Background: Thrombosis, well known as a condition of the elderly, is occurring in the otherwise healthy adolescent population. Immobility is a significant risk factor for venous thromboembolism (VTE), and adolescents who play video games are immobile for extended periods of time. Some are presenting with VTE. When other risk factors such as obesity are present, the risk of VTE formation increases. We provide a review of published case reports regarding gaming and thrombosis.Methods: We searched PubMed, Scopus, Web of Science, and EBSCO for articles published through July 2019, using the keywords “computer game thrombosis,” “computer game pulmonary embolism,” “computer game deep vein thrombosis,” “video game thrombosis,” “video game pulmonary embolism,” and “video game deep vein thrombosis.”Results: Of the 26 articles we identified, we included 12 articles in our review that report a total of 15 cases, of which 2 resulted in fatalities. Modifiable risk factors included cigarette use, being overweight, birth control use, and prolonged immobility. Anticoagulation was the principal treatment modality in patients presenting with gaming thrombosis.Conclusion: We strongly encourage screening gamers for possible VTEs if clinically warranted. A retrospective study of cases from the Pediatric Health Information System administrative database showed that from 2001 to 2007, adolescents accounted for 30% of admissions for VTE, and other articles report similar findings across the pediatric and adolescent population.6Venous thromboembolism (VTE), a blood clot that starts in a vein, typically has 2 types: deep vein thrombosis (DVT), which occurs when the clot occurs in a deep vein such as the leg, and pulmonary embolism (PE), which occurs when a DVT breaks off and enters the arteries of the lungs. Approximately 300,000 to 600,000 cases of VTE occur annually in the United States, approximately 33% of patients will have a recurrence within 10 years, and 10% to 30% of patients die within 1 month of diagnosis.7 The most common reversible factors in both adolescents and adults are obesity and sedentariness.10 Prolonged immobility in adolescents is often attributable to playing video games. In this review, we summarize the reported cases related to thrombosis in gamers and describe the clinical implications.Numerous risk factors, both reversible and irreversible, can foster a hypercoagulable state and thus predispose patients to the development of a VTE. The Virchow triad illustrates 3 of the important factors—hemostasis, hypercoagulability, and vessel wall injury—that alter blood flow and, in turn, cause or facilitate thrombus formation.We searched PubMed, Scopus, Web of Science, and EBSCO for articles published through July 2019, using the keywords “computer game thrombosis,” “computer game pulmonary embolism,” “computer game deep vein thrombosis,” “video game thrombosis,” “video game pulmonary embolism,” and “video game deep vein thrombosis.” To be included in this review, articles had to meet the following inclusion criteria: (1) must be full-text articles; (2) must involve humans; (3) must be a randomized controlled trial, prospective trial, retrospective analysis, case series, or case report; and (4) must include clinical findings at presentation. The senior author (SKA) identified relevant articles reporting the outcomes of interest. The lead author (KAR) extracted data from all reports, and SKA resolved any discrepancies. We checked the reference lists of each relevant article for additional resources that met the selection criteria.22 Among these 15 patients, recurrent VTE occurred in 3 patients, and 2 patients died as a result of VTE.21 One of the cases was reported postmortem, so an approximate duration of gaming is provided. Five patients lived in the United States, 3 in New Zealand, 3 in Singapore, 2 in Korea, and 1 each in Austria and the United Kingdom. Three patients had DVT, 5 had PE, and 7 had both DVT and PE. Concerning risk factors, 2 patients used cigarettes, 2 patients had factor V Leiden (FVL) heterozygosity, 5 patients had a sedentary lifestyle, 2 patients had a family history of VTE, 1 patient had May-Thurner syndrome, 1 patient had Klinefelter syndrome, 1 patient was taking birth control, 1 patient had Guillain-Barré syndrome (GBS), and 1 patient had prior bariatric surgery. Seven of the 15 patient cases reported body mass index (BMI) data, and all 7 patients had a BMI >25 kg/m2. Twelve of the 15 patients received anticoagulation treatment . Many individuals—young and old—spend hours playing video games online and offline.21 Of the 15 cases included in this review, the number of hours a patient spent gaming in a single setting ranged from 3 to 80 hours. Additionally, 9 of the 15 patients had modifiable risk factors including cigarette use, being overweight, birth control use, and prolonged immobility.21 One patient was taking a second-generation oral contraceptive; individuals taking oral contraceptive pills have a 2 to 4 times greater risk of VTE than those who do not use this contraceptive method.28Lee reported the first death from gaming thrombosis in 2004, and the second death was reported in 2015.29 The Ageno et al metaanalysis assessing the association between cardiovascular risk factors and VTE found that “compared with control subjects, the risk of VTE was 2.33 for obesity , 1.51 for hypertension , 1.42 for diabetes mellitus , 1.18 for smoking , and 1.16 for hypercholesterolemia .”30 The authors concluded that cardiovascular risk factors are associated with VTE. The Tromsø study showed that heavy smokers (>20 pack-years) had an increased risk for total VTE (hazard ratio [HR] 1.46) and provoked VTE (HR 1.75) compared to never smokers.31 Similarly, the Multiple Environmental and Genetic Assessment of risk factors for VTE (MEGA study) reported that overweight and obese individuals were associated with a 1.7- and 2.4-fold increase in VTE, respectively, compared to individuals of healthy weight.32Goldhaber and colleagues investigated the risk factors for PE in women and reported that obesity, cigarette smoking, and hypertension were the major contributors.19Many of the patients included in this review presented with risk factors identified in these studies. Thus, educating and counseling patients on lifestyle modification to prevent cardiovascular and VTE complications is important. Of note, 5 patients received lifestyle management counseling, 1 was enrolled in a weight management program, and 1 received physical therapy after DVT and PE occurrence.34Several patients included in this review had nonmodifiable risk factors. As previously noted, 2 patients had FVL heterozygosity, an abnormal version of factor V that is not affected by activated protein C (APC). Because FVL is not affected by APC, clot formation is prolonged, and these patients would seem to have a greater risk for thrombosis; however, the evidence is conflicting as to whether the presence of FVL heterozygosity increases risk for VTE.35One patient presented with GBS, a condition in which the body's immune system attacks peripheral nerves, potentially leading to muscle weakness or loss of sensation in the extremities. This patient had 3 weeks of immobility secondary to GBS and had a sedentary lifestyle at baseline. A retrospective study of 73 patients admitted to a hospital for GBS showed that 5 patients had a DVT postdischarge, and 4 were anticoagulated with prophylactic low molecular weight heparin; the authors concluded that full anticoagulation may be needed for patients with GBS.36 However, a clot that breaks off and travels to the lungs can become life threatening, which is possibly what occurred in this case because this patient presented with a PE.One patient presented with May-Thurner syndrome in which the left iliac artery is compressed by the right iliac artery, thus predisposing the patient to left extremity DVT. This condition is generally not life threatening, as >70% compression is needed to result in a DVT.39One patient presented with Klinefelter syndrome which is associated with VTE and is comparable to inherited thrombophilias. The mechanism behind the association between Klinefelter syndrome and VTE is multifactorial, and the current literature on this topic is limited.40 According to the CHEST guideline, direct oral anticoagulants are preferred to vitamin K antagonists; in turn, vitamin K antagonists are preferred to low molecular weight heparin. For treatment of pediatric patients, the American Society of Hematology (ASH) guidelines suggest 6 to 12 months of anticoagulation therapy with either a vitamin K antagonist or low molecular weight heparin in patients with unprovoked VTE.41For treatment of VTE, the Antithrombotic Therapy for VTE Disease: CHEST Guideline and Expert Panel Report states that adults should receive anticoagulation for 3 months, and if a patient has a low or moderate risk of bleeding, anticoagulation should be continued without a scheduled stop date.11For the patients included in this review, the duration of therapy varied and was not always consistent with guideline recommendations. Of the 15 cases, 4 adults received 6 months of anticoagulation, and 4 pediatric patients received 3 months to 2.5 years of anticoagulation. The remaining cases did not specify the duration of therapy. Six patients received heparin preparations either secondary to surgery for removal of the clot or as bridging therapy to warfarin. One patient was prescribed aspirin after a 6-month course of warfarin.In terms of prevention, lifestyle management, such as interrupting the duration of prolonged gaming by walking around every few hours and changing positions, can help prevent a VTE. Such preventive measures are especially important if a patient has risk factors such as being overweight or obese. Patients should quit smoking to reduce their chances of developing VTE.This clinical review is based on case reports that are not without limitations. Consequently, this information should be interpreted with caution because the majority of the cases, albeit a small number, were reported from countries outside of the United States and therefore may not generalize to the US population.When an adolescent presents with a VTE, asking the patient or the patient's parents about any history of gaming and the number of hours spent gaming is an important aspect of the workup. Clinicians should follow the current CHEST or ASH guidelines to treat the VTE because specific gaming thrombosis guidelines are not available. Because gaming is prevalent, it should be recognized as a public health issue and a health burden. Well-defined diagnostic guidelines and criteria for gaming disorder are needed to establish appropriate treatment.


