This study aimed to estimate the prevalence of eating disorders (EDs) in musicians, and to evaluate their relation to perfectionism, stress, anxiety and depression.It examined: (1) the prevalence of EDs using the Eating Disorder Examination Questionnaire (EDE-Q), body mass index (BMI) and self-reports, (2) psychological risk factors using the Depression Anxiety Stress Scale (DASS-21) and perfectionism inventory and (3) demographic details, information about musical and career development, lifestyle, eating habits and health. A survey was distributed worldwide and a total of 301 English-speaking musicians aged 18 years and older participated.Our screening tools for EDs showed a high prevalence of EDs in musicians: the EDE-Q Global Score (EDE-QGS) showed pathological values in 18.66% of the musicians and when questioned about lifetime prevalence, 32.3% of the musicians answered positively. The median BMI was within the normal range. Regarding general mental health, the DASS-21 showed that depression and stress were severe, anxiety was extremely severe and the perfectionism inventory composite score was 26.53. There was no significant difference on the EDE-QGS between musicians who perform different types of music, but music students, professionals, soloists and musicians travelling overseas had a higher percentage of pathological EDE-QGS. Perfectionism was higher in classical musicians and there was a low positive correlation between EDE-QGS and the risk factors: perfectionism, depression, anxiety, stress, peer pressure and social isolation.EDs are prevalent in musicians and possible risk factors are their increased perfectionism, depression, anxiety and stress due to the demands of their job.The online version of this article (doi:10.1007/s40519-017-0414-9) contains supplementary material, which is available to authorized users. Of all the mental illnesses, eating disorders (EDs) have the highest mortality rate . Eating Adequate nutrition and a balanced diet are crucial to having a healthy lifestyle, particularly for musicians whose careers are at stake. Eating Disorders can affect them in many ways, e.g. voice dysfunction due to vomiting or gastroesophageal reflux disease (GERD) as part of BN , 10, 34,In addition, musicians have an increased likelihood of developing an ED, because EDs are related to various developmental and biopsychosocial factors which are present in many musicians’ lifestyle and training. A musician’s unpredictable work schedule, performing and low income are major factors which both from a mental  and practical  aspect draw them into a vicious circle of unhealthy eating , 35, 42.Musicians may avoid reporting any health issues such as an ED, because of pressure from managers and fear of losing a long-awaited career . TherefoAlthough EDs have been extensively researched in dancers , there hThe purpose of this study was to investigate EDs in musicians, considering that they are prevalent among performing artists , 5, 44. A total of 306 musicians took part in this study. Of the 306 musicians, three saved but did not complete the survey, one was excluded due to the specific age requirements and one was excluded due to answering only one question (answered positively on having an ED) leading to the analysis of 301 data sets. Some questions were not answered by all 301 musicians; therefore, percentages were calculated using the total of musicians who had answered the particular question, not all the 301 musicians (see tables for more details). Participants were self-identified musicians of 18 years old and older of both gender, multinational, at all stages in their careers  and they were recruited between 5th March and 1st June 2016. We used a random sample to avoid any selection bias. This cross-sectional study was undertaken at University College London (UCL) and was approved by the UCL Research Ethics Committee.The “Eating disorders in musicians” self-administered survey consisted of 28 questions. The self-constructed questions (Q1-16b) included information about the participants’ musical development, lifestyle, eating habits, health and demographics. To find the lifetime prevalence of EDs in musicians, we asked if they have ever suffered from any ED including definitions of each one according to DSM-5 . Any curAll questions were uploaded on UCL Opinio 7.3 (online survey software) as one web-based survey in the English language and the advertisement was sent with the survey link to musicians worldwide together with an informed consent form as a welcome message on the Opinio website. Confidentiality and anonymity were maintained and it is not possible to identify participants from any publications. Participants had the right to withdraw with no penalty. The information was used and reported solely by UCL and was not distributed to any third party. The benefit for participants was that the results and copies of any reports or other publications arising from their participation would be sent by the researchers to all those who forwarded an email address.p < 0.05 and all analyses were performed using the Statistical Package for Social Sciences version (SPSS) 23.0 . Most of the data were categorical, so Chi squared test and descriptive analyses were used. To adjust for confounders, we used multiple linear regression. All results were reported as mean ± SD and confidence interval (CI) unless otherwise stated.Significance levels were set at There were a total of 119 male (39.5%) and 182 (60.5%) female participants aged 18-75 years . Regarding the music they perform or teach, musicians responded with: classical (85.8%), jazz (19.5%), pop (15.9%), rock (14.2%), folk (3.9%), rap (2.3%), metal (1.9%), world music (1.6%) and other (17.8%). Of the participants, 82.8% were instrumentalists, 31.3% were singers, 4.5% were composers, 2.2% were musicologists, 1.5% were conductors and 2.4% other.The stages they were in their musical careers were: music students (37.3%), music teachers (36%), professional performers (36.3%), amateur performers (35.6%), other (9.6%). Most frequently they perform: solo (46.9%), within small group (55.8%), within large group (32.3%), other (4.7%).To identify EDs, we used the EDE-Q, the BMI and a lifetime prevalence question “Have you ever suffered from any of the following ED?”.2 (IQR = 20.08–26.23) (Table The self-reported BMI median was 23.14 kg/mp < 0.001). For female musicians, the mean score for each subcategory was: restraint (1.82 ± 1.68), eating concern (1.27 ± 1.43), shape concern (2.67 ± 1.77), and weight concern (2.32 ± 1.7). For male musicians, the mean score for each subcategory was: restraint (0.939 ± 1.29), eating concern (0.52 ± 0.848), shape concern (1.65 ± 1.53), and weight concern (1.34 ± 1.4).The global score of the EDE-Q mean and SD was 1.67 ± 1.43  and the specific items of EDE-Q: restraint (1.49 ± 1.60), eating concern (0.99 ± 1.29) , shape concern (2.28 ± 1.75) (14 musicians did not answer this category of questions), weight concern (1.95 ± 1.66) (15 musicians did not answer this category of questions). The EDE-QGS was pathological in 18.66% of the musicians in our study. In the subcategory, “restraint” 59 musicians (19.6%) had pathological scores, in “eating concern” 41 musicians (13.6%), in “shape concern” 106 musicians (35.2%) and in “weight concern” 78 musicians (25.9%). A total of 83 musicians (27.6%) should be seen for further examination regarding EDs, after showing higher scores in at least two EDE-Q subcategories. The BMI scores were pathological in 45 individuals of this subgroup (54.2%). The EDE-QGS mean was higher in female (2.02 ± 1.49) compared to male (1.09 ± 1.1) musicians  compared to travelling overseas 14.8%) . Most musicians (61.7%) reported that their eating habits do not affect their careers or performance , general mental health demographic details, information about musical and career development, lifestyle, eating habits and health. Consistent with our hypotheses, both our screening tools for EDs  showed a high prevalence of EDs in musicians. Regarding the lifetime prevalence of EDs, 32.3% of all the musicians questioned answered “yes” and all EDE-Q subcategories showed high scores in both female and male musicians. Music students, professionals, soloists and musicians travelling overseas had a higher percentage of pathological EDE-QGS and there was a low positive correlation between EDE-QGS and the risk factors: perfectionism, depression, anxiety, stress, peer pressure and social isolation.Musicians in our study who reported a pathological EDE-QGS were 18.66% compared to 8% in musical theatre students in another study and more musicians reported a pathological score in all the EDE-Q subcategories compared to musical theatre students . FurtherOur participants’ BMI was in the normal range which has also been found in music students , 15, andAlthough we found that there is a higher lifetime prevalence of EDs and a higher percentage of pathological EDE-QGS amongst professional compared to amateur musicians, others have found that a competitive environment alone does not necessarily lead to greater expression of AN . There wMost musicians in our study (61.7%) reported that their eating habits do not affect their careers or performance, compared to 75% of the touring musicians in another study, who reported that certain foods did affect their performance . In our Musicians reported severe levels of stress and depression, extremely severe levels of anxiety, increased levels of perfectionism and their mean score for peer pressure was 2.44 and for social isolation 2.63 , which is consistent with the description of how stressful a musician’s life is . There wAlthough this study has many strengths, several limitations must be acknowledged.We could not study musicians under 18 years old who probably have a higher percentage of EDs, particularly the adolescents . There mOnly musicians who had access to the internet were able to complete the online survey which restricted entries. Also, snowball sampling may have occurred although avoided as much as possible, by sending the survey to many different institutes simultaneously and worldwide. The survey was also just in English, so only English-speaking musicians were able to participate.Instead of or in addition to the EDE-Q, we could have used other questionnaires, e.g. the EAT. Although the EAT is more popular, the EDE-Q is the gold standard in assessing EDs . Still, Participants self-identified as being simply musicians and of either professional or amateur or student status. A clarification regarding their professional status would have given us perhaps more accurate results. Also, there could have been nonmusicians who participated identifying themselves as having strong music identity. We could in this case have added a measure of musician identity such as the musicians’ version of the Athletic Identity Measurement Scale (AIMS) . We coulThe increased EDs in musicians could be due to their increased levels of perfectionism , because their main aim is to perform perfectly , 24, 33.Our results have implications for clinicians in the prevention and therapy of EDs in musicians. Clinicians can be made further aware of the increased prevalence of EDs in musicians, which is possibly due to extra pressures, and therefore, provide special care to optimize their health, well-being and in turn their performance.Given that this is one of the first and largest scale studies regarding EDs of adult musicians, there is potential for additional research. Future studies could compare musicians with and without EDs with behavioural tests and neuroimaging to see if there are any differences in brain structure. Qualitative studies could explore why musicians have more EDs, whether the pathological mechanism involves the reward system (musicians want happiness and autonomy) and whether it is because they like to be in control  , 25, 38.Supplementary material 1 (DOCX 42 kb)Below is the link to the electronic supplementary material.


