motivated by the importance of this breed as a genetic resource, two focused, interconnected breeding programs were initiated: one for milk traits  other for beef traits .Nevertheless, because of the selection process based on genetic evaluation, further reduction in the number of sires used by breeders, due to the selection process based on genetic evaluation ranks, has become a concern. In the medium—and long-term, this could result in the reduced genetic variability of the population and increased levels of inbreeding ,9. From Genetic diversity and population structure have been studied in bovine populations using microsatellite markers ,16,17,18The main objective of this study was to ascertain genetic diversity and population structure in a meta-population of the Guzerá breed. A meta-population was defined in the current study as the fraction of the population to which we had access, that is, the subpopulation made up of herds that take part in the breeding programs and among which some degree of gene flow occurs. From the results of this study, we expect to effectively contribute to the sustainability of this breed by minimizing the loss of diversity and allowing long-term genetic progress.This study used blood samples from 744 animals, representing around 10% of the animals in each of the 15 major Guzerá herds located in Minas Gerais state, in southeastern Brazil. The majority of herds are concentrated in the Minas Gerais State, which provides proven genetic material from Guzerá nationwide. Currently, some herds exploit Guzerá animals for dual-purpose (dairy and beef), using proven sires from both breeding programs. Other herds use Guzerá strictly for beef purposes . CurrentA herd was defined as the group of animals present in a unique breeding location or farm. Many farms take part in the traditional breeding programs, owning herds composed of their own lineages or a mix composed of their own and external lineages from the whole Brazilian Guzerá livestock. Furthermore, the sampled herds have different selection objectives (selection lines), which were defined here as the group of animals selected for beef, dairy or dual-purpose. Two approaches were applied to understand the structure of this meta-population. In the first approach, each farm was considered a subpopulation; in the second approach, each lineage was considered a subpopulation. The comparison of genetic diversity among different lineages or farms was used to infer the population structure and gene flow in subpopulations.Genomic DNA extraction, PCR amplification, and capillary electrophoresis were developed as described previously . The micAllele frequencies were extrapolated from direct counting. The number of alleles present in the population, numbers of individuals genotyped, expected (He) and observed heterozygosities (Ho), Polymorphism Information Content (PIC), and non-exclusion probabilities were calculated for each locus using the Cervus 3.0  softwareThe Hardy–Weinberg equilibrium (HWE) was tested to support the assessment of the degree of allelic and genotypic diversity in the population. Departures from the HWE could indicate the impact of artificial selection (due to non-random mating) or reduced population size. Heterozygote deficiency was accessed using a multi-sample version of the Score test or U test with the GenePop 4.0 software . The mulis), fixation index (Fst), overall fixation index (Fit)) were calculated following the variance-based method of Weir and Cockerham (1984) [is and pairwise Fst values were calculated for each subpopulation using the Molkin v3.0 software [To estimate genetic differentiation and relationships among subpopulations, Wright (1978)  allele fm (1984) , using tm (1984) . Differesoftware . The AMOsoftware .n = 1000) was performed to evaluate the robustness of the clusters. Additionally, a Neighbor-Net graph , based on the Fst estimated for lineages as subpopulations, was constructed using the SplitsTree4 [Pair-wise standard genetic distances (D)  were calitsTree4  program.The program structure was used to obtain the cluster assignment of lineages through a Bayesian approach, implementing the Markov Chain method with increasing numbers of inferred populations . The tesThe contribution of each lineage to meta-population genetic diversity was assessed using the method proposed by Caballero and Toro (2002)  with theThe microsatellite panel was evaluated in terms of the number of alleles, He, Ho, PIC, and non-exclusion probabilities, presented in The mean observed heterozygosity ranged from 0.47 to 0.85, diverging numerically from the expected values for some loci, mainly for DIK4513. The average Ho was 0.73, slightly below expected (0.77). Such differences are further discussed in light of the HWE tests. Polymorphic information content (PIC) ranged from 0.57 (BM1237) to 0.88 (DIK5183). Combined with the very small non-exclusion probabilities , these rp < 0.05) results were obtained from the HWE test for each locus  between herds [ntiation . These vis was slightly lower (−0.007) and the Fst was slightly higher (+0.007) than those for farms. The Fst for the lineage approach was 0.034, compared to 0.027 for subpopulations grouped