Animal welfare science embraces all factors that might affect the physical and emotional state of the animal, its ability to cope, and its overall quality of life. In recent years, awareness of farm animal welfare has increased among veterinary practitioners—a major professional figure influencing a farm’s routine, farm workers, consumers, and the general public. In particular, the farm worker’s knowledge of animal welfare is an essential component of the rearing system. The aim of this study was to examine attitudes toward and awareness of select animal welfare issues among farm workers and practitioners. A survey was performed based on anonymous questionnaires filled out by dairy farm workers and veterinary practitioners. The results demonstrated that farm workers’ enjoyment of their work is of great importance, as is their cows’ welfare. The survey showed the farm workers’ awareness of their influence on the cow during milking, the effects of stress on milk production, and the possible effect of human behavior on heifers and cows. The main areas where animal welfare might be improved were farmers’ awareness of learning, memory, and pain masking in cattle, and knowledge transfer from veterinary practitioners to the farm workers. The survey answers further emphasized the crucial importance of communication and understanding between farm workers and their practitioners.Attitudes toward practical dairy cow welfare issues were evaluated based on a questionnaire answered by 500 dairy farm workers and 27 veterinary practitioners. Primarily, the effect of demographic characteristics on attitudes toward cattle welfare was tested. Professionally, five themes were identified: effect of welfare awareness on productivity, knowledge of cattle’s senses and social structure, effects of man–animal interactions on milk yield, pain perception and prevention, and knowledge transfer from veterinary practitioners to farm workers. Farms with a higher welfare awareness score also had higher annual milk yield, with an annual mean difference of 1000 L of milk per cow between farms with higher and lower awareness scores. Veterinary practitioners showed high awareness of cows’ social structure, senses, and pain perception. Farm workers were aware of the influence of man–animal interactions during milking and stress effects on milk yield, and the possible effect of man’s behavior on heifers and cows. Practitioners and farm workers had different views regarding pain perception, mostly involving mutilation procedures. All veterinary practitioners advocated the use of pain alleviation in painful procedures, but only some of them instructed the farm workers to administer it. The survey results emphasize the variation in welfare knowledge and practical applications across farms, and the interest of both the animals and their managers to improve applied knowledge of best practice. The Israeli dairy sector consists of about 135,000 Israeli-Holstein cows on 700 dairy farms. The dairy sector is well organized by the Israeli Milk Board and the Israeli Cattle Breeders’ Association. Based on the Israeli Herd Book data, which includes nearly 90% of the dairy cattle, the 2020 average annual milk production was 12,120 kg/cow, with 3.73% fat and 3.25% protein [Animal welfare remains an area of consistent public concern, with acceptance of animal sentience enshrined in the legislation of many countries . A draftMany data are collected through precision dairy farming ,6. HowevUnderstanding the differences between human and bovine senses can contribute to the way in which the cattle are treated. For example, cows hear a wider range of frequencies than humans do  . Dairy fResearch into animals’ mental status, i.e., their sensory and cognitive abilities, has developed greatly in recent years . CognitiDairy farm workers’ behavior seems to affects cows’ behavior and milk yield . Cows caFindings demonstrate that learning at an early age can impact cows’ behavior as adults . For exaFreedom from pain is one of the main aspects of animal welfare . Among vIn light of the above, we performed a survey of dairy farm veterinary practitioners and workers to examine current attitudes to, and awareness of, cattle’s mental needs, and to identify the areas where further awareness of welfare issues can be improved.A total of 41 Israeli dairy cow veterinary practitioners working with the corporation Hachaklait Veterinary Services Ltd. received an anonymous questionnaire through internal software in 2018. The questionnaire, which is provided in A questionnaire was written by the authors, translated into Arabic, English and Thai, and distributed by hand to dairy farm workers on each farm. The questionnaire is provided in Dairy farms were categorized as cooperatives or private family farms (as mentioned in the introduction). In total, 61 cooperative and 113 family dairy farms were selected from all geographic regions, representing 37% and 20% of each category, respectively, in the national herd book, which consists of all the Israeli dairy farms’ database. The first part of the survey asked general demographic questions: responder’s gender, age, seniority in farming (length of time working on a dairy farm), academic background, past guidance on welfare topics, role on the farm, extent of enjoyment from the work, and extent of the importance of animal welfare. The second part asked questions related to five themes: effect of welfare awareness on productivity, knowledge regarding cattle’s senses and social structure, effect of man–animal interactions on milk yield, pain perception and prevention, and knowledge transfer from veterinary practitioners to farm workers. Answers were single choice only. Adaptation to each sector resulted in slight differences between the questions for the veterinary practitioners and those for the farm workers. Specific questions were chosen based on an assessment of the concurrently existing knowledge and suspected gaps. Previous audits and guidance by the extension unit of the Ministry of Agriculture in recent years revealed the subjects where knowledge was suspected to be lacking.To facilitate the ranking of extent of enjoyment from work and personal importance of welfare issues, we used a 1–5 scale . To evaluate the farm workers’ perception of animals’ pain, respondents were asked to score dairy cows’ expression of pain on a scale of 1 (do not hide) to 5 (hide excessively).Data from the completed questionnaires were collected and analyzed; 18 questions received “correct” and “incorrect” answers, and questions 5 and 13 were excluded. “Correct” answers were given 5.555 points, whereas “incorrect” was given zero. n = 166): a mean WAS of the workers on each farm was calculated, so every farm received a final farm welfare assessment score, designated FWAS. The effects of these FWAS were analyzed with the farms’ production data (taken from the Israeli Herd Book).An overall welfare assessment score (WAS) was given to each respondent to examine the effect of attitude and awareness on different parameters. Additional analysis was performed on farms reporting to the Herd Book . Statistical analysis of the data was performed using SAS software . All variables’ means were calculated using the PROC MEANS procedure of SAS. In addition, we used a general linear mixed model (GLM) and the PROC MIXED procedure of SAS to test the effects of different parameters on the participants’ answers (both veterinary practitioners and farm workers) and their WAS. The entire model for the participants’ final grade was: Herd + welfare guidance + duty on the dairy farm + seniority + error, where Herd = 166 dairy farms; welfare guidance = yes/not at all; duty on the dairy farm = milking personnel, responsible for nursery, responsible for health, and general manager; seniority ≤ 4 years, 4–14 years, 15–29 years, and >30 years. All variables except Herd were considered fixed effects. Herd was included as a