In China, both population aging and kidney damage has become emerging public health challenges. Despite the number of elders is huge, data on kidney damage in this population are scarce. The present study aimed to describe the prevalence of kidney damage among older adults in Wuhan, China.2 or the presence of proteinuria. Decreased renal function was defined as an eGFR < 60 mL/min/1.73 m2. Proteinuria was defined as urine protein ≥1+ and without urine WBC or nitrite positive. The associated risk factors of eGFR decline and kidney damage were analyzed by multivariate logistic regression.To describe the prevalence of kidney damage among Chinese elderly, the health screening data of 350,881 adults older than 65 years in Wuhan, China were collected and analyzed. Kidney damage was defined as eGFR less than 60 mL/min per 1·73 mThe age-standardized prevalence of kidney damage, decreased renal function and proteinuria was 17.2, 13.5 and 5.3%. Among the patients, up to 74.4% was stage 3. The prevalence of kidney damage and eGFR decline were higher in suburbs than in urban (18.3% vs 16.0 and 14.6% vs 12.4%). Factors independently associated with kidney damage were age, female, BMI, abdominal circumference, hypertension, diabetes, stroke and coronary heart disease.Kidney damage has become an important public health problem in Chinese elderly. More attention should be paid to elderly lived in suburbs or rural area in our further work. Population aging is an emerging worldwide public health challenge in the twenty-first century, which is especially prominent in low- and middle-income countries . It is eAging is associated with an increased prevalence of chronic diseases, such as diabetes, hypertension, heart disease and stroke. Patients with these chronic diseases suffer from chronic kidney disease (CKD) risk multiplier. Except that, age is an independent risk factor of CKD . In deveDelayed diagnosis of CKD in the elderly potentially lead to further damage and increase the risk of death or disability . Early iThe design of this study was a population-based study of kidney damage among individuals older than 65 years in Wuhan, China. Wuhan consists of 17 districts and has 854, 833 elderly. Under a scheme launched by Wuhan municipal government, Wuhan Center for Disease Control and Prevention provided health screening for 372,990 older adults in 11 urban districts and 6 suburban districts from May 2012 to March 2013. The participants were voluntary and informed consent. The health screening included health status questionnaire , blood pressure, height, weight, waist circumference and core laboratory tests, containing blood tests, urinalysis, liver function, kidney function, fasting plasma glucose (FPG), blood lipids, abdominal B ultrasound, ECG and chest X-ray. All blood and urine samples were analyzed at the central laboratory, which successfully completed a standardization and certification program. The individuals with abnormal physical or laboratory tests at screening were recommended to the related departments to review and treat in the general hospital.Urinary protein was measured from a fresh morning spot urine sample with the Automatic Urine Analyzer . The result was reported as negative, trace, +, ++, +++ or ++++. Proteinuria was defined as urine protein ≥1+ and without urine WBC or nitrite positive.To measure the estimated glomerular filtration rate (eGFR), blood was collected by venepuncture after an overnight fast of at least 10 h. Serum creatinine was measured with Jaffe’s kinetic. The Modification of Diet in Renal Disease (MDRD) equation for Chinese CKD patients was used to calculate the eGFR .2) = 175 × Scr(mg/dL)-1.24 × age(year)-0.179[female× 0.79].eGFR , G2 ,G3, G4 , G5 (eGFR< 15 ml/min/1.73 m2).Decreased renal function was defined as an eGFR < 60 mL/min/1.73 mAll participants were measured the blood pressure by a mercury sphygmomanometer after quiet rest for at least 15 min. Systolic blood pressure (BP) ≥ 140 mmHg or diastolic BP ≥ 90 mmHg for two times, or with a history of hypertension and use of antihypertensive medication was defined as hypertension.The fasting plasma glucose≥7 mmol/L, and/or 2-h postprandial plasma glucose≥11.1 mmol/L, or with the history of diabetes was defined as diabetes.Hyperlipidemia was defined as total cholesterol≥6.22 mmol/L and (or) triglycerides ≥2.26 mmol/L.Obesity was defined as body mass index (BMI) > 28, and central obesity was defined as a waist measurement > 85 cm for men or > 80 cm for women.P-value less than 0.05 was taken as the threshold for statistical significance.Data management and analyses were performed on SAS software, version 9.13 . Continuous data were presented as mean ± SD. Mean comparison was performed by t test. The age-standardized prevalence of kidney damage, decreased renal function and proteinuria were calculated using the direct standardization method. The standardized population is based on the population composition of the 6th National Census in 2010. The data comes from the National Bureau of Statistics of the People’s Republic of China. The ratio was compared by Chi-square test. The associated risk factors of eGFR decline and kidney damage was analyzed by multivariate logistic regression, and A total of 372,990 elders participated in the examination. 350, 881(94.07%) cases with complete data were included for the current study, and the average age was 71.9 ± 5.6 years. 163,454 (46.58%) were male and 187,427 (53.42%) were female. 172,028 (49.03%) lived in urban districts and 178,853(50.97%) were from suburbs. The average age of urban participants was 72.2 ± 5.6 years, and the participants from suburban areas were 71.6 ± 5.6 years. Table p = 0.059). In suburbs, the proportion of eGFR< 60 mL/min/1.73 m2 was 11.0% in males, which was lower than in females (15.7%) (p = 0.000). In total, 11.4% male participants and 13.5% female participants had decreased renal function (p = 0.000). The eGFR of 43,949 participants was lower than 60 mL/min/1.73 m2, accounting for 12.5%. The proportion of decreased renal function in suburbs participants was higher than in urban participants  (Table p = 0.000). In urban, 0.2% was G5 in participants aged 80 years or older and in other groups was only 0.1%. In suburbs, the percentages of G5 had no difference in various groups. After adjusted for age, 11.8% participants from urban were G3, which was lower than in suburbs . There was no difference of G4 and G5 percentages in urban and suburbs after adjusted for age. In total, 12.9% of participants was G3, 0.5% G4 and 0.1% G5 after adjusted for age (p = 0.000). The age-standardized prevalence of decreased renal function in urban was 12.4%, in suburbs was 14.6 and in total was 13.5%. . In suburbs, 4539 (5.3%) males and 4629(5.0%) females had proteinuria (p = 0.000). In all participants, the percentage of proteinuria in males was 5.4%, which was higher than in females . In total, 17,858 (5.1%) participants had proteinuria and the prevalence of proteinuria in urban and suburbs was the same . In other groups, the prevalence of proteinuria was higher in suburbs than in urban .The prevalence of proteinuria was increased with age both in urban and suburbs. In group of age 70~74 years, 5.2% participants from urban had proteinuria, which was higher than in suburbs (p = 0.002). The prevalence of kidney damage in females from urban was 14.8%, which was lower than in suburbs . In total, 15.2% males and 17.0% females had kidney damage (p  = 0.000). The prevalence of kidney damage in urban was 15.1%, which was lower than in suburbs   Table . Fifty-sp = 0.517). But in the groups of ages 65–69 years, 70-74 years and 75-79 years, the kidney damage prevalence in urban was lower than in suburbs (p = 0.000). In total, 11.8% participants ages 65–69 years, 15.7% participants ages 70–74 years, 20.5% participants ages 75–79 years and 26.1% participants ages 80 years or older had kidney damage  patients had eGFR decline, but without proteinuria; 12,594 (22.3%) patients with proteinuria and normal renal function; only 5264 (9.3%) patients had both proteinuria and decreased renal function. 42,081 (74.4%) was stage 3, 1545 (2.7%) stage 4 and only 323 (0.6%) was stage 5 .Age, sex, blood urea nitrogen, serum creatinine, blood cholesterol, serum triglyceride, fast blood glucose, BMI, abdominal circumference, history of hypertension, diabetes, hyperlipidaemia, obesity, stroke and coronary heart disease were analyzed by the multivariate logistic regression and age, female, BMI, abdominal circumference, history of hypertension, diabetes, stroke and coronary heart disease were all independently associated with eGFR decline and kidney damage Table .Table 7Although much is known about kidney damage in elderly, a great deal remains to be researched. To our knowledge, this study is the first large-scale population-based study to research the prevalence of kidney damage in Chinese elderly. In this study population, the age-standardized prevalence of kidney damage, decreased renal function and proteinuria was 17.2, 13.5 and 5.3%. Based on these results, 20.4 million Chinese aged 65 years or older have kidney damage, and 16.0 million have decreased renal function. Several studies have reported the high prevalence of chronic kidney disease in Chinese –14. HoweIn this study, we found 15.2% males and 17.0% females had kidney damage. It suggested that the prevalence of kidney damage was gender-different and females had a higher prevalence of kidney damage than males. The same phenomenon was revealed in some other large studies . In CanaOur results revealed the prevalence of decreased renal function, proteinuria increased in parallel with age, which was consistent with the results from Australia . The stu2 [Among the kidney damage participants, proteinuric kidney damage only accounted for 22.3%, but up to 74.4% patients was stage 3. In Canada elderly, the percentage of CKD stage 3 was higher than CKD stage 4–5 in any age groups (by 5-years) and in any sex . In Aust2 . It sugg2 .The data from the current study suggest that the prevalence of proteinuria in urban and suburbs residents was the same, but the prevalence of decreased renal function in suburbs residents was higher than in urban residents. The prevalence of kidney damage in suburbs residents was higher than in urban residents in any age groups. Zhang LX et al. reported that more rural residents had CKD than urban residents in Chinese general population (average age 49.6 ± 15.2 years) . The resThe present study is the first large-scale population-based study to research the prevalence of kidney damage in Chinese elderly. But it had several limitations. First, uric protein was only detected by dipstick urinalysis, which has less favorable diagnostic properties than urine albumin-creatinine ratio (ACR) for the assessment of proteinuria . To reseOur results show that kidney damage has become an important public health problem in Chinese elderly. Among the older patients, stage 3 is prominent and special attention should be paid to which in our further work. We also found that decreased renal function in suburbs residents was higher than in urban residents, improve the income and medicare amount of which in future may alleviate the social financial burden effectively.