according to farm , FARM2 (LINE6), FARM3 (LINE10), FARM4 (LINE1), and FARM5 (LINE12).Estimated values for Fis, Fst, and genetic distances.Farms selecting for dual-purpose were originally beef farms or newly formed. For example, FARM11 (LINE9) was also closed for many years and, only from the end of the 20th century, started to use bulls of different lineages. On the contrary, FARM7 and FARM15 were recently formed by animals of different lineages. In the dual-purpose selection group, the correspondence between farms and lineages was far less strict. These historical aspects affected both the genetic diversity, FFor example, some farms  showed lower levels of heterozygosis, probably resulting from the intensive or exclusive use of their own sires or a small number of sires from few origins. At the same time, other farms  showed high heterozygosis, attributable to the broad use of planned mating and/or semen from animals of different origins or lineages.is using the lineage approach ranged from −0.109 to 0.061. This indicates that genetic diversity also varied among lineages. Some lineages were developed as part of the selection strategy of farms. In those cases, higher Fis were observed for both the farms and the lineages developed within them. For example, some lineages  showed higher Fis and lower heterozygosity, probably resulting again from the intensive or exclusive use of their own sires or a small number of sires from few origins.Estimated values for FOther lineages  showed high heterozygosity. LINE8 originated from LINE7, hence the high genetic similarity between them, and it has also been composed of other lineages resulting in its high heterozygosity. LINE14 came from FARM15, which contains a herd recently formed from a large genetic basis, hence its high degree of heterozygosity.st estimates are shown in st ranged from 0.0029 (FARM15 × FARM1) to 0.048 (FARM6 × FARM3) using the farm approach. They ranged more widely, from 0.007 (LINE8 × LINE11) to 0.087 (LINE7 × LINE10), using the lineage approach. In general, higher average Fst values were estimated for beef lineages. This means that less gene flow has occurred (pairwise Fst), both in the meta-population and in the whole (average Fst). This contrasts with the higher gene flow among dual-purpose lineages expressed by the lower average Fst estimates. One exception can be observed regarding LINE6 (FARM2), which has the lowest pairwise Fst average among the beef lineages (0.018 in the lineage and 0.015 in the farm approaches). Guzerá beef cattle are more extensively bred, generally using little artificial insemination and thus less exchange of genetic material between herds or lineages. However, considering the results of this study, beef herds kept a larger amount of the genetic diversity in Guzerá breeds. The pairwise Fst estimates for the dual-purpose selection lines reveal high genetic variability. This is probably a result of the wide genetic basis for these subpopulations. One clear exception was observed among the dual-purpose selection lines: the average Fst estimated for LINE7 was 0.052, the highest value among all the subpopulations considering dairy, dual-purpose, and beef selection lines. This subpopulation was closed for many generations, and this Fst value reinforces the available information on the breeding policy for this lineage [In general, the lower average F lineage . It shouNei’s standard genetic distances ranged from 0.05  to 0.031  for the farm grouping approach a. In theIn the dendrogram, LINE4, LINE6, and LINE11 were clustered to the same branch. Indeed, LINE11 was derived mainly from LINE4, LINE8, and LINE6 b. In addst values obtained using the farm (0.025) and lineage (0.034) subpopulations were lower than almost all the results shown in The contribution of each subpopulation to the overall genetic diversity is presented in An interesting result was obtained with respect to meta-population structure . If lineDespite the historical events that the Guzerá breed went through in the last decades, such as a strong founder effect and several population bottlenecks, the breed nevertheless preserved a good amount of genetic diversity. In the cluster analysis, it was possible to recover both the origin of the lineages and the selection purposes. Reasonably, in the best model (K = 3), dual-purpose selection lines clustered with the beef selection lines from which they originated. Also, gene flow between the different lineages was detected in the clusters formed. Modeling the genetic structure of the Guzerá meta-population helped to understand the impact of planned crosses on the genetic diversity and the distribution of the genetic diversity among lineages selected for different purposes . The separation of animals for a new selection purpose, if not implying a stronger selection pressure, decreases the risk of loss of diversity. Taken together, these results suggest that, despite the population structure produced by selection strategies based originally on relatively closed farms/lineages, the amount of genetic diversity conserved in the Guzerá breed is high. In particular, beef farms/lineages emerge as a reservoir for both genetic selection and conservation programs.