random effect.A second analysis was performed to test the effect of FWAS on cow milk production over 305 days. Only the 166 farms reporting to the Herd Book were included. We separated the dairy farms into four groups according to their FWAS values: group 1, ≤50; group 2, 50.1–67; group 3, 67.1–89; group 4, 89.1–100. The entire model was: 305 d milk yield, 305 d ECM, Fat %, Protein %, SCC = Herd + dairy farm size + FWAS group + error, where Herd = 166 farms reporting to the Herd Book; dairy farm size ≤ 300 or >300 cows; and FWAS group = four groups based on FWAS as delineated above. The results are presented as least squares (LS) mean values ± SE. To compare levels within a variable, we ran the Bonferroni adjustment for multiple comparisons.A total of 27 veterinary practitioners, representing 66% of the bovine practitioners working at Hachaklait Veterinary Services, answered the questionnaire (24 men and 3 women). Hachaklait Veterinary Services works with nearly 90% of the dairy farms in Israel. Of the surveyed practitioners, 20 declared that they had undergone some animal welfare seminars or lectures, while the other 7 had little exposure to the topic. No correlation was found between the practitioner’s gender and the overall scoring, or between age, seniority, or training experience and their personal score. The extent of the practitioners’ enjoyment from their work is shown in n = 124), whereas in the family farms, the most frequent (39%) was the oldest category . A significant part of the workers  on the cooperative farms have less than 4 years of work experience at the farm, while at the family farms, 44% work more than 30 years in farming (n = 75).A total of 500 Israeli farm workers from 174 dairy farms participated in the survey: 324 farm workers were from cooperative dairy farms, and 176 were from private family farms; 443 were men, 57 women; 79 (16%) of the responders were workers from Thailand. In the cooperative farms, the most frequent category (39%) was the young workers , i.e., those who enjoyed their work were also concerned about animal welfare.The first question referred to the extent of personal enjoyment from work at the farm, and the results are presented in n = 129) reached a mean WAS of 62.7 ± 3.2, similar to the workers aged 31–41 years (n = 92) who had a mean WAS of 62.5 ± 3.3. The workers aged 42–56 years (n = 113) had the highest WAS, 66 ± 3, and the older workers  scored slightly lower, with an average WAS of 60 ± 3.3, (p = 0.09).The younger workers  tended to score slightly lower than those working on cooperative dairy farms (n = 274), with mean WAS of 61.2 ± 2.9 and 64.4 ± 2.8, respectively (p = 0.08). Farm workers’ formal education significantly affected the survey score: farm workers with no academic or technical education (n = 224) scored 60.3 ± 2.7, those who graduated from professional or technical studies (n = 122) scored 61.6 ± 2.9, and academic graduates (n = 83) had a WAS of 66.5 ± 3.2 (p = 0.01).Farm workers on private family farms (n = 222) had a mean WAS of 59.3 ± 3, whereas farm workers who attended external courses (n = 207) scored 66.4 ± 2.7 (p < 0.0001). In addition, the extent of guidance on “cattle welfare” significantly affected workers’ knowledge, as expressed by their WAS , and finally, the milkers, who had the lowest WAS .p < 0.0001; Seniority of less than 4 years resulted in relatively low WAS. Numerous years of experience had a positive effect on the level of farm workers’ knowledge regarding animal welfare issues: from 4–14 years and 15–29 years of experience, up to 30 years of seniority. However, after 30 years, the mean WAS decreased , 8.4% (n = 41) did not know, 8.2% (n = 40) answered that “there is no hierarchy in cow herds,” and 5.7% (n = 40) answered “no.” Both parties were asked about the possible effect of separating a young cow for artificial insemination on its conception rate; 51% (n = 238) of the farm workers answered “it should not have any effect,” 37.4% (n = 175) thought it might have a negative effect, and 11% (n = 52) assumed that it might have a positive effect. However, 63% (n = 17) of the practitioners thought that inseminating a cow that is restrained away from the others would negatively affect conception rate, 37% (n = 10) said that it would not have any effect, and no practitioner thought that it might have a positive effect on conception.Another question on the survey, directed to the farm workers, referred to the social hierarchy in cattle: “Will the cow’s level of dominance affect its position in the herd?”; 77.6% of the workers answered “yes”  of the farm workers thought that the way in which they lead the cows to the milking parlor can affect milk yield, whereas 14% (n = 66) thought that this has no effect at all. The results showed that 74.5% (n = 353) of the farmers administer medications to the cows in a designated shed (as instructed), while 17% (n = 65) do not use a specific location; 12% (n = 56) of the workers treated the cows in the milking parlor, which goes against professional recommendations. The question of whether high milk production signifies good welfare is elaborated on in Farm workers’ answers to questions exploring the effect of man–animal interactions on milk production are listed in Veterinary practitioners’ and farm workers’ results are given in Regarding the need for pain relief following mutilation procedures, all practitioners and 71% of the farm workers thought that disbudding (using caustic paste) should be performed with pain relief medication. Only 63% of the farm workers thought that cold branding is a painful procedure, whereas 92.6% of the practitioners believed that this procedure requires pain medication.n = 91) of farms moved their calves into groups at the age of 1 week, 22.7% (n = 107) at 2 weeks, 20% (n = 92) at 1 month, and 38.5% (n = 182) at the age of 2 months (at weaning). When asked about the ideal age for group rearing  of veterinary practitioners stated that they had instructed farm workers to apply medical therapy in a designated shed, rather than in the milking parlor, to prevent a negative association between pain and milking. However, only 74.5% of the farm workers followed those instructions. Moreover, 92.6% of the practitioners thought that cold branding requires pain medication, but only 63% of them instructed the farm workers to use it .The practitioners were asked what they do when a repeated welfare problem at the farm remains unsolved; 28% answered that they only make a remark to the farm worker, 34% said that they insist on solving the problem without further professional assistance, and 38% said that they turn to other professionals for a solution.The survey results demonstrated the importance of guidance and knowledge acquisition on farm workers’ perception of animal welfare issues. In a survey performed in the UK, 56% of the farmers stated that they had gained a major part of their farming skills from their family, and only 18% had learned their skills from agricultural courses . In thisMost dairy farmers, animal and dairy scientists, and many veterinarians argue that high levels of milk production and good health are clear evidence of high standards of welfare . EnhanceIn general, both veterinary practitioners and farm workers demonstrated high WAS on topics related to cattle characteristics, such as social structure of adult cows, calves’ social needs, and the cows’ senses. The effect of human behavior on milk production was also well understood, mainly due to extensive training efforts regarding milking management by the Israeli Dairy Board through the years . HoweverMost practitioners and farm workers agreed that cows can identify specific humans connected to a positive or negative experience; 78% of the practitioners thought that cows remember previous events from an early age, and they all thought that experience with humans would affect the nature of the adult cow. Although only 60% of