Eisenia andrei exposed to soils from a gradient of CCA wood preservative contamination near Hartola, Finland. In soils contaminated with 1480–1590 mg Cr/kg dry soil, 642–791 mg Cu/kg dry soil, and 850–2810 mg Ag/kg dry soil, uptake and elimination kinetics patterns were similar for Cr and Cu. Both metals were rapidly taken up and rapidly excreted by Eisenia andrei with equilibrium reached within 1 day. The metalloid As, however, showed very slow uptake and elimination in the earthworms and body concentrations did not reach equilibrium within 21 days. Bioaccumulation factors (BAF) were low for Cu and Cr (< 0.1), but high for As at 0.54–1.8. The potential risk of CCA exposure for the terrestrial ecosystem therefore is mainly due to As.The aim of this study was to determine the bioavailability of metals in field soils contaminated with chromated copper arsenate (CCA) mixtures. The uptake and elimination kinetics of chromium, copper, and arsenic were assessed in the earthworm The online version of this article (10.1007/s11356-019-04908-6) contains supplementary material, which is available to authorized users. According to a report from the European Soil Data Centre of the European Commission, the estimated number of potentially contaminated sites in Europe is over 2.5 million. Metals (37.3%) and mineral oil (33.7%) are the most frequent soil contaminants across Europe . Since the toxicity of metals depends on the concentration in the body, uptake and elimination kinetics are important and relevant tools for evaluating the bioavailability of metals.Little is known about the bioavailability of CCA metals in mixtures, and therefore great uncertainty exists about their potential risk in soils. Measures of bioavailability can be used as a guideline for the risk assessment of soil contamination  were as follows: Cr 12.5–1592, As 10.1–2812, and Cu 5.1–79. In Finland, the background levels and the range (mg/kg) for Chromium, Arsenic, and Copper are 31 (6–170), 1 (0.1–25), and 22 (5–110), respectively Oxide, 26.6% CrPicea abies L.) stand of Oxalis-Myrtillus site type  with a SCHOTT pH meter, type CG842. Soil moisture content was determined by drying soil samples at 105 °C for 24 h. Particle size distribution was determined using laser size grain analysis, which is based on the forward scattering of monochromatic coherent light as described by Konert and Vandenberghe (Soil water holding capacity (WHC) was determined following ISO . Organicenberghe .Eisenia andrei) were exposed to contaminated soils from the four test sites: high (H), medium (M), low (L), and control (C). Each soil had six replicates per sampling time. One earthworm was placed in a glass container containing 35–50 g of test soil (ww). The earthworms were taken from a synchronized culture at the Vrije Universiteit, Amsterdam, The Netherlands. Only adults with a well-developed clitellum were used. Before starting exposures, the earthworms were acclimatized in OECD artificial soil  was added. The acids (HCl 36.5–38.0% and HNO3 69.0–70.0%) were supplied by J.T. Baker for trace metal analysis. After closing, the bottles were placed in an ultrasonic bath (Transsonic 820/H Elma®) for 3 × 3 min at a temperature of about 45–50 °C. After sonification and cooling, the samples were filtered (Whatman no. 41) into a 25-mL volumetric glass bottle, diluted with high purity ELGA water to a volume of 25 mL, and stored in plastic bottles for the analysis with Inductively Coupled Plasma-Optical Emission Spectrometry . All equipment was rinsed with acid before use. Reference materials for contaminated soils, SRM 2710 and SRM 2711, both certified by the National Institute of Standards and technology (NIST), were included in the analysis. Recoveries from the certified reference sample SRM 2710 were 96% for As and 92% for Cu; recovery of Cu from the reference material SRM 2711 was 96%. No certified reference values were given for Cr concentration in the SRM 2710 sample. The procedure has been described by Väisänen et al. (About 500 mg (dw) soil was weighed into 50-mL plastic bottles and 10 mL aqua regia . Earthworm samples were placed for 1 h in a water bath at 70–80 °C. After cooling, the extract was filtered and diluted with high purity ELGA water to a volume of 25 mL . The wavelengths with the axial plasma viewing used in the determination were 193.696 nm, 283.563 nm, and 324.752 nm for As, Cr, and Cu, respectively. Quality control was performed by the analysis of two certified reference materials, DOLT-4 Dogfish liver and TORT-2 Lobster hepatopancreas. High recoveries were obtained for all the elements of interest. The certified and measured concentrations (mg/kg) and recoveries of metals (%) from the DOLT-4 Dogfish liver were as follows: As 9.66 ± 0.62, 7.75 ± 0.08, and 80.2; Cu 31.2 ± 1.1, 30.3 ± 0.4, and 97.1; Cr 1.4 , 1.4 ± 0.4, and 100. The certified and measured concentrations (mg/kg) and metal recoveries (%) from the TORT-2 Lobster hepatopancreas were as follows: As 21.6 ± 1.8, 22.9 ± 0.3, and 105.8; Cu 106 ± 10, 97 ± 2, and 92, and Cr 0.77 ± 0.15, 1.0 ± 0.2, and 130, respectively.The earthworms were digested individually in 4 mL aqua regia , C0 is the initial (background) copper/chromium/arsenic concentration in the earthworms at t = 0 (mg/kg dry body weight), k1 is the uptake rate constant (kg soil/kg earthworm/day), k2 is the elimination rate constant (day−1), Cexp1 is the copper/chromium/arsenic exposure concentration during the uptake phase (mg/kg dry soil), t is the exposure time (days), and tx is the day on which animals were transferred to clean OECD artificial soil (day 21). Both Eqs. E. andrei was estimated using the following equation  of chromium and copper in the high and medium contaminated areas were similar, while they were low in the low contaminated and control areas Table . Total AEarthworm survival was high during the 42-day experimental period, with only 3 animals dying in the control (83 worms) and 2, 7, and 11 dead worms out of 83 in low, medium, and high contaminated soils, respectively.In the low contaminated soil, no uptake of chromium, copper, and arsenic was seen, with earthworm body concentrations remaining more or less constant at approximately 0.9, 7.7, and 22 mg/kg body weight, respectively throughout the uptake and elimination phases Fig. . In the k1) and elimination (k2) rate constants for medium and high contaminated soils, and they were indicating very fast kinetics. For As, k1 and k2 in both the high and medium contaminated soils were very small, indicating slow kinetics. Bioaccumulation factors (BAF) were well below 0.1 for Cr and Cu, but between 0.54 and 1.8 for As, and did not differ much for the medium and high contaminated soils.There was little difference between Cr and Cu uptake (k1) based on water- or CaCl2-extractable concentrations  wood preservative. In an earlier study, Karjalainen et al. , copper (Cu), and arsenic (As) to the earthworm n et al.  indicate2O and 0.01 M CaCl2 extracts. This was expected because the field soil was contaminated decades ago. The metal contaminant pool requires time to diffuse into micro or nanopores and to be absorbed into organic matter and adsorbed onto soil particles , which has influenced speciation and mobility of the metals present. Under acidic conditions, the toxic and mobile Cr(VI) will be reduced to the stable and less toxic Cr(III)  form, but when pH increases and redox potential decreases, As(III) is the dominant form in soils  for Cu in different UK field soils. In our study, very fast elimination of Cr and Cu from E. andrei occurred with k2 = 7.6–24.6 and 2.2–2.4 day−1, respectively. These k2 values agree with the data of Peijnenburg et al. (In the medium (M) and high (H) contaminated soils, fast uptake kinetics of chromium and copper with fast elimination rates were seen in the earthworm i et al.  found sig et al. , Spurgeog et al. , and vang et al. . Nahmanig et al.  found th2+ and Cr3+). Their size is similar and they have very similar chemical characteristics. Both are Lewis acids which bind strongly to organic matter forming similar type organic ligands, which may explain why these two metals have similar uptake and elimination patterns in E. andrei like other essential metals. Fast uptake reaching equilibrium within a few days for other essential metals like Zn and non-equilibrium for non-essential metals were reported by Spurgeon and Hopkin . There are several mechanisms for Cr(VI) reduction to Cr(III) via intermediates like Cr(V) and Cr(IV) , H2O2, and hydroxyl (·OH) radicals, collectively called oxygen species (ROS), play a major role as a messenger for NF-kB activation in Jurket cells  calculated to estimate metal bioavailability in the different soils was low (< 0.1) for Cr and Cu due to fast excretion from the earthworms. BAFs for Cu in 2O and CaCl2 extractions (Table E. andrei upon exposure to the medium (M) and high (H) contaminated Hartola field soils, and steady-state was not reached after 21-day exposure. Similar uptake patterns have been reported in field soil  contaminated field soil, no uptake of arsenic was seen, with earthworm body concentrations remaining more or less constant at approximately 22 mg/kg body weight, throughout the uptake and elimination phases. This matches with the low availability of As in this soil seen from Hk1 values for As were 0.011 and 0.0065 kg soil/kg worm/day, respectively, which agrees with the k1 of 0.0046 kg soil/kg worm/day reported by Peijnenburg et al. , reaching steady-state after 1–3 days of exposure of contaminated soils from a mine tailing. The elimination rate constant k2 = 12.7 day−1 (20 °C and 50%WHC) was much higher than in the present study. This fast kinetics of As is totally opposite to our findings, and is probably due to the different soil types, especially the high pHCaCl2 (6.04–7.44) and low organic matter contents (~ 1.5–4.3%) of the soils used in the study of González-Alcaraz and van Gestel  4.71–7.18, while the history of As contamination in the mining area has been very long and As speciation form probably was different from our study. In the mining area, arsenic is accumulated as an arsenobetaine form  in CCA-contaminated field soils. All three metals were available at the site, but the uptake and elimination patterns in E. andrei of Cr, Cu, and As were quite different. Uptake and elimination for the essential metals Cr and Cu were very fast with equilibrium being reached within 1 day, probably due to active regulation of the body concentrations by the earthworms. For As, uptake and elimination kinetics were very slow leading to relatively high bioaccumulation factors (BAF), suggesting potential risk of metal biomagnification in the food chain. When assessing the ecological risk of CCA-contaminated soils in Hartola, Finland, focus should especially be on the high bioavailability of As, and consider its possible transfer in the food chain. Further research is needed to study the consequences of exposure to multiple metals at this site.Little is known about the bioavailability of the metals in the CCA mixture, leading to great uncertainty about the potential risk of CCA-contaminated soils. In this study, we used a toxicokinetics approach to assess the bioavailability of copper, chromium, and arsenic to earthworms (ESM 1(DOCX 439 kb)