Background: Accumulating evidence suggests that coronavirus disease 2019 (COVID-19) is associated with hypercoagulative status, particularly for critically ill patients in the intensive care unit. However, the prevalence of venous thromboembolism (VTE) in these patients under routine prophylactic anticoagulation remains unknown. A meta-analysis was performed to evaluate the prevalence of VTE in these patients by pooling the results of these observational studies.Methods: Observational studies that reported the prevalence of VTE in critically ill patients with COVID-19 were identified by searching the PubMed and Embase databases. A random-effect model was used to pool the results by incorporating the potential heterogeneity.Results: A total of 19 studies with 1,599 patients were included. The pooled results revealed that the prevalence of VTE, deep venous thrombosis (DVT), and pulmonary embolism (PE) in critically ill patients with COVID-19 was 28.4% [95% confidence interval (CI): 20.0–36.8%], 25.6% (95% CI: 17.8–33.4%), and 16.4% (95% CI: 10.1–22.7%), respectively. Limited to studies, in which all patients received routine prophylactic anticoagulation, and the prevalence for VTE, DVT, and PE was 30.1% (95% CI: 19.4–40.8%), 27.2% (95% CI: 16.5–37.9%), and 18.3% (95% CI: 9.8%−26.7%), respectively. The prevalence of DVT was higher in studies with routine screening for all patients, when compared to studies with screening only in clinically suspected patients .Conclusion: Critically ill patients with COVID-19 have a high prevalence of VTE, despite the use of present routine prophylactic anticoagulation. The novel coronavirus SARS-CoV-2)-infected disease COVID-19) occurred in Wuhan in December 2019. Since then, the worldwide spread of the disease began   and CochThe PubMed and Embase databases were systematically searched using a search strategy of combined terms. The combined terms were entered into PubMed as a single search, as (“coronavirus” OR “severe acute respiratory syndrome coronavirus 2” OR “SARS-CoV-2” OR “novel coronavirus” OR “nCoV” OR “2019-nCoV” OR “COVID-19”) AND (“pulmonary embolism” OR PE OR “deep venous thrombosis” OR DVT OR “venous thromboembolism” OR VTE OR thrombosis OR embolism OR thrombus OR thrombli OR embolization OR thromboembolism). We used this keyword search strategy instead of those searched as “text words” or as “Mesh terms” or “Emtree” to retrieve more comprehensive records. The Cochrane's Library database was not searched because this study was a meta-analysis of observational studies rather than clinical trials . The search was limited to human studies published in the English or Chinese language. The reference lists of the original and review articles were also manually screened. The final literature search was performed on July 4, 2020.Studies that fulfilled the following criteria were included: (1) published as full-length articles in the English or Chinese language in peer-reviewed journals; (2) designed as observational studies that include critically ill patients with COVID-19, who were treated in the ICU, or a subgroup data of critically ill patients was reported; (3) included adult patients (≥18 years of age) that were screened for the events of VTE, DVT, or PE, with imaging studies such as duplex ultrasound scanning (DUS) and/or computed tomography pulmonary angiogram (CTPA); and (4) reported the prevalence of VTE, DVT, or PE during the ICU stay. If studies with overlapping participants were encountered, reports with a larger sample size were included. Abstracts, reviews, preclinical studies, and studies published in preprints that were not peer-reviewed were excluded.The literature search, data extraction, and quality assessment were independently performed by two authors according to the predefined inclusion criteria. Discrepancies were resolved by consensus. Data on the characteristics of these studies were extracted: name of the first author, year of publication, country where the study was conducted, sample size, mean age, proportion of males, number of patients with chronic lung disease, prevalence of diabetes, status for prophylactic anticoagulation before screening, strategy for VTE diagnosis, and number of patients with each VTE outcome. The modified Newcastle–Ottawa Scale (NOS)  was usedP-values and were logarithmically transformed to stabilize variance and normalize the distribution. For studies that did not report the prevalence data, data regarding the incident cases of VTE outcomes and overall enrolled patients were extracted. The Cochrane's Q test and I2 test were used to evaluate the heterogeneity among the include cohort studies  were calculated from the 95% CIs or  studies . A signid result . FurtherThe process for the literature search and study identification is summarized in A total of 19 studies with 1,599 critically ill patients with COVID-19 were included –33. The P = 0.31, Eleven studies with 947 critically ill patients with COVID-19 , 30–33 rP for subgroup difference = 0.94, P for subgroup difference < 0.001, Fourteen studies with 1,312 critically ill patients with COVID-19 , 28–33 rP for subgroup difference = 0.70, Twelve studies with 1,024 critically ill patients with COVID-19 , 30–33 rIn the present meta-analysis of observational studies, it was found that the prevalence of VTE, DVT, and PE in critically ill patients with COVID-19 was 28.4, 25.6, and 16.4%, respectively. Limited to studies, in which all patients received routine