the farm workers agreed that cows remember past episodes, 80% thought that their interactions with humans would affect their temperament as adults.Awareness of the importance of calves’ social rearing has progressed greatly in recent years. In our survey, 90% of practitioners thought that calves should be reared in pairs or groups until weaning, and only 4% preferred isolated rearing until weaning. The main reason for individual rearing of calves in Israel is disease prevention . Farm woLooking at the perception of cows’ pain in both groups, survey results demonstrated that farm workers tend to think that cows are less likely to mask pain, as opposed to the veterinary practitioners, who are more familiar with the instinctive pain-masking prey behavior in cattle. Nevertheless, in the case of visible pain, such as severe lameness, 80% of the farm workers understood that this would affect milk production. Thomsen et al.  found thIn summary, this study found that there is considerable variation in welfare knowledge and practical applications, and this shows there is scope to improve dairy calf and cow welfare. Furthermore, many of the aspects of welfare knowledge link to good farm and animal husbandry, and there is good evidence that welfare knowledge is associated with levels of cow productivity across farms. Therefore, it is in the interest of both the animals and their managers to improve the knowledge for application of best practices.The aim of our survey was to explore the attitudes toward practical animal welfare issues on dairy farms. The high enjoyment obtained by farm workers and veterinary practitioners from their work and their perceived importance of cattle welfare constitute the elementary foundations for continual improvement. Professionally, farm workers were relatively proficient on the effect of welfare awareness on productivity, knowledge regarding cattle’s senses and social structure, and the effect of man–animal interactions on milk yield. There is room for improvement in three main areas: farm workers’ awareness of cows’ learning and memory, farm workers’ awareness of pain masking, and knowledge transfer from veterinary practitioners to farm workers. Since the veterinary practitioner is a leading professional on the farm, a great deal lies on his or her shoulders. Although farm workers give high importance to animal welfare issues, some seem to hold to traditional habits and changing their perceptions is rather challenging. The results highlight the importance of communication and understanding between farm workers and their veterinary practitioners. In light of the gaps demonstrated by the survey, reasons for insufficient knowledge transfer have yet to be understood. In conclusion, the results emphasize the variation in welfare knowledge and practical applications across farms, and it is in the interest of both the animals and their managers to improve the applied knowledge of good practices.


Worldwide, South Korea had the second highest incidence rates of colorectal cancer (CRC) in 2018. To inform public health policy to prevent CRC, we aimed to identify major modifiable lifestyle factors underlying the alarming increase in CRC incidence. We obtained information on CRC statistics from the Korea National Cancer Incidence Database and on the distribution of dietary and lifestyle factors known to modify CRC risk from the Korea National Health and Nutrition Examination Survey. To examine time trends between 2001 and 2013, we calculated annual percent changes of CRC incidence rates and of prevalence of etiologic factors by sex and age. Across all sex and age groups, the most commonly diagnosed cancer was rectal cancer while the most rapidly increasing cancer was distal colon cancer. For the lifestyle factors examined, decreases in exercise were observed across all age groups of both sexes. Yet, obesity and alcoholic drinks appear more relevant CRC contributor to men, smoking to women aged 30–49 years, and processed meat intake to adults aged 30–49 years. The heterogeneous results suggest that dietary and lifestyle target to prevent CRC be tailored by sex and age. In South Korea, the leading cause of death is cancer2, of which CRC death ranked third after lung and liver cancer deaths3. With CRC imposing a large burden on human health both globally and locally, identification of etiologic factors driving a rise in CRC incidence rates in South Korea has important implications for public health.In recent decades, South Korea has experienced a rapid increase in colorectal cancer (CRC) incidence rates. Worldwide, South Korea had the second highest incidence rate of CRC in 2018, with the rate estimated to be 44.5 cases per 100,000 persons per year4. Of note, accumulating evidence suggests that etiology of CRC may be heterogeneous by anatomical subsite of cancer , with a risk factor associated possibly more strongly with a subsite than with the other subsites5. For example, obesity is a stronger risk factor for colon cancer than for rectal cancer6. Thus, populations that have undergone varying degree of changes in the prevalence of CRC risk factors show differential distribution of CRC cases by anatomical subsite5. In the United States, which has a greater obesity prevalence, proximal colon cancer is the most common CRC subsite7, while in South Korea rectal cancer accounts for the highest proportion5.Development of CRC is largely attributable to the constellation of dietary and lifestyle factors associated with westernization such as poor diet characterized by high intakes of red and processed meat and alcoholic drinks and low intakes of fiber-rich foods, low physical activity, and obesity5, we conducted the analyses separately for men and women and for early-onset and late-onset CRC.Considering that major risk factors driving increases in CRC incidence rates could differ across countries, public health targets to curb the rising CRC should prioritize etiologic factors based on their prevalence and time trends within the population. Therefore, to identify predominant factors underlying the escalating incidence rates of CRC in South Korea, we conducted an ecological study by examining temporal trends in CRC incidence rates in relation to concurrent changes in the distribution of dietary and lifestyle factors established to be implicated in colorectal carcinogenesis. Additionally, to address potential etiologic heterogeneity by sex and age at CRC diagnosisCRC incidence rates showed a significantly increasing trend from 2001 to 2011 in men and women . Yet, the degree of annual increase on average was higher for early-onset CRC, with AAPC of the 2001–2013 interval for early-onset vs. late-onset being 3.6% (p < 0.01) vs. 3.4% (p < 0.01) in men  and age , rectal cancer was the most commonly diagnosed cancer but distal colon cancer had the largest annual % increase on average. Yet, concurrent changes in the distribution of known CRC etiologic factors were heterogeneous by sex and age groups. In men, those aged 30–49 years showed decreases in physical activity and increases in intakes of processed meat and alcoholic beverages, and BMI; those aged 50 years or older experienced decreases in physical activity and increases in alcohol consumption, and BMI. In women, those aged 30–49 years showed decreases in physical activity and increases in intakes of processed meat and alcohol beverages and smoking; those aged 50 years or older experienced decreases in physical activity for women aged 50 years or older. Despite the overall upward trends in CRC incidence rates and in the prevalence of the aforementioned etiologic factors, a significant downturn in CRC incidence rates from 2011 through 2013 was observed especially in men.10. Physical activity has beneficial effect against obesity and diabetes, which are established risk factors for CRC12. In the Health Professionals follow-up study that examined the risk of digestive system cancer by type