Ganoderma lucidum using response surface methodology (RSM). Interactions were studied with three variables, viz. glucose, peptone and culture time using a Central Composite Design (CCD). The CCD contains a total of 20 experiments with the first 14 experiments organized in a fractional factorial design, with the experimental trails from 15 to 20 involving the replications of the central points. A polynomial model, describing the relationships between the yield of GA-Me and the three factors in a second-order equation, was developed. The model predicted the maximum GA-Me yield of 11.9 mg·L−1 for glucose, peptone, culture time values of 44.4 g·L−1, 5.0 g·L−1, 437.1 h, respectively, and a maximum GA-Me yield of 12.4 mg·L−1 was obtained in the validation experiment, which represented a 129.6% increase in titre compared to that of the non-optimized conditions. In addition, 11.4 mg·L−1 of GA-Me was obtained in a 30-L agitated fermenter under the optimized conditions, suggesting the submerged culture conditions optimized in the present study were also suitable for GA-Me production on a large scale.Enhanced ganoderic acid Me  yield was attained with the medicinal fungus  Ganoderma lucidum (Leyss. ex Fr.) Karst (“Ling-zhi” in Chinese and “Reishi” in Japanese) is a famous medicinal mushroom that has been used as a traditional medicine for a long time in Asia  for the total model indicated the model was highly significant. The goodness of fit of the model was examined by the coefficient of determination (R2 = 0.9257), which implied that more than 92% of the sample variation was attributable to the variables and only 7.43% of the total variance could not be explained by the model. The adjusted determination coefficient (Adj. 2R = 0.8570) was also satisfactory to confirm the significance of the model. The results of CCD experiments for GA-Me production are presented in P-value, the more significant is the corresponding factor. X3 (culture time) had extremely significant effect on GA-Me production (P < 0.01), followed by x2 (peptone concentration), suggesting culture time and peptone concentration are very important for GA-Me production in submerged fermentation of G. lucidum.t-test and P-value, which is listed in t-test and smaller the P-value, the more significant is the corresponding coefficient. The polynomial model for GA-Me yield YGA-Me was regressed by all the terms and was expressed by Equation (1). The regression equation showed that the GA-Me yield was an empirical function of test variables in coded unit. YGA-Me is the predicted GA-Me yield, x1 glucose, x2 peptone, and x3 is culture time.The significance of each coefficient was determined by Student’s x3) had a strong positive linear effect on the response (P < 0.01) of YGA-Me as it had the largest coefficient, followed by peptone (x2), which showed a significant negative linear effect (P < 0.01). However, glucose (x1) had no significant effect on GA-Me production at the tested concentrations (P > 0.05), and the above three variables also indicated negative quadric effects on GA-Me yield (P < 0.05). No significant interactions were noted between any two of the three variables (P > 0.05).Equation (1) also reveals that culture time  was obtained, and the content of GA-Me in mycelial cell was 42.7 μg·100 mg−1 DW , and that in fermented supernatant  it was 7.6 mg·L−1, that is, a 12.4 mg·L−1 of GA-Me production yield was obtained in G. lucidum cultures, which represented a 129.6% increase in titre compared to the the non-optimized culture conditions (5.4 mg·L−1), and was also in agreement with the predicted value (11.9 mg·L−1). The good agreement between the predicted and experimental results verifies the validity of the model and the existence of an optimum point. The feasibility of the regression model was further tested in a 30-L scaled fermenter under the optimized culture condition. A maximum yield (11.4 mg·L−1) of GA-Me was obtained (Triplicate experiments were carried out to verify the availability and accuracy of the model (Equation (1)) for GA-Me production in flask shake culture. The results are shown in obtained , suggestG. lucidum [G. lucidum is recognized as a promising alternative for efficient production of GAs [G. lucidum, many studies were about total GAs production [et al. studied the production of individual ganoderic acids  by G. lucidum cell culture [−1 , and the GA-Me content was higher than that obtained in the present study. However, the GA-Me yield was very low in shaking flask culture with a lactose-based culture medium, and the content of GA-Me in the mycelium was about 10.1 μg·100 mg−1 DW, which is lower than that in the present work. Nevertheless the content of GA-Me in fermented supernatant (extracellular GA-Me) was not determined by Xu et al. In the present study, we obtained 7.6 mg·L−1 of GA-Me from fermented supernatant, and 4.8 mg·L−1 of GA-Me from mycelia, suggesting some quantity of GA-Me was excreted into the fermented broth during the culture process, a phenomenon also observed in several previous studies [−1, and 9.1 g·L−1 of mycelium pellets and a 11.4 mg·L−1 titre of GA-Me was obtained, suggesting GA-Me could be also produced under the optimized culture conditions in a large-scale agitated bioreactor.Until now, GAs were mainly extracted from the solid cultivated fruiting bodies of  lucidum . It usua lucidum . Consequn of GAs ,18,19. Aoduction ,17,18,19 culture . It was  studies ,23,24,25 studies . HoweverG. ludicum SCIM 0006 were obtained from the Strain Collection of Industrial Microorganisms (SCIM), Central South University of Forestry & Technology , and maintained on potato-dextrose-agar slants, stored at 4 °C.Stock cultures of G. lucidum SCIM 0006 was grown in a 250-mL flask containing 80 mL seed culture medium (see below) at 30 °C for 8 days with shaking at 160 rpm. This was then inoculated at 12% (v/v) into the fermentation medium incubated at 28 °C for different days for optimization of GA-Me production. The seed culture medium composed of (in g·L−1): glucose (40.0), peptone (4.0), KH2PO4 (0.75), MgSO4·7H2O (0.45) and vitamin B1 (0.01). The fermentation medium composed of (g·L−1): glucose (36.595–3.41), peptone (3.326–68), KH2PO4 (0.75), MgSO4·7H2O (0.50) and vitamin B1 (0.01). The basic fermentation conditions were as follows: glucose (38 g·L−1), peptone (4.5 g·L−1), KH2PO4 (0.75), MgSO4·7H2O (0.45) and vitamin B1 (0.01); culture time 300 h; culture temperature 28 °C [re 28 °C .The bioreactor culture was carried out by a similar method described in our previous work  in an agSamples collected from flasks were filtered using a 40-mesh stainless sieve and the mycelium was harvested. Mycelial biomass was collected by centrifuging the mycelium at 8,000 rpm for 15 min, washing the precipitated cells for three times with distilled water, and drying at 60 °C until it reached to a constant weight .−1) includes the contents of GA-Me from both mycylia and fermented broth. The determination of GA-Me was made by a similar method described in a previous work [−1 with a linear gradient of solvent A  and solvent B (water). The gradient, starting at sample injection, was linear from 80% to 100% A in 20 min and the elution continued for an additional 20 min at 100% A. The sample injection volume was 20 μL, GA-Me was detected at 245 nm. Standard curves of peak area, as a function of the concentration of GA-Me was prepared for quantitative analysis [The GA-Me yield ) as follows:iY is the predicted response variable; 0β, iβ, iiβ, ijβ are constant regression coefficients of the model, and ix, jx  represent the independent variables (fermentation parameters) in the form of coded values.The preliminary trails indicated that glucose, peptone and fermentation time were the significant variables for GA production. Hence, these three variables were chosen to obtain the optimum levels. A central composite design (CCD) was used in the optimization of GA-Me production. l points . A matheR2. The significance of each coefficient was determined using Student’s t-test.The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination The SAS statistical package  was usedG. lucidum in submerged culture was studied. From the present study, it is evident that the use of a statistical culture condition optimization approach and response surface methodology was helpful to locate the optimum levels of the significant conditions with minimum effort and time. The optimization of the culture conditions not only resulted in a 129.6% higher GA-Me concentration than using non-optimized medium, but also in a successful large scale fermentation process. In addition, GA-Me is not only present in G. lucidum mycelia but also in fermented broth.Enhanced production of individual GA-Me, an important bioactive triterpene, by


Bixa orellana have been reported to be traditionally used in the treatment of diarrhea by local people in the district of Khulna in Bangladesh. The aim of this study was to investigate the effects of the hydroethanolic extract of Bixa orellana leaves on castor oil-induced diarrhea in mice.  The treatment of diarrheal diseases is a serious problem in developing countries, where population generally uses medicinal plants. The leaves of Bixa orellana was macerated in ethanol/water mixture (20/80) for 48 hours and then filtered. The filtrate obtained was lyophilized, and the solutions to be administered to the animals were prepared. To induce diarrhea, animals orally received castor oil (1 mL/100 g bw). To determine the effective doses, each mouse received, 30 minutes after the administration of castor oil, one of the single oral doses of hydroethanolic extract of Bixa orellana leaves: 0, 25, 50, 100, and 200 mg/kg bw. The mass, number, and frequency of stool diarrhea were measured and recorded per hour for five hours. The effect of the hydroethanolic extract of Bixa orellana leaves on the intestinal transit was evaluated by measuring the distance traveled by the charcoal meal in thirty minutes. The effects of the aqueous extract of hydroethanolic extract of Bixa orellana leaves on intestinal secretion were evaluated by measuring the volume of the intestinal content and by dosing the electrolytes  in the intestinal content by the colorimetric method.  The powder of the leaves of P < 0.01) decreases, respectively, 35.52%, 54.47%, 74.80%, and 87.80% in the severity of diarrhea. The extract at 100 and 200 mg/kg bw showed a significant (P < 0.01) decrease of castor oil-induced enteropooling (61.08% and 65.41%), and only the 200 mg/kg bw exhibited significant (P < 0.01) reduction on intestinal transit (24.46%) as compared to standard drug.  The extract produced significant ( The hydroethanolic extract was found to be effective against castor oil-induced diarrhea in experimental mice at 50, 100, and 200 mg/kg bw which provides evidence that could justify its traditional use. In developing countries, diarrheal diseases are a major cause of suffering and millions of deaths every year. These diseases represent the most serious problem in these countries, affecting more children and are considered the second leading cause of death in children under five , 2. DiarOxalis barrelieri  for tannins, Liebermann-Burchard test for terpenoid and sterol, Shinoda test for flavonoids, frothing test for saponins, Molisch test for polysaccharides, UV lamps for coumarins, and Borntrager's test for anthraquinones.The qualitative phytochemical screening of the hydroethanolic extract of nd tests –19: Dragin vivo experiments were done according to the guidelines of the European Union on Animal Care (CEE Council 86/609)  is the concentration of chloride ions in the intestine content; Abs T is the absorbance of the test tube; Abs S is the absorbance of the standard tube; [S] is the standard concentration of chloride ions (100 mEq/L).One thousand microliters . Sodium ions react with the selective chromogen and produce a chromophore whose absorbance is directly proportional to the concentration of sodium in the sample.μL) of the reagent was put in all the tubes. We added 10 μL of distilled water in the blank tube, 10 μL of standard solution (150 mEq/L) in the standard tube, and 10 μL of samples in the test tubes. Each mixture was incubated in a water bath for five (5) minutes at 37°C, and then, the absorbance was read at 630 nm with a spectrophotometer (UNICO© ULTRA VIOLET). The concentration of sodium ions in each tube was determined by the following formula:+] is the concentration of sodium ions in the intestine content; Abs T is the absorbance of the test tube; Abs S is the absorbance of the standard tube; [S] is the standard concentration of chloride ions (150 mEq/L).Five hundred microliters . The potassium ion reacts with tetraphenol in a specially prepared buffer to form a colloidal suspension. The amount of turbidity produced is directly proportional to the potassium concentration in the sample.