prophylactic anticoagulation, the prevalence for VTE, DVT and PE was 30.1, 27.2, and 18.3%, respectively. The prevalence of DVT was higher in studies with routine screening for all patients, when compared to studies with screening only in clinically suspected patients (47.5 vs. 15.1%). Taken together, these results demonstrate that critically ill patients with COVID-19 have a high prevalence of VTE, despite the use of the present routine prophylactic anticoagulation. These findings highlight the importance for the awareness of the high incidence of VTE for clinicians in managing critically ill patients with COVID-19. Furthermore, these results suggest that the present routine prophylaxis for VTE in critically ill patients with COVID-19 may be inadequate.Results of the meta-analysis revealed that critically ill patients with COVID-19 have a high prevalence of VTE, despite the use of the present routine prophylactic anticoagulation. In a previous large-scale cross-sectional study that included 11,111 critically ill medical patients who received chemoprophylaxis, the overall prevalence of VTE was 6.5% , which iFurthermore, it was found that the prevalence of VTE and its components was not significantly reduced in the meta-analysis limited to studies of all patients receiving prophylactic anticoagulation. Similar to studies on other critically ill patients, the prevalence of VTE has been confirmed as an independent predictor of in-hospital mortality in critically ill patients with COVID-19 . TherefoThe mechanisms of the vulnerability of critically ill patients with COVID-19 to VTE events remain undetermined. Several possible mechanisms have been suggested according to previous clinical and preclinical studies, including cytokine storm with activation of leukocytes, endothelium, and platelets resulting in upregulation of tissue factor, activation of coagulation, thrombin generation and fibrin formation, and deranged coagulation with imbalances in plasminogen activator inhibitor-1, tissue factor pathway inhibitor, and activated protein C that promotes fibrin generation and limits fibrinolysis, hypoxic vaso-occlusion, and direct viral effects with cell activation –52. FutuThe present study has limitations, which should be considered when interpreting the results. First, the sample size of the included studies was generally small, and most of the studies were retrospective. Accordingly, selection and recall biases may exist, which could confound the findings. Moreover, to the best of our knowledge, no consensus has reached for the validated tool for quality evaluation of studies with non-comparative longitudinal design. Therefore, the validity of the modified NOS applied in our study for non-comparative longitudinal studies remain to be determined. Second, heterogeneity was considerable among the included studies. Although the present subgroup analysis in DVT revealed that the screening strategy  could significantly affect the results, there were other factors that may also contribute to the heterogeneity, such as the disease status of patients, previous history of chronic lung disease, concurrent medications, and the use of venous catheters. The investigators were unable to evaluate the potential influences of these factors on the prevalence of VTE, because these data are rarely reported. In addition, detailed information of prophylaxis and diagnosis (including screening) of VTE are important factors that may affect the prevalence of VTE in critically ill patients with COVID-19. However, these data were also rarely reported in detail in the included studies. Third, the subgroup analysis for the influence of the prophylactic status of patients on the prevalence of VTE should be cautiously interpreted, because these results were based on the meta-analysis of data at the study level. Furthermore, the investigators could not directly compare the prevalence of VTE in patients with and without prophylactic coagulation due to lack of access to individual-patient data. Moreover, because of the everyday rapidly increased publications regarding COVID-19 due to the pandemic, it is impossible to include the newest studies in our meta-analysis. Accordingly, the data of the manuscript could only reflect the situation of early pandemic of COVID-19. Finally, the influences of drugs and doses of prophylactic coagulation on the prevalence of VTE in critically ill patients with COVID-19 could not be determined, since none of the included studies specifically reported the related data. For future studies, clinical trials are preferably warranted.In conclusion, the present meta-analysis demonstrated that critically ill patients with COVID-19 have a high prevalence of VTE, despite of use of the present routine prophylactic anticoagulation. Clinicians should always be aware of the high incidence of VTE in these patients, and the optimization of prophylactic coagulation for these patients may be needed to improve the prognosis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.CW was responsible for the preparation of the manuscript. CW, YL, XC, and WZ were responsible for study selection and data collection. CW and YL developed the search strategy and conducted the search in consultation with CF. CF provided support for statistical analysis, conceived the project, and will guarantee the content of the review. All authors contributed to the article and approved the submitted version.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.