and intensity of physical activity, aerobic exercise, regardless of its intensity, was found to be particularly beneficial, with the optimal benefit observed at approximately 30 MET-hours/week13. Walking is the most popular form of aerobic exercise worldwide and the amount of walking South Koreans engaging in has been suboptimal and decreased over time, as shown in our results.In our study, decreases in physical activity were a common change that occurred along with increases in CRC incidence across all age and sex groups. Although ecologic study design precludes causal inference, there are numerous biological mechanisms that suggest a role for physical activity in colorectal carcinogenesis. Physical activity helps reduce exposure time of intestinal mucosa to carcinogens including secondary bile acid14. Regarding alcohol drinking, in a recent meta-analysis of cohort studies, light (5–≤ 15 g/day) and moderate (> 15–≤ 30 g/day) drinking were significantly associated with 6% and 19% increased CRC risk, respectively, in men, but not in women15. The differential association by sex may be in part explained by higher level of alcohol intake in men. In South Korea, from 2001 to 2013, absolute alcohol consumption in men was high and also rapidly increased from 8.5 to 19.4 g/day. In women, on the contrary, the absolute intake remained very low (< 5 g/day) and increased only minimally (from 1.1 to 3.3 g/day) over the same time period , which are added as preservatives or formed endogenously in the gastrointestinal through the reaction across nitrite, amines, and amides from processed meat18. The endogenous formation of NOCs by processed meats is further facilitated by heme–iron in red meat20. NOCs act as genotoxins inducing DNA mutations and thus promoting carcinogenesis22.Among South Korean adults aged 30–49 years, increases in processed meat consumption was notable, which may have contributed to increases in early-onset CRC. By the International Agency for Research on Cancer Monograph Working Group, processed meat has been classified as “carcinogenic to humans” (Group 1)23, rectal cancer was more prevalent than colon cancer in South Korea. Smoking may be a potential contributor, considering its heterogeneous associations by anatomical subsite of the colorectum and lagged effect. Smoking was generally more strongly associated with rectal cancer  than colon cancer 5. Of note, the effect of smoking on rectal cancer outcomes emerged with a long latent period5. For instance, in a large prospective study of US veterans, RR of rectal cancer mortality comparing current smokers versus nonsmokers was only 1.1 after 16 years of follow-up24, but increased to 1.4 upon 26 years of follow-up25. The long latency suggests that higher prevalence of rectal cancer observed in South Korea in recent years may be associated with increases in smoking in distant past. Indeed, there was a rapid growth in the annual consumption of cigarettes in South Korea from the 1970s to 1980s 27.Unlike the distribution of CRC in high incidence countries31. Among South Koreans, the average calcium intake from food sources is low  kimchi and tofu, in which calcium has lower bioavailability than dairy products, constitute a large part of the intake32. In addition, calcium supplements are not widely used (< 15%). According to a dose–response meta-analysis of prospective observational studies, each 300 mg/day increase in total calcium intake  was associated with an approximately 8% reduced risk of CRC34. The association was observed well beyond 1000 mg/day of calcium intake32, which suggests that efforts to increase calcium intake might help curb the rising incidence of distal colon cancer in South Korea. Of note, given evidence that calcium helps reduce visceral adiposity35 calcium may confer additional benefit in preventing CRC among men in South Korea, for whom visceral fat was suggested as an important contributor to rising incidence of CRC.Rapid increase in distal colon cancer was notable in the epidemiology of CRC in South Korea. A potential explanation might relate to low calcium intake in South Korea. Considerable evidence suggests that the benefit of calcium intake against CRC might be confined to primarily distal colon cancer but generally less so to cancers in other segments of the colorectum36. Individuals with positive results from fecal occult blood test, the primary modality for mass screening, are entitled to either endoscopy or double-contrast barium enema36. From 2005 to 2008, the proportion of South Koreans who received endoscopy screening increased from 18.0 to 20.5%37. Of note, distal colon cancer predominantly occurs in men than in women, and it is readily detected by endoscopy due to its tendency toward polypoid morphology rather than flat morphology, which is frequently found in proximal colon cancer38. Furthermore, in South Korea, men were associated with a higher endoscopy uptake than women37. Taken together, endoscopy screening may have conferred a greater protection against CRC in men, which manifested as a downward trend in CRC incidence rates in the 2011–2013 interval. The time lag between the introduction of the nationwide CRC screening program and decreases in CRC incidence relates to the initial rise in cancer detection due to increasing screening39. In view of the highest annual % increase in distal colon cancer in South Korea and greater effectiveness of endoscopy in detecting distal colon cancer than proximal colon cancer, efforts to increase participation rates in CRC screening program may be of particular importance to lower CRC incidence in South Korea.Despite the overall upward trends in CRC incidence rates over the 2001–2013 period, a sudden downturn in the rates from 2011 through 2013 was evident in men. In light of concurrent declines in distal and rectal cancer rates during the 2011–2013 interval among men in later adulthood, screening effect may provide a potential explanation. In South Korea, since 2005, the National Cancer Screening Program has provided individuals aged 50 years or older with CRC screening at no or minimal cost biennially until 2011 and then annually from 201240, and our study did not intend to estimate relative risk between a specific factor and CRC risk but rather examined consistency or inconsistency of temporal trend between multiple established etiologic factors and CRC incidence. Other concern in our study includes measurement error in the dietary data. The 24-hour recall is generally considered reliable for estimating population average intake due to cancellation of random errors across individuals. Yet, until before 2007, the dietary data in South Korea were collected at a specific season rather than throughout the year, failing to account for seasonal variability. This, along with changes in food/nutrition composition data over time, might have generated systematic errors in estimating average dietary intake.Our study has major weaknesses that limit the establishment of causal relationships. First, as an ecological study based on population-level averages, any association observed between changes in the level of lifestyle factors and changes in CRC incidence cannot be ascribed to associations between individual-level characteristics. Second, while colorectal carcinogenesis spans several decades, our study juxtaposed temporal trends over the same specified period due to lack of national statistics on the lifestyle factors in earlier periods. Thus, our study could not address temporality or potential lag-time between change in lifestyle factors and change in CRC risk. However, only few factors such as folate, calcium, and smoking have been found to act early on colorectal carcinogenesis41 and recent increase in early-onset CRC43, our study to identify major contributors to CRC specific to sex and age groups provides a scientific basis for tailored public health intervention strategy for effective CRC prevention. Finally, statistics