μL) of the reagent was put in all the tubes. We then added 20 μL of distilled water in the white tube, 20 μL of standard solution in the standard tube, and 20 μL of samples in the test tubes. Each mixture was incubated in a water bath for five (5) minutes at 37°C, and the absorbance of the contents of each tube was read at 630 nm with the spectrophotometer (UNICO ULTRA VIOLET). The concentration of potassium ions in each tube was determined by the following formula:+] is the concentration of potassium ions in the intestine content; Abs T is the absorbance of the test tube; Abs S is the absorbance of the standard tube; [S] is the standard concentration of chloride ions (5 mEq/L).Five hundred microliters OG=29X ± S.E.M). Data were analyzed by one-way ANOVA followed by Dunnett's t-test using computerized GraphPad InStat (DATASET1.ISD). Values were considered significant at P < 0.05 in comparison with the control.Data were expressed as mean ± standard error of mean  decreased in animals treated with the hydroethanolic extract of Bixa orellana leaves at 50 mg/kg (HEBo50), 100 mg/kg (HEBo100), and 200 mg/kg (HEBo200). The inhibition rate of diarrhea was 84.00%, 32.00%, 56.00%, 76.00%, and 88.00%, respectively, in mice treated with Loperamide 5 mg/kg bw (Lop5), Bixa orellana leaf hydroethanolic extract at 25 mg/kg (HEBo25), 50 mg/kg (HEBo50), 100 mg/kg (HEBo100), and 200 mg/kg (HEBo200). The frequency of stool emission was, respectively, 3.0, 0.6, 1.8, 0.8, 0.6, and 0.2/h for diarrheal control, Lop5, HEBo25, HEBo50, HEBo100, and HEBo200  hours after the administration of castor oil, diarrheal controls emitted 0.25 ± 0.02 g and 3.00 ± 0.71 N/h of diarrheal stools. These diarrheal stools significantly ( HEBo200 .Bixa orellana leaf hydroethanolic extract at 50 mg/kg (HEBo50), 100 mg/kg (HEBo100), and 200 mg/kg (HEBo200). The inhibition of intestinal transit was 37.04%, 27.75%, 33.54%, and 37.94%, respectively , the group treated with atropine sulfate 0.3 mg/kg (AT0.3), and the groups treated with the ectively .Bixa orellana leaf hydroethanolic extract at 50 mg/kg (HEBo50), 100 mg/kg (HEBo100), and 200 mg/kg (HEBo200) , the normal control (NC), the treated group with Loperamide 5 mg/kg (Lop5), and the test groups treated with HEBo200) .Bixa orellana hydroethanolic extract at 50 mg/kg (HEBo50), 100 mg/kg (HEBo100), and 200 mg/kg (HEBo200). The inhibition of secretion was 35.14%, 61.08%, 65.41%, and 62.16%, respectively , the diarrheal control (DC), the Loperamide-treated group, 5 mg/kg (Lop5), and the test groups treated with ectively .P < 0.01) in treated animals and were 91.27 ± 8.17 mEq/L, 103.18 ± 6.14 mEq/L, 108.61 ± 5.12 mEq/L, and 106.39 ± 3.08 mEq/L, respectively, for animals treated with Loperamide or with Bixa orellana leaf hydroethanolic extract at 50 mg/kg, 100 mg/kg, and 200 mg/kg. Sodium ion concentrations in animals treated with Bixa orellana leaf hydroethanolic extract or Loperamide were significantly (P < 0.01) low compared to diarrhea control (129.60 ± 8.24 mEq/L) and were, respectively, 107.82 ± 4.46 mEq/L, 106.91 ± 6.47 mEq/L, and 106.33 ± 4.85 mEq/L at 50, 100, and 200 mg/kg or 103.16 ± 3.82 mEq/L for Loperamide 5 mg/kg. Potassium ion concentrations were 10.51 ± 2.74 mEq/L, 5.75 ± 1.01 mEq/L, 11.05 ± 3.41 mEq/L, 9.89 ± 0.68 mEq/L, 10.54 ± 0.43 mEq/L, and 10.02 ± 2.91 mEq/L, respectively, in normal control, diarrheal control, and in animals treated with Loperamide or with hydroethanolic extract of Bixa orellana leaves at 50 mg/kg, 100 mg/kg, and 200 mg/kg  reduced the amount and frequency of castor oil-induced diarrheal stools in mice, unlike the methanolic extract which showed maximal inhibition (85%) at 500 mg/kg. Our extract could act by inhibiting one or more of the mechanisms of induction of diarrhea by castor oil.Depending on its etiology, diarrhea may be characterized by increased secretion of electrolytes (secretory diarrhea), increased luminal osmolality (osmotic diarrhea), decreased electrolyte absorption, and/or increased intestinal motility responsible for reduced transit time. Many antidiarrheal agents can therefore act by increasing the transit time by inhibiting gastrointestinal motility, inhibiting intestinal secretions, and/or increasing the intestinal absorption of water and electrolytes. Laxatives such as castor oil induce diarrhea by increased motility and/or gastrointestinal secretions via its active ingredient, the ricinoleic acid. Ricinoleic acid modifies the permeability of the intestinal mucosa to electrolytes  by inhibactivity  and stimactivity . In thisBixa orellana extract (200 mg/kg bw) significantly inhibited intestinal peristalsis almost as the reference drug, atropine. Atropine is a competitive antagonist of acetylcholine muscarinic receptor [3 and Diacyl glycerol (DAG) which are responsible for the increase of cytosolic calcium and smooth muscle contraction [Bixa Orellana [μ receptors located on the smooth intestinal muscle and thus reducing intestinal peristalsis [receptor  located traction , 29, 30.Orellana , phytochistalsis –35.Bixa orellana leaves at 100 and 200 mg/kg bw reduced the volume and mass of the intestinal contents and maintained normal the value of the stool osmotic gap (greater than 50 mEq/L) almost like the reference drug, Loperamide. The antidiarrheal action of Loperamide can be mediated by an antisecretory mechanism. Loperamide is an opioid that affects intestinal absorption and inhibits its secretion by acting on the δ receptor. The antisecretory effect of Loperamide can be explained by the inhibition of the mechanisms leading to the synthesis of cAMP and cGMP, by the activation of Na+/K+ ATPase [2+ ions, the Na+/K+ ATPase catalyzes the release of Na+ and the entry of two K+ ions. The Na+/K+ ATPase is expressed in all intestinal epithelial cells and acts to maintain a low intracellular concentration of Na+ and a high intracellular concentration of K+ [+/K+ ATPase pump with ricinoleic acid inhibits all these active transport mechanisms in the intestine. This could indicate that the effect of the hydroethanolic extract of Bixa orellana leaves could be acted by an antisecretory mechanism similar to that of Loperamide by inhibiting the synthesis of cAMP and cGMP or by activating the Na+/K+ ATPase activity. These results could be explained by the presence in our hydroethanolic extract of antidiarrheal compounds such as flavonoids, tannins, terpenes, saponins, and sterols which are known for their antidiarrheal activities either as a transit inhibitor or as an antisecretory agent [α2-adrenergic and calcium systems to exert their inhibitory effect on intestinal functions [Castor oil induced secretory diarrhea in mice because the osmotic gap was less than 50 mEq/L , 36. The+ ATPase  or by st+ ATPase , 36. In on of K+ . The inary agent –34. Indeunctions .Bixa orellana leaves inhibited diarrhea, reduced stool frequency, and inhibited intestinal peristalsis as well as water and electrolyte secretions. These effects prove the antidiarrheal activity of the hydroethanolic extract of Bixa orellana leaves and would justify the use of this plant in the treatment of diarrheal diseases. The precise mechanisms of this hydroethanolic extract of Bixa orellana leaves, which are responsible for the antidiarrheal activity, would be clarified by further studies.The hydroethanolic extract of


Of the five Kampo medicines, YKS showed the strongest inhibition (IC50 = 1.94 mg/ml), which was attributed to Uncariae Uncis Cum Ramulus. Unfortunately, we could not find the active ingredients responsible for its action. Finally, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance issued by the Japanese Ministry of Health, Labor, and Welfare and the US Food and Drug Administration. The Igut/IC50 values for the five Kampo medicines were ≤3.4. As these values were <10, they were evaluated as having a weak P-gp inhibitory effect that does not require further verification in humans, suggesting that the DDI risk due to P-gp inhibition for these Kampo medicines is low. The results should provide useful clinical information on the safety and efficacy of the combined use of Kampo and Western medicines.In modern medical care in which Kampo and Western drugs are often combined, it is extremely important to clarify drug–drug interaction (DDI) to ensure safety and efficacy. However, there is little evidence of DDI in Kampo medicines. Therefore, as part of our studies to clarify the DDI risk for Kampo medicines, we evaluated the effects of five Kampo medicines  that are widely used in Japan, on drug transporter P-glycoprotein (P-gp) using a Caco-2 permeability assay. These Kampo medicines inhibited the P-gp transport of digoxin through a Caco-2 cell monolayer. The ICThe online version of this article (10.1007/s11418-018-1222-x) contains supplementary material, which is available to authorized users. Many reports have demonstrated that various metabolizing enzymes and transporters have important roles in the pharmacokinetics of xenobiotics –4. P-glyCaco-2 cells derived from human colon carcinoma cells, which express P-gp and metabolizing enzymes such as peptidase, are widely used as an in vitro model of human small intestinal mucosa to predict the absorption of orally administered drugs  because Many traditional Japanese Kampo medicines approved by the Japanese Ministry of Health, Labor, and Welfare (MHLW) have been used for the treatment of various diseases in Japan for many years. In modern medical care in which Kampo and Western drugs are often combined, it is important to clarify DDI to ensure the safety and efficacy of their combined use. However, there is little evidence of DDI in Kampo medicines. Yokukansan (YKS), rikkunshito (RKT), hangeshashinto (HST), goshajinkigan (GJG), and shakuyakukanzoto (SKT) are Kampo medicines which have been used for treating the behavioral and psychological symptoms of dementia, including aggression and insomnia , functioIgut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance of the MHLW -Digoxin (26.3–39.8 Ci/mmol), [3H]-mannitol (17.2 Ci/mmol), and ULTIMA Gold scintillation fluid were obtained from PerkinElmer Life and Analytical Sciences . Digoxin was obtained from Alfa Aesar . Mannitol was obtained from Wako Pure Chemical Industries . Verapamil hydrochloride was obtained from Enzo Life Sciences .-mannitol (0.058 µmol/l) was loaded into apical chambers, and the monolayer permeability of mannitol was measured in the same procedure as that of the following test substances. The integrity of a cell monolayer was confirmed by measuring the TEER, and mannitol permeability was used for evaluation of the test substance.The transepithelial electrical resistance (TEER) to evaluate the integrity of the cell monolayer was measured using the Millicell ER-2 . For further verification of the tight junctions in the monolayer, warm HBSS-HEPES buffer containing mannitol (10 µmol/l) and [3H]-digoxin (0.025–0.038 µmol/l) was added into either the apical or basolateral chamber in the absence or presence of various concentrations of test substances or verapamil (50 µmol/l). Aliquot solution in the apical or basolateral chamber was collected after incubation at 37 °C for 120 min with slow rotation in the horizontal direction and addition of an ULTIMA GOLD scintillation cocktail. The mixture was immediately injected into a liquid scintillation counter  to measure the radioactivity.To evaluate the test substance effect on the permeability of digoxin via the cell monolayer, warm HBSS-HEPES buffer containing digoxin (10 µmol/l) and [Papp) of digoxin was calculated as follows:Q/dt is the slope of the linear portion of the permeated amount versus time curve (µmol/l/s), A is the effective surface area of the Transwell insert (0.3 cm2), C0 is the initial concentration of the digoxin applied at t = 0 (µmol/l), and V is the volume of the receiver chamber (ml).The apparent permeability  or from the least square regression line. The concentration–response correlative formula and correlation coefficient (R2) for each crude drug constituting YKS were determined by regression analysis using Excel .The efflux ratios in each experiment were expressed as the mean ± standard deviation (SD). The statistical significance was evaluated by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. A The viabilities of Caco-2 cells treated with various concentrations of test substances are summarized in Supplementary Table S1. From the results, the concentrations of test substances that showed little cell toxicity  were used in the digoxin transport assay using a Caco-2 cell monolayer.2. Therefore, Caco-2 cell monolayers showing a TEER value of ≥250 Ω cm2 were first selected for this experiment. Next, the flux of mannitol across the cell membrane was examined to verify the integrity of the monolayer. The permeability of mannitol was < 1.5 × 10−6 cm/s, which indicated that the tight junctions of the cell monolayer were completed.The TEER value of the Caco-2 cell monolayer guarantees tight junction formation, which depends on the cell source, passage number, number of seeded cells, and culture conditions . Based oFurthermore, to confirm the expression and function of P-gp in the completed cell monolayer, transport of digoxin via the cell monolayer was investigated. The efflux ratio, an index for permeability of digoxin (6.98), was inhibited (0.832) by co-treating a P-gp inhibitor, verapamil (50 µmol/l). The inhibition rate was 88.1%, which shows that P-gp was expressed in the cell monolayer and functioned.F5,18 = 69.3, P < 0.001; RKT, F5,12 = 129.0, P < 0.001; SKT, F5,12 = 12.0, P < 0.001; HST, F6,14 = 35.2, P < 0.001; GJG, F5,12 = 32.0, P < 0.001). The IC50 values of YKS, RKT, SKT, HST, and GJG calculated by non-linear regression analysis were 1.94, 3.36, 10.80, 6.37, and 6.34 mg/ml, and the Igut/IC50 values as P-gp inhibition index were 3.4, 2.4, 0.5, 1.4, and 1.4, respectively. However, the IC50 values of SKT and HST were calculated as the estimated values, because both medicines did not show 50% inhibition in the examined concentration range.Figure F3,12 = 229.0, P < 0.001; Poria, F3,12 = 58.4, P < 0.001; GR, F3,12 = 471.0, P < 0.001; UUCR, F2,9 = 577.0, P < 0.001). The correlativity was observed as y = − 0.031x + 95.264 and the correlation coefficient as (R2) = 0.9537 for ALR, y = − 0.033x + 96.634 and R2 = 0.9379 for Poria, y = − 0.046x + 104.790 and R2 = 0.9757 for GR, and y = − 0.191x + 97.706 and R2 = 0.9926 for UUCR, where y is the efflux ratio and x is the drug concentration. From these formulas, the IC50 values were calculated to be 1,470 µg/ml for ALR, 1,430 µg/ml for Poria, 1,190 µg/ml for GR, and 249 µg/ml for UUCR. Other crude drugs  had low correlation, and the IC50 values could not be determined in the concentration range examined.Next, the P-gp inhibition rates of seven crude drugs constituting YKS showing the strongest suppression of P-gp were examined. As shown in Fig. 50 concentration  compared to those in the control, and the IC50 values were 16.5 and 56.0 µmol/l, respectively.Next, the effects of GM and RP, which are UUCR alkaloids in YKS, were evaluated using a Caco-2 permeability assay Fig. . GM (>10The concentrations of GM and RP were 0.238 nmol/ml (0.238 µmol/l) and 0.179 nmol/ml (0.179 µmol/l) in the 1.94 mg/ml of YKS, respectively. To clarify the contributions of both ingredients to the inhibitory effect of YKS, although the inhibition rate at the concentration of each ingredient contained in the YKS extract were examined in Fig. We examined the inhibitory effect of the five Kampo medicines  widely used in clinical treatments for various diseases in Japan on P-gp transport of digoxin across a Caco-2 cell monolayer. Djuv and Nilsen  suggeste50: 10.80 mg/ml). This result suggests that the GR level contained in each Kampo medicine may not contribute much to the P-gp inhibitory action of the five Kampo medicines, including YKS. On the other hand, UUCR is a crude drug that was contained only in YKS among the Kampo medicines examined in the present study, among which the inhibitory action of UUCR was stronger than the others  that were available for this study in order to to clarify the components contributing to the inhibitory activity of UCCR, and found that both ingredients had P-gp inhibitory activity. However, the P-gp inhibition rate at the GM and RP concentrations in the YKS extract powder was slight (≤1.5%), suggesting that other ingredients are involved in the inhibitory activity of UCCR. Because many other active alkaloids have been identified in YKS in addition to GM and RP , it is nIgut/IC50 ≥10 are likely to be P-gp inhibitors. For test substances evaluated as P-gp inhibitors it is stated that more detailed DDI tests should be conducted in humans. Therefore, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance. The rates of YKS, RKT, SKT, HST, and GJG examined in the present study were 3.4, 2.4, 0.5, 1.4, and 1.4, respectively. These values were <10, which was evaluated as a weak P-gp inhibitory effect that does not require further verification in humans, suggesting that the DDI risk due to P-gp inhibition for these Kampo medicines may be low. Furthermore, it has been reported that the inhibitory potential of YKS and RKT against P-gp was low in an ATPase assay [Both the Japanese MHLW  and US Fse assay  and did se assay . Ito et se assay  and Sorase assay  also evase assay , and it All Kampo medicines examined  inhibited the P-gp transport of digoxin in a Caco-2 permeability assay but to a low degree according to the criteria in the DDI draft guidance of the MHLW and FDA. These results suggest that the risk of drug interaction due to inhibition of P-gp in combination therapy with Western medicines is extremely low for these Kampo medicines. This finding should provide useful clinical information on the safety and efficacy of combinations of Kampo medicines and Western medicines.Supplementary material 1 (DOCX 23 kb)Below is the link to the electronic supplementary material.


Robot-assisted laparoscopic radical prostatectomy (RLRP) can increase intracranial pressure (ICP) related to a change in position. Increasing ICP may result in various ocular complications, which are rare but serious, such as a corneal abrasion and ischemic optic neuropathy. We performed a prospective observational trial using ultrasonographic measurements to compare optic nerve sheath diameter (ONSD) related to ICP between patients who received either propofol or sevoflurane and underwent RLRP. n = 16; group S: sevoflurane, n = 16). ONSD, end-tidal partial pressure of CO2, and blood pressure were measured 10 min after induction of anesthesia (T0), 30 min (T1), 60 min (T2), and 90 min after changing to the steep Trendelenburg position and introducing a pneumoperitoneum (T3) and 10 min after returning the patient's position to supine (T4) during surgery.  Thirty-two male patients scheduled to undergo RLRP were assigned into groups according to the anesthetic agent used , respectively. The mean left ONSDs were 38.4 and 40.8 mm at 30 min (p=0.021) after changing to the Trendelenburg position. The ONSDs between the two groups were significantly different during surgery.  No significant differences were observed in the demographic data of the patients, surgery time, or intraoperative variables, including hemodynamic and respiratory variables, at any of the time points. The mean right ONSDs in the propofol and sevoflurane groups were 37.3 and 40.1 mm at 30 min ( ONSD increased more in the sevoflurane group than in the propofol group during RLRP. Intravenous anesthetics could alleviate the increase in ICP during RLRP. Prostate cancer is the second most common cancer in men worldwide. Robot-assisted laparoscopic radical prostatectomy (RLRP) has become a very popular treatment because it has better outcomes, less blood loss, and decreased recovery time compared to other options . HoweverMeasuring optic nerve sheath diameter (ONSD) by ultrasonography is a very useful method for evaluating intracranial pressure (ICP); it is strongly correlated with invasive ICP measurements estimated using intraventricular and intraparenchymal devices .All inhaled anesthetics have a direct, dose-dependent vasodilator effect on the cerebral vessels that increases cerebral blood volume and blood flow . SevofluIn the present study, we hypothesized that ONSD in the sevoflurane group would increase more than that in the propofol group.http://cris.nih.go.kr, 9/11/2018, KCT 0003332). After obtaining written informed consent, we enrolled 32 male patients between 19 and 79 years of age with an American Society of Anesthesiologists (ASA) physical status of I–III who were undergoing RLRP. Patients with a previous neurological disease or cerebrovascular disease that could increase ICP or patients with a history of ophthalmological disease or surgery were excluded.This study had a prospective, randomized, double-blind design and was approved by the Institutional Review Board of the National Cancer Center (NCC2017-0065). It is registered with the Korean Clinical Trials Registry  between 35 and 40 mmHg. The random assignments were generated by using a computer with block sizes of 2 and 4 with a 1 : 1 assignment using the random block size permutation method. Patients were randomized into two groups: group S or group P. In group S, anesthesia was maintained with 1-2 vol% sevoflurane and an infusion of remifentanil. In group P, anesthesia was maintained with propofol and an infusion of remifentanil. The end-tidal concentration of sevoflurane or propofol was adjusted to maintain a bispectral index score of 40–60, and the targeted effect-site concentration of remifentanil was adjusted to 1.0–3.0 ng/mL using a target-controlled infusion (TCI) device.After preoxygenation, anesthesia was induced with remifentanil and propofol followed by rocuronium (0.6 mg/kg) to facilitate tracheal intubation. After endotracheal intubation, the patients were mechanically ventilated with a 50% oxygen-air mixture, using a tidal volume of 8 mL/kg ideal body weight at a respiratory rate of 10–14/min to maintain end-tidal COThe vaporizer and TCI pump were concealed from the anesthesiologist by using a screen. The blinded anesthesiologist measured ONSD, evaluated as described previously . Briefly2, blood pressure, and heart rate (HR) were measured at each time point. We also measured surgery time and the volume of infused fluid. In pilot study, the mean standard deviation of ONSD was 38.83 ± 2.23 mm in the sevoflurane group and 36.17 ± 3.31 mm in the propofol group. The difference was about 3 mm in the pilot study. However, the difference of about 10% was considered clinically significant. We assumed that the mean difference was 4.0 mm for the primary endpoint of ONSD at 30 min after changing to the Trendelenburg position with a standard deviation in both groups of 2.8. We calculated that 14 patients in each group would achieve 95% power to reject the null hypothesis of equal means with a significance level of 0.05 using a two-sided two-sample equal-variance t-test. We recruited 16 patients per group, considering a dropout rate of 10%. The primary outcome in this study was the group difference in ONSD at 30 min after the head-down position. The secondary outcomes included were as follows: the group difference in ONSD at 10 min after induction of anesthesia, 60 min and 90 min after head-down position, and 10 min after supine position; end-tidal carbon dioxide pressure; blood pressure at 10 min after induction of anesthesia, 30 min, 60 min, 90 min after head-down position, 10 min after supine position and; infused intravenous fluid at the end of surgery.ETCOt-test was used to evaluate the difference between the propofol and sevoflurane group in regard to changes in ONSD, systolic blood pressure, diastolic blood pressure, mean blood pressure, HR, and ETCO2. The difference between the time points in each group was tested with the paired t-test, and the differences between the two groups over time were compared based on a mixed-effect model. All statistical analyses were performed using SAS version 9.4 .The independent Thirty-two patients scheduled for RLRP were included and evaluated. The patients' characteristics are shown in p=0.638) at T0, 37.3 and 40.1 mm (p=0.003) at T1, 36.7 and 38.8 mm (p=0.036) at T2, 36.3 and 38.9 mm (p=0.011) at T3, and 35.1 and 38.1 mm (p=0.008) at T4, respectively. The mean left ONSDs were 37.3 and 37.2 mm (p=0.950) at T0, 38.4 and 40.8 mm (p=0.021) at T1, 37.4 and 40.2 mm (p=0.019) at T2, 36.8 and 39.7 mm (p=0.017) at T3, and 35.9 and 39.1 mm (p=0.003) at T4 (p < 0.01). ONSDs of the propofol group were significantly increased only in the left from 37.3 (T0) mm to 38.4 mm (T1) (p=0.042).ONSD was measured at 10 min after induction of anesthesia (T0), 30 min (T1), 60 min (T2), and 90 min (T3) after changing to the Trendelenburg position and introducing a pneumoperitoneum, and 10 min after the position changes to supine (T4) in 3) at T4  The ONSD3) at T4 . ONSD inWe investigated changes in ONSD according to the anesthetic agent. We observed significant differences between the sevoflurane and propofol groups. Whitely et al.  reportedMeasuring ONSD is a noninvasive method that correlates significantly with changes in ICP . ONSD vaA prolonged steep Trendelenburg position and increasing intra-abdominal pressure can result in increased cerebral venous pressure, ICP, and cerebral blood flow. Weber et al.  reportedICP can be affected by carbon dioxide, blood pressure, infused fluid, and operation time . In our Many studies have demonstrated that sevoflurane has a dose-dependent cerebral vasodilatory effect and that propofol either decreases or does not affect ICP. In our study, ONSD did not increase in the propofol group. We hypothesized that the use of propofol would not affect ICP in patients at risk for elevated ICP, such as those in the steep Trendelenburg position, and would not increase ONSD. We confirmed this in our study.2 at baseline, which may have affected ICP. However, because EtCO2 has a strong relationship with PaCO2, we maintained EtCO2 partial pressure at 35–40 mmHg over the whole period and adjusted ventilator settings in both groups. No significant difference in EtCO2 was detected between the two groups.Some limitations of this study should be mentioned. We only measured PaCOIn conclusion, ONSD during RLRP increased in the sevoflurane group but not in the propofol group. Intravenous anesthetics could alleviate the increase in ICP during RLRP. We recommend using intravenous anesthetics when performing this surgery in a position that could cause an elevation in ICP, such as the prolonged steep Trendelenburg position and when placing an artificial pneumoperitoneum, because sevoflurane has a cerebral vasodilatory effect although a sufficient compensatory mechanism may weaken the intracranial effects.