on CRC incidence are accurate because CRC cases were ascertained from the South Korea Central Cancer Registry, a nationwide cancer database, which captures over 90% of incidence cancer cases in South Korea44.Yet, our study has several strengths. As a country with the second highest CRC incidence rates in the world, an investigation to identify major contributing factors to this epidemic furthers our understanding of colorectal carcinogenesis. Given sex difference in CRC riskIn conclusion, patterns in CRC incidence and major etiologic contributors may be highly heterogeneous by sex and age in South Korea. While decreasing physical activity appears to be a common driver of increasing CRC across all sex and age groups of South Koreans, obesity and alcoholic drinks may be more relevant CRC contributors to men, and processed meat to middle-aged adults. Efforts to increase calcium intake and CRC screening uptake may help control increasing distal colon cancer in South Korea. Our study provides some supporting evidence for tailored public health campaigns to reduce CRC, necessitating more rigorous epidemiologic studies than ecologic study on this research topic.45. Based on the codes of the 10th revision of the International Statistical Classification of Diseases and Related Health Problems46, CRC was defined as C18-C20; and further classified by anatomical subsite into proximal colon cancer (C18.0–18.4), distal colon cancer (C18.5–18.7), and rectal cancer (C19–20)32. Colon cancer cases coded as C18.8 (overlapping lesions of the colon) or C18.9 (colon not otherwise specified) were excluded  from the subsite analysis. National population statistics were obtained from Korean Statistical Information Service by the Ministry of the Interior and Safety47.Information on the number of CRC cases, anatomical subsites, date of diagnosis, and sex was obtained from the Korea National Cancer Incidence Database, sourced from a nationwide hospital-based cancer registry constructed by the Korea Central Cancer Registry4.Established etiologic factors of CRC that are modifiable, such as risk factors  or protective factors , were selected based on the 2017 extensive summary report by World Cancer Research Fund (WCRF) and American Institute of Cancer Research (AICR)50. To elaborate on this further, dietary data were obtained from 24-hour diet recall. Processed meat, defined as any red meats preserved by smoking, salting, curing, or adding other chemical preservatives, included bacon, ham, sausage, etc. Red meat included beef, pork, lamb, goat from domesticated animals4. Alcoholic drinks included beer, soju (South Korean distilled spirits), makgeolli (South Korean rice wine). Whole grains included brown rice, barely, pearl barley, sorghum, oat, rye, buckwheat, millet, etc. Foods containing dietary fiber included whole grains, fruits, vegetables, and legumes. Dairy products included milk and any foods made from milk such as cheese and yogurt. Body fatness were indicated by body mass index (BMI) and waist circumference. Obesity was defined as BMI ≥ 25 kg/m2. Physical activity was estimated by the proportion of individuals who walked a total of ≥ 30 minutes per day (in bouts of at least 10 minutes\ duration) at least 5 days per week. Smoking was estimated by the proportion of current smokers with lifetime smoking of at least 100 cigarettes.Annual distributions of these dietary and lifestyle factors in South Korea were estimated based on responses from the Korea National Health and Nutrition Examination Survey, a series of nationally representative cross-sectional surveys conducted by the Korea Disease Control and Prevention Agency49.For each pre-specified group of age at diagnosis , annual incidence rates of cancer  were calculated for the entire colorectum and by subsites from 2001 to 2013. For cases diagnosed over a wider age range , in order to compare annual incidence rates from 2001 to 2013 without being affected by changes in population age structure over time, age-standardized rates (ASRs) were calculated through direct standardization by averaging age-specific incidence rates weighing to mid-year age structure of South Korean population in 2005, the recommended standard population by Statistics Koreahttps://surveillance.cancer.gov/joinpoint/) provided by the U.S. National Cancer Institute. In brief, joinpoint regression analysis fits a series of joined straight lines to the trend data , wherein each joinpoint indicates a statistically significant change in linear slope 51. In finding the best fitting joinpoint model, we allowed a maximum of 2 joinpoints and calculated the annual percent change (APC)  in ASR and prevalence for each line segment and the average annual percent change (AAPC)  for the whole period. The AAPC, as a weighted average of APCs with weights equal to the length of each line segment, quantifies the average trend over the whole period accounting for changes in trends during the interval51. The unit for APC and AAPC were expressed as % change per year. Of note, information on calcium supplement use was available from 2010 and thus, only the prevalence was estimated.Time trends of CRC incidence rates and distributions of aforementioned etiologic factors were described using the Joinpoint Regression Program, Version 4.8.0.1 .Supplementary Information


To study the expression of growth factors in the regulation of tissue repair after peritoneal damage tissue response to peritoneal damage.Experimental study in 35 male Wistar rats determining the evolution over time of the tissue response to aseptic peritoneal damage. A standardized bowel and peritoneal lesions were created in the right lower quadrant by laparotomy. Then, tissular expression of growth factors was evaluated by multiplex polymerase chain reaction at seven timepoints between 6 h and 30 days, postoperatively.Tissular responses of granulocyte-stimulating factors , connective tissue growth factor (Ctgf), epidermal growth factors and receptor , fibroblast growth factors , heparin binding EGF-like growth factor (Hbegf), hepatocyte growth factor (Hgf), insulin-like growth factor-1 (Igf1), mitogenic transforming growth factors , and vascular endothelial growth factor A (Vegfa) were biphasic with a first expression peak at day 3, followed by a more pronounced peak at day 14.We observed a long-lasting, widespread response of tissular growth factors for at least two weeks after peritoneal damage. To be clinically effective, the prophylaxis of postoperative adhesions might be needed for an extended period of time. In recent decades, growth factors have attracted a lot of attention in the study of the pathogenesis of various diseases and conditions because they play a crucial role in the processes of growth, development, and maintenance of cell populations. Growth factors are biologically active polypeptides that function as regulatory signals controlling cell proliferation and differentiation, and they promote cell survival , 2]..2].Most growth factors are multipotent and can affect various cell types. Although their names often reflect specific cellular types, their action might not be limited to this cell population. For example, vascular endothelial growth factor (VEGF) stimulates not only vascular growth. Still, it is also involved in the proliferation, migration, differentiation, and mobility of fibroblasts, both in physiological and reparative regeneration processes , 4], , . SimilarThe general role of growth factors in the regulation of physiological and reparative regeneration processes response has been extensively documented. FGF and VEGF are the master regulators of connective tissue formation; they activate the repair in the damaged tissue by stimulating fibroblast migration and the growth of granulation tissue. However, some peculiarities are depending on the organ localization of its formation and tissue environment .Growth factors play an essential role in repairing