Petunia integrifolia × P. axillaris were phenotyped for days to anthesis (DTA), flower count (Flower_C), flower diameter (Flower_D), flower length (Flower_L), plant height (Plant_H), plant spread (Plant_S), and plant size (Plant_Z) in 2014 and 2015. Transgressive segregation was observed for all traits in both years. The broad-sense heritability on a 2-year basis varied from 0.38 (Flower_C) to 0.82 (Flower_L). Ten QTL were consistently identified in both years and by two mapping strategies [multiple QTL mapping (MQM) in MapQTL and inclusive composite interval mapping (ICIM) in IciMapping]. Major QTL explained up to 30.2, 35.5, and 47.1% of the total phenotypic variation for Plant_S, Flower_L, and Flower_D, respectively. These findings should be of significant values for introgression of desirable genes from wild petunias into commercial varieties and future genetic improvement of this important flower.Petunia is a very important flower in the global floriculture industry and has played a critical role as a model in plant genetic studies. Owing to limited genetic variability in commercial germplasm, development of novel petunia phenotypes and new varieties has become increasingly difficult. To enrich petunia germplasm and facilitate genetic improvement, it is important to explore genetic variation in progenitor species that may contain highly valuable genes/alleles. In this study, an interspecific recombinant inbred population (168 recombinant inbreds) derived from  P. integrifolia and P. axillaris. The team phenotyped the petunias in an open-air sub-tropical field rather than in an artificial environment like previous studies. They identified and characterized 17 genetic loci for seven important aesthetic traits, ranging from flower count to plant size. The petunia species had very different genetic backgrounds, likely stemming from their different geographic origins. The two species each can contribute novel genes for enhancing cultivated petunia cultivars.Uncovering the genetics behind desirable traits in petunia plants could help improve commercial cultivars. While petunia plants are a key feature of the global horticultural industry, the limited genetic variability in commercial plants makes it difficult to improve desired plant traits. Zhanao Deng at the University of Florida, US, and co-workers conducted a genome-wide QTL identification study using a crossbred population combining two petunia  species,  Since then, further growth has occurred in almost every continent. It is projected that the global floriculture market will continue to grow at a compound annual growth rate of 5.4% over the period from 2016 to 20202.Cultivated flowers serve a very important role in human life and health, the global economy, and the beautification and protection of the environment. Flower production has become one of the most dynamic and sophisticated sectors of the global horticulture industry. It was estimated that the worldwide production value of cultivated flowers or floricultural production reached 60 billion dollars in 20033, the lack of genetic diversity and lack of novel genes/alleles in the commercial germplasm pool have been limiting plant breeders’ progress in genetic improvement and new cultivar development5.To sustain the global flower production industry, continuous introduction of new cultivars with improved or novel characteristics is essential. Towards this, plant breeders constantly seek to identify novel genes/alleles and combine them into new or improved cultivars. In many crops, including widely produced and used flowers and other ornamental plants6. On the other hand, wild and ancestor species often perform poorly in horticultural aspects compared to elite germplasm7. Wild accessions may carry undesirable genes for the improvement of commercial cultivars9. When a wild species is crossed with an elite cultivar, the inferior alleles can be simultaneously dragged into cultivars, reducing the plant performance of new cultivars10. Numerous rounds of backcrossing are required to reduce or eliminate the inferior donor alleles from elite cultivars, which is a laborious and time-consuming process. Genetic mapping, identification of quantitative trait loci (QTLs), and marker-assisted selection have been used to facilitate the introgression of desirable alleles from wild species to elite cultivars11. Over the past two to three decades, several molecular marker systems have been used in such efforts. Genotyping by sequencing (GBS) is a recently developed strategy for large-scale marker discovery12. It has been made possible by rapid advances in next-generation sequencing technology. With this strategy, it is possible to sequence hundreds of barcoded samples in a single sequencing lane simultaneously and to reveal single-nucleotide polymorphism (SNP) sites throughout the whole genome. The high output of SNP discovery by GBS has greatly facilitated the construction of high-density, high-resolution genetic linkage maps. GBS has been widely used to construct high-coverage linkage maps and conduct QTL analyses in multiple important agronomic crops13.Identification and utilization of desirable genes/alleles from wild or progenitor species have been suggested as an effective approach to overcoming this limitation. Enormous efforts have been made in some major agronomic and horticultural crops to characterize wild and ancestor germplasm and identify favourable genes/alleles from the germplasm through phenotyping, genetic mapping, and introgressionPetunia hybrida) is a very important flower in the global floriculture production. It is cultivated all over the world and is one of the most important Solanaceae utilized for ornamental purpose14. Garden petunia is often among the most popular flowers planted in outdoor gardens in many countries15. In the United States, it ranks first in wholesale value among annual bedding plant crops16. Cultivated petunia originated from the cross between Petunia axillaris and Petunia integrifolia17. As petunias have been commercially bred with limited germplasm sources for the past 150+ years, the genetic diversity among current commercial cultivars has been low, resulting in high similarities among commercial cultivars and loss of some useful traits19. Several studies have indicated that wild P. axillaris and P. integrifolia carry traits that may be beneficial to commercial petunia, such as faster development rates8, superior freezing tolerance19, longer flower longevities20, or arthropods resistance21. Consequently, interest in introgressing traits from progenitor species to elite petunia cultivars has been strong21.Garden petunia . A SNP-based linkage map was recently reported in petunia and employed to identify QTL controlling petunia plant development rates (as well as the number of branches and flower buds and days to anthesis (DTA)) under varying temperatures25. All reported petunia QTL studies were conducted in the greenhouses using container-grown plants. No or few QTLs have been reported for important aesthetic traits in petunia, including plant size (Plant_Z) and flower count (Flower_C).Several genetic linkage maps were developed in petunia, using restriction fragment length polymorphism markers, amplified fragment length polymorphism markers, and simple sequence repeat (SSR) markersP. integrifolia × P. axillaris for 2 consecutive years in 2014 and 2015, (2) estimated the heritability for these traits, and (3) identified and located QTL controlling these traits using a high-density SNP bin map developed by the GBS technology.In this study, we (1) characterized and phenotyped seven important plant and flower aesthetic traits , flower length (Flower_L), plant height (Plant_H), plant spread (Plant_S), and Plant_Z) in an open field using a recombinant inbred population derived from a cross between H2) estimate for each trait based on combined 2-year data are presented in Table P. axillaris , Flower_L (98–141% longer), and Plant_H (53–70% higher), but lower values than P. integrifolia in Flower_C (73–78% lower) and DTA (6–9% lower or 5 or 6 days earlier). P. axillaris and P. integrifolia had similar values for Plant_S and Plant_Z.Phenotypic data including mean value, mid-parents value, and data range for DTA, Flower_C, Flower_D, Flower_L, Plant_H, Plant_S, and Plant_Z of the parents and their recombinant inbred lines (RILs) and the broad-sense heritability  into three categories, low (<0.30), moderate (0.30–0.60), and high (>0.60). According to these criteria, Plant_H (H2 = 0.70), Flower_D (H2 = 0.79), and Flower_L (H2 = 0.82) exhibited high H2. Moderate H2 was observed for Flower_C (H2 = 0.38), DTA (H2 = 0.40), Plant_S (H2 = 0.58), and Plant_Z (H2 = 0.58). Transgressive segregation was observed for all traits studied, including DTA, Flower_C, Flower_D, Flower_L, Plant_H, Plant_S, and Plant_Z  were 0.233 (2014) and 0.312 (2015) for the former and 0.331 (2014) and 0.194 (2015) for the latter. Between floral traits, Flower_D was negatively correlated with DTA , but it was positively correlated with Flower_L . And between plant traits, Plant_H, Plant_S, and Plant_Z were all correlated with each other; the correlation coefficients were positive between Plant_H and Plant_Z  and between Plant_S and Plant_Z  but were negative between Plant_H and Plant_S . In addition, there were several pairs of traits that were only correlated in either 2014 or 2015, such as DTA and Plant_S (r = 0.156 in 2015), Flower_C and Flower_D (r = −0.298 in 2014), and Flower_L and Plant_S (r = 0.156 in 2014).Significant correlations were observed between several trait pairs in both 2014 and 2015 (Table 7) for an F7 RIL population. Overall, ~80% of the RILs in the population had a heterozygosity level below 4.86%, and ~90% of the RILs had heterozygosity level below 7.39%.We calculated the heterozygosity level in each RIL based on the number of heterozygous marker loci out of the total number of marker loci analysed (518). The heterozygosity level in the 168 RILs ranged from 0 to 21.80% Fig. , averageWe also calculated the heterozygosity level for each of the 518 marker loci by dividing the number of heterozygous RILs by the total number of RILs analysed (168). The percentage of heterozygosity per locus ranged from 0% to 23.67%. The average heterozygosity level for the 482 SNP marker loci was 2.70%, while the average heterozygosity for the 36 SSR marker loci was at least one-fold higher, reaching 6.64%. There were 12.74% of the marker loci (66) that had 5–10% heterozygosity, and 1.16% of the marker loci (6) had heterozygosity above 10%. These six loci are all of the SSR type. To show a genome-wide landscape of the heterozygosity residues, the genetic position of each marker locus with its heterozygosity level was plotted on the genetic linkage map , qDTA2.1 (LG 2), and