the peritoneal damage and have a significant impact on the development of adhesions in the abdominal cavity, for example, after surgery. Transforming growth factor-beta (TGF-β) expression has been documented from the third postoperative day and increases gradually until day 14 . ConversHowever, the expression of growth factors in the formation of adhesions after peritoneal damage has received relatively little attention. The purpose of this study was to investigate the response of the peritoneal tissue to injury by examining the expression of a large panel of growth factors during the development of the adhesive process.In an experimental study in 35 male Wistar rats, we determined the tissue response to aseptic peritoneal damage over time. A standardized bowel and peritoneal lesion were created in the right lower quadrant by laparotomy. Then, tissular expression of growth factors was evaluated by multiplex polymerase chain reaction (PCR) at seven timepoints between 6 h and 30 days, postoperatively.The study protocol was approved by the Institutional Review Board of the Irkutsk Scientific Center of Surgery and Traumatology, Irkutsk, Russia. The experiments were carried out under the rules of animal welfare proposed by the International Guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).Thirty-five, nine-month-old male Wistar rats weighing 220–250 g were purchased from a commercial provider. The animals were narcotized using ketamine 50 mg/kg, droperidol 2.5 mg/kg and atropine 0.4 mg/kg.2, by scratching the surface over 2–3 mm with a scalpel tip in the longitudinal and transverse directions, as described previously   postoperatively, and peritoneal tissues were taken for further analysis. The serous-muscular layer of the cecum in intact animals served as a control (n=5).2 were taken under sterile conditions from the damaged area of the cecum. Biopsies were plunged into RNAlater solution  for 12 h, and then frozen at −20 °C until further analysis.Biopsies of 2 × 2 mmThe biopsies were broken up by grinding in a mortar with liquid nitrogen to prevent thawing of the tissue. Samples were homogenized by passing the solution 10-fold through the needle of a 5-mL syringe until complete homogeneity and transparency were obtained. In order to avoid microbial or RNase contamination, preanalytical sample preparation was carried out under sterile conditions.RNeasy Mini Kit was used  for RNA extraction. Total RNA was extracted from tissues according to the manufacturer’s instructions. The concentration of total RNA was determined using a spectrophotometer . To additional purify RNA preparations from DNase commercial kit “Rnase-Free DNase Set”  was used.2 First Strand Kit”, Qiagen GmbH, Германия, Cat. No. 330401), according to the instructions of the manufacturer. The possible genomic DNA impurities were removed during the procedure.cDNA was generated from 300 ng total RNA using a commercial kit , using a customized array . A list of genes is given in http://www.qiagen.com/Products/Genes and Pathways/Data Analysis Center Overview Page / RT2 Profiler PCR Arrays Data Analysis Center). This software takes into account multiple comparison testing.Data are presented as mean and CI 5–95%. Statistical analysis of gene expression was performed using the software provided with the kit , connective tissue growth factor (Ctgf), epidermal growth factors and receptor , fibroblast growth factors , heparin binding EGF-like growth factor (Hbegf), hepatocyte growth factor (Hgf), insulin-like growth factor-1 (Igf1), mitogenic transforming growth factors , and vascular endothelial growth factor A (Vegfa).First, we examined the granulocyte and macrophage recruitment in the damaged area. As shown in A similar expression profile over time with an early and a later expression peak was also observed for connective and epidermal growth factors. Expression of connective tissue growth factor (Ctgf), e exceeded the indices of intact animals on postoperative 3 and 14 by a factor 3.53 and 5.80, respectively . Two simWhen expression of fibroblast growth factors , was examined, two activity peaks were also found on days 3 and 14 for Fgf2 and 10, while expression of Fgf7 was characterized by a single peak on day 14  . The expWe also examined neoangiogenesis in the wound. Unexpectedly, the increase of VEGF expression during the reparative response was relatively moderate. Here again, two activity peaks were seen on days 3 and 14, but the maximum activity increased only by 2.74-fold on day 14 as compared to intact animals. A long-lasting increase of insulin-like growth factor 1 was noted on days 14 and 30 . ExpressAlthough postoperative adhesions remain an unsolved challenge in modern surgery, expression of growth factors in the formation of adhesions after peritoneal damage has received little attention. The purpose of this study was to investigate the response of the peritoneal tissue to injury by examining the expression of growth factors over time during the development of the adhesive process. For this purpose, to induce an aseptic inflammatory response, we created standardized peritoneal damage in rodents and determined the expression of a large panel of growth factors. We included granulocyte-stimulating factors , connective tissue growth factor (Ctgf), epidermal growth factors and receptor , fibroblast growth factors , heparin binding EGF-like growth factor (Hbegf), hepatocyte growth factor (Hgf), insulin-like growth factor-1 (Igf1), mitogenic transforming growth factors , and vascular endothelial growth factor A (Vegfa) in our analysis. The hope was to detect common patterns of expression of these growth factors over the wound healing process.Our main finding is that tissular expression of growth factors during peritoneal wound repair is a biphasic process, with an early phase culminating on day 3 after injury, and a late phase reaching its maximum two weeks after this injury. These results are in line with the expression of proinflammatory cytokines in the abdominal cavity , 21]. N. N21]. NPeritoneal adhesions frequently develop after abdominal surgery and can cause a significant burden, such as chronic abdominal pain, small bowel obstruction, or infertility. The etiology of peritoneal adhesions is multifactorial and involves a dysbalance between coagulation, inflammation, and fibrinolysis. Cellular components such as monocytes, macrophages, fibroblasts, and mesothelial cells are involved in this process. Moreover, growth factors and secreted signaling molecules play a key role in peritoneal adhesions development . PeritonPrevention of postoperative adhesions is an active area of research. Various drugs and methods such as icodextrin, barriers, and others have been clinically tested, but their effectiveness is debated . AgainstWe make the hypothesis that a single intraoperative administration of antiadhesive agents might be insufficient to prevent the formation of postoperative adhesions. Such a single administration would have an effect on the first active phase of growth stimulation immediately after surgery but is unlikely to influence the second growth phase we demonstrated to last at least two weeks postoperatively. This observation calls indeed for long-term preventive strategies, with demonstrated target effect during at least two weeks after surgery. Our hypothesis is supported by the fact that gels and other biomaterials appear to be the most effective adhesion prevention agents for use during gynecological surgery . Gel forFuture research should evaluate the ability of novel preventing approaches to reduce the expression of growth factors over a more extended period. We propose to use our established experimental model of peritoneal injury and the gene panel above as a surrogate marker of efficacy. Such an approach might be useful for speeding up the validation of novel prophylactic strategies against postoperative adhesions.Click here for additional data file.