qDTA6.1 (LG 6). The QTL qDTA2.1 and qDTA6.1 were consistently detected in both years, and the percentage of phenotypic variance explained (PVE) by qDTA2.1 and qDTA6.1 ranged from 10.2%  to 21.3% . The early flowering alleles  at these loci advanced flowering by 3.37 and 6.26 days, respectively, and they were from P. axillaris. The QTL qDTA1.1 was only detected in 2015, and its PVE was 8.6%. For this locus, the beneficial allele for early flowering (3.38 days) was from P. integrifolia.Days to anthesis (DTA): Three QTLs controlling DTA were qFlower_C1.1 (LG 1) and qFlower_C2.1 (LG 2). The QTL qFlower_C1.1 was consistently detected in both 2014 (PVE = 14.9%) and 2015 (PVE = 11.4%), while qFlower_C2.1 (PVE = 17.8%) was identified only in 2014. The P. axillaris allele at qFlower_C1.1 increased the Flower_C by 53–68 per plant, while the P. integrifolia allele at qFlower_C2.1 increased the Flower_C by 54 per plant.Flower count (Flower_C): Two QTLs controlling Flower_C were qFlower_D2.1 (LG 2) and qFlower_D3.1 (LG 3) were detected for Flower_D. Both QTL were consistently detected in 2014 and 2015. QTL qFlower_D2.1 seems to play a more important role, with a PVE of 47.1% in 2014 and 44.3% in 2015. QTL qFlower_D3.1 had a PVE of 10.3% in 2014 and 10.8% in 2015. Alleles at these loci that contributed to larger Flower_D are from P. axillaris, with an additive effect ranging from 0.31 to 0.75 cm per allele.Flower diameter (Flower_D): Two QTLs qFlower_L1.1 (LG 1), qFlower_L2.1 (LG 2), qFlower_L3.1 (LG 3), and qFlower_L4.1 (LG 4), were detected controlling Flower_L. QTLs qFlower_L1.1 and qFlower_L2.1 were detected in both years, and their PVE ranged from 5.5%  to 35.5% . Two QTLs qFlower_L3.1 (2014) and qFlower_L4.1 (2015) were detected only in 1 year, and their PVE were 10.4% and 5.6%, respectively. All beneficial alleles at these loci accounting for longer flowers are from P. axillaris, and their additive effect ranged from 0.25 to 0.88 cm in Flower_L per allele.Flower length (Flower_L): Four QTLs, qPlant_H1.1 and qPlant_H2.1 were consistently detected in 2 years. The former had a PVE of 16.3% in 2014 and 41.1% in 2015, and the latter displayed a PVE of 19.6% in 2014 and 10.6% in 2015. P. axillaris alleles at these loci exhibit additive effects, increasing Plant_H by 0.04 to 0.08 m per allele.Plant height (Plant_H): Two QTLs qPlant_S1.1 and qPlant_S2.1 controlling Plant_S were identified in both years. The locus qPlant_S1.1 had a smaller PVE of 12.9% in 2014 and 10.8% in 2015, while the QTL qPlant_S2.1 exhibited a larger PVE of 30.2% in 2014 and 23.0% in 2015. All alleles at these loci for larger Plant_S were from P. axillaris, and their additive effects ranged from 0.10 to 0.24 m per allele.Plant spread (Plant_S): Two putative QTLs qPlant_Z1.1 (LG 1) and qPlant_Z2.1 (LG 2). QTL qPlant_Z2.1 was consistently detected in both years, having a PVE of 29.2% and 29.6% in 2014 and 2015, respectively. QTL qPlant_Z1.1 was only detected in 2014, and its PVE was 13.0%. And the alleles at these loci for larger Plant_Z were from P. axillaris, and their additive effects were between 0.02 to 0.03 m per allele.Plant size (Plant_Z): Two putative QTLs identified were qDTA2.1, qDTA6.1, qFlower_C1.1, qFlower_D2.1, qFlower_L1.1, qFlower_L2.1, qPlant_H1.1, qPlant_H2.1, qPlant_S2.1, and qPlant_Z2.1) that were detected in both 2014 and 2015 and 2 QTLs (qDTA6.1 and qFlower_L4.1) that were observed in either 2014 or 2015. All QTLs detected by ICIM were located very close to the QTLs detected by MQM, except for two QTLs, qFLower_L1.1 and qPlant_H1.1, which were 9 and 13 cM away from the QTLs by ICIM, respectively.When two environments year 2014 and 2015) were jointly considered and the ICIM mapping algorithm was used, a total of 17 QTL were detected for the 7 petunia traits  > 3.0). The QEI at C-Flower_D-2.1 explained 7.38% of the phenotypic variation and the QEI at qFlower_L2.1 accounted for 6.92% of the phenotypic variation. QEI effects increased the Flower_D in 2014 by 0.34 cm and the Flower_L in 2014 by 0.28 cm.Two QTLs, Significant QTL × QTL interactions were observed for two traits, DTA and Plant_Z Table . The int7 RIL population derived from the cross between P. integrifolia and P. axillaris  in the Fis Table . All traH2 across different growing environments and/or seasons can be reliably selected in breeding. In this study, Plant_H (H2 = 0.70), Flower_D (H2 = 0.79), and Flower_L (H2 = 0.82) were found to have high heritability. Similar results were reported in previous petunia studies29. Four traits, including Flower_C (H2 = 0.38), DTA (H2 = 0.40), Plant_S (H2 = 0.58), and Plant_Z (H2 = 0.58), have moderate H2, suggesting that these traits are more sensitive to environmental changes. Moderate H2 was observed for DTA in previous studies9. Similar H2 estimates were also reported for Plant_S, Plant_Z, and Flower_C in a P. axillaris × Petunia exserta RIL population30.Traits with higher r = 0.233 in 2014, and r = 0.312 in 2015) and between Plant_H and Flower_L   suggested that selecting large flowers (in diameter) could compromise Flower_C. The moderate correlation coefficient  between Flower_D and Flower_L indicated that flowers with larger corolla sizes tended to be longer. While the negative correlation between Plant_H and Plant_S  indicated that selecting higher wider plants may sacrifice Plant_H, other pairs of traits only exhibited significant correlation in either of 2014 and 2015. This might be due to phenotypic variability between years, especially for some low heritable traits, such as DTA and Flower_C.In both 2014 and 2015, positive correlation was observed between Plant_S and Flower_C  in the F7 interspecific petunia population  and 29.6% (2015) in MQM, and 23.3% in ICIM; qFlower_L2.1, PVE = 28.5% (2014) and 35.5% (2015) in MQM, and 14.6% in ICIM; qFlower_D2.1, PVE = 44.3% (2015) and 47.1% (2014) in MQM, and 20.4% in ICIM].To identify consistent, useful QTL in petunia, we phenotyped a large number of RILs over two growing seasons and replicated each RIL several times in each growing season. By using MQM in MapQTL, a total of 17 putative QTL controlling seven important petunia plant and flower traits were observed. Twelve of these QTL were also confirmed by ICIM in the IciMapping software. Ten QTL were detected in both 2014 and 2015 and by both mapping strategies (MQM and ICIM). Some of these consistent QTL explained large proportions of phenotypic variance .In each year, the large RIL population as well as their parents was phenotyped for seven plant and floral traits of the most significance to the use of petunia as a bedding and garden plant, including DTA, Flower_C, Flower_D, Flower_L, Plant_H, Plant_S, and Plant_Z. DTA were calculated as follows: DTA (days) = date of first flower anthesis − date of seed sowing. When approximate 50% of progeny came into flowering, all flowers on each plant were counted weekly for 7 weeks to obtain Flower_C data. Three fully opened flowers per RIL and parent were randomly selected to collect data for Flower_D and Flower_L. Flower_D was measured from one side of the flower’s petal to the opposite side; Flower_L was measured from the base of the flower’s calyx to the top of the flower’s corolla. Plant_H and Plant_S were recorded near the end of the growing season (early June to mid-June). Plant maximal spreads were measured along the longest axis between two opposite margins of the plant. While plant minimal spread was taken along a straight line between two plant margins that were perpendicular to the maximal Plant_S. Two directional Plant_S  were collected and then averaged to represent Plant_S. Plant_H was measured from the bed surface to the highest point of the plant. The value of Plant_Z was determined by Plant_H, plant maximal spread, and plant minimal spread and calculated using the formula: Plant_Z (mH2) estimates were calculated using the following statistical model: yijk = µ + Gi + Ej + Gi × Ej + Bh(j) + εijk, where yijk represents the measured phenotypic value of the studied trait for individual plantijk, µ the population mean value for the specific trait, Gi the genetic effect, Ej the environment effect, Gi × Ej the effect of interactions between genotype and environment, Bh(j) the block effect, and εijk the random error. All components , and εijk) in this model were treated as random effects.The statistical software JMP Pro 10.0.2  was used to calculate progeny distribution for each trait studied and Pearson’s correlation coefficients and to estimate the broad-sense heritability for each trait. All board-sense heritability (25. The genotyping data consist of data from 482 SNP and 36 SSR markers. Molecular marker genotypes were categorized into either being heterozygous or homozygous. The level of heterozygosity (%) in each RIL was calculated by dividing the total number of heterozygous marker loci in each RIL by the total number of marker loci analysed. The level of heterozygosity at each marker locus was obtained by dividing the total number of heterozygous RILs by the total number of RILs analysed. The resulting data were plotted in the software Matplotlib43 to show a genome-wide landscape of heterozygosity retention with and among LGs.The heterozygosity level of each RIL and at each marker locus was calculated using the marker genotyping data described by Guo et al.25 was used for QTL identification and localization in this study. The genetic map contained 518 bins (482 SNPs and 36 SSRs) spanning a total genetic distance of 220.2 cM across petunia’s seven chromosomes. Molecular markers in this genetic map could be located to 620 scaffolds, 0.74% of the total number of scaffolds in the assembled P. axillaris genome (https://solgenomics.net/organism/Petunia_axillaris/genome)42. Nevertheless, these 620 scaffolds contain 747,650 kb of nucleotides, which is approximately 53.4% of the P. axillaris genome (1.4 Gb). The software MapQTL 6.044 was employed for QTL analysis. Putative QTL regions were first determined by interval mapping and the resulting highest scored markers were then highlighted and labelled. These stamped markers were subsequently treated as cofactors and run in MQM. The LOD thresholds for putative QTL were determined by permutation tests (1000 times per run) with the significant threshold at 95th percentile of LOD scores. Only QTL with a LOD score more than the LOD threshold value were declared and retained in the analysis and reported here. To verify the QTL detected in MapQTL and to estimate the QEI and QTL × QTL (epistasis) effects, the ICIM model software IciMapping 4.145 was used; the LOD threshold value 3.0 was used to declare significant QTL and QEI, and the LOD cutoff of 5.0 was used to declare the presence of epistasis.The genetic linkage map described by Guo et al.Supplemental Table S1Supplemental Table S2Supplemental Table S3