Pinus pinaster Ait.) were produced in Escherichia coli to enable study of their molecular and kinetics properties. The results reported here provide a molecular basis for the regulation of arginine and ornithine metabolism at the enzymatic level, suggesting that the reaction catalyzed by OTC is a regulatory target in the homeostasis of ornithine pools that can be either used for the biosynthesis of arginine in plastids or other nitrogenous compounds in the cytosol.The amino acids arginine and ornithine are the precursors of a wide range of nitrogenous compounds in all living organisms. The metabolic conversion of ornithine into arginine is catalyzed by the sequential activities of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASSY) and argininosuccinate lyase (ASL). Because of their roles in the urea cycle, these enzymes have been purified and extensively studied in a variety of animal models. However, the available information about their molecular characteristics, kinetic and regulatory properties is relatively limited in plants. In conifers, arginine plays a crucial role as a main constituent of N-rich storage proteins in seeds and serves as the main source of nitrogen for the germinating embryo. In this work, recombinant PpOTC, PpASSY and PpASL enzymes from maritime pine ( Nitrogen (N) is an essential constituent of proteins, nucleic acids and many other compounds essential for life such as hormones, vitamins, porphyrins and a wide range of specialized metabolites. Although N is highly abundant in the atmosphere as dinitrogen, only combined forms such as nitrate, ammonium or amino acids can be assimilated by plants. Because these forms of N are usually present in low abundance in nature, N availability is a major limiting factor for plant growth and development . When anThe biosynthesis of arginine can be divided into two pathways: the conversion of glutamate into ornithine and the synthesis of arginine from ornithine through the sequential action of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASSY) and argininosuccinate lyase (ASL) ,5.The compartmentation of arginine biosynthesis in plants was controversial in early studies, and the cytoplasmic and mitochondrial localization of several enzymes was reported , as prevStorage proteins are of key importance for the nutrition of conifer germinating seeds. In maritime pine, arginine is one of the most abundant amino acid residues in the storage proteins representing with glutamine/glutamate the majority of the total amino acid content . StorageE. coli as recombinant proteins, and their molecular, kinetic and regulatory properties have been determined. The results reported here provide new insights into the short-term biochemical regulation of the arginine biosynthetic pathway.Current research efforts are addressed to further understand how arginine biosynthesis is regulated during pine embryogenesis. The availability of genomic resources has greatly facilitated the performance of functional studies in maritime pine ,22. To fPpOTC, PpASSY and PpASL genes [In a previous paper, full-length cDNAs of SL genes  were cloSL genes . Here, aThrough a multiple alignment analysis of PpOTC from different plant and animal species, the characteristics of C-terminal ornithine binding motifs FMHCLP and DVWASMG were identified (consensus DXXXSMG)  Figure . The motE. coli were established to enable the characterization of their molecular and kinetic attributes. For this purpose, the ORFs without the sequence corresponding to the chloroplast transit peptide (CTP) were subcloned into a pDEST17 vector. The CTPs were removed according to theoretical predictions based on the TargetP-2.0 server [PSORT tools [With the aim of deepening the understanding of the metabolic role of these enzymes, experimental protocols for the production of active recombinant PpOTC, PpASSY and PpASL enzymes in 0 server  and WoLFRT tools . The proPurified preparations of recombinant PpOTC, PpASSY and PpASL enzymes were used to estimate the molecular mass of holoenzymes by gel filtration chromatography through a calibrated column with protein markers of a known size . The rec0.5 value was estimated to be 1.28 mM. On the other hand, our analysis showed negative cooperativity of PpOTC assayed with increasing concentrations of carbamoyl-P, as determined by the Hill plot (nH = 0.48) with a S0.5 value of 3.48 mM  were submerged in water with constant aeration for 24 h. Soaked seeds were spread between two layers of vermiculite  for 30 days, adding only distilled water for seed germination and seedling growth. Cotyledons of seedlings were harvested and quickly frozen in liquid nitrogen. Frozen cotyledons were pulverized, and total RNA was extracted and isolated following the protocol described by de la Torre et al. . RNA wasOligonucleotides for the corresponding ORF cloning were designed by retrieving maritime pine OTC, ASSY and ASL  sequences from maritime pine data available at Congenie database . Oligonu® cloning . Sequences were inserted into the pDONR207 vector via Gateway® BP II clonase mix  reaction and later inserted into the pDEST17 expression vector via Gateway® LR clonase II mix  reaction following manufacturer’s indications. pDEST17 with inserted cloned sequences were sequenced to confirm correct insertion of cDNA sequence.Cloned cDNAs were 984 nucleotides in length for PpOTC, 1218 nucleotides for PpASSY and 1398 nucleotides for PpASL. attB1 and attB2 ends were added by PCR using specific primers , necessaEscherichia coli BL21 AI (Thermo Fisher Scientific™), RIL  and LEMO  strains were transformed with the pDEST17 plasmid for recombinant protein production. Different growth and production conditions were tested until reaching the optimals or overproduction of each recombinant protein  for PpOTC and 0.5 mM IPTG for PpASSY. No IPTG was required for PpASL production. Temperature was set to 30 °C for induction of PpOTC, 25 °C for PpASSY and 37 °C for PpASL. Cultures were maintained under their optimal temperature and gentle shaking (60–75 rpm) for 8 h (PpOTC) and 5 h (PpASSY and PpASL). Bacterial cultures were centrifuged at 5000× g and pellets were frozen. Proteins were purified by HisTag affinity onto Ni-agarose resin . Aliquots from the different purification steps were analyzed by SDS-PAGE to follow the progress of purification  acetone, 5 N H2SO4 and 5% (w/v) ammonium molybdate in proportions (2:1:1)). Absorbance was measured at 430 nm, 12 s after the arrest of the reaction. The concentrations used ranged from: 375 μM to 4 mM, for ornithine and between 250 μM and 30 mM for carbamoyl-P. Pi contents were calculated based on a standard curve using commercial sodium phosphate .PpOTC activity was determined by quantification of the Pi released with molybdate, that turns into the colored compound phosphomolybdate. The protocol used is the one described by Bernhart and Wreath , with miThe ASSY enzyme catalyzes the following reaction:2 and 2.4 μg of purified PpASSY. Incubation was performed at 25 °C for a range of times from 1 to 8 min. Aliquots of 10 μL were taken each minute and the enzyme reaction stopped by adding 10 μL of an EDTA-NaOH solution containing 400 mM EDTA, 1.4 M NaOH. A volume of 200 μL of a molybdate solution containing 60% (v/v) acetonitrile, 5% (v/v) 12 N HCl and 20 mM sodium molybdate was added, and the resulting solutions were incubated at room temperature (RT) in darkness for 10 min. A volume of 80 μL of a solution containing 60% (v/v) acetonitrile, 5% (v/v) 12 N HCl and 500 mM ascorbic acid  was added to a final volume of 300 μL, incubated at room temperature for 10 min and absorbance read at 790 nm. The concentrations used ranged from 50 μM to 2 mM for both citrulline and aspartate. PPi contents were calculated based on a standard curve using commercial sodium pyrophosphate .PpASSY activity was determined by measuring of the released PPi and its reaction with molybdate, forming 18-molybdopyrophosphate. This compound turns blue when reduced in presence of ascorbic acid and has a peak of absorbance at 790 nm, as described previously by Katano H. et al. . The reaThe ASL catalyzes the reaction:PpASL activity was determined by measuring absorbance of fumarate at 240 nm. The reaction mixture contained in a volume of 100 μL 50 mM Tris-HCl (pH 8), 2 mM argininosuccinate and 13.2 μg of the purified PpASL. The reaction was incubated at 25 °C for 8 min, measuring the absorbance each 30 s. The concentrations used ranged from 25 μM to 2 mM of argininosuccinate. Fumarate contents were calculated based on a standard curve using commercial fumaric acid . m and Vmax values were determined using PRISM5 software  (GraphPad Software LLC).For the three enzymes, apparent Kβ-amylase (200 kDa), alcohol dehydrogenase (150 kDa) and carbonic anhydrase (29 kDa) . The fractions were collected, and the elution of the holoenzymes was determined by measuring absorbance at 280 nm and further confirmed by western blot analysis [Desalted aliquots of purified recombinant enzymes  were loaded separately onto a size-exclusion ENrich™ SEC 650 column (BioRad) in order to determine the size of the multimer. The column was calibrated using commercial protein standards: thyroglobulin (669 kDa), apoferritin (443 kDa), analysis .


Degeneration of intervertebral disc is a major cause of lower back pain and neck pain. Studies have tried to unveil the regulatory network using either transcriptomic or proteomic analysis. However, neither have fully elucidated the exact mechanism of degeneration process. Since post-transcriptional regulation may affect gene expression by modulating the translational process of mRNA to protein product, a combined transcriptomic and proteomic study may provide more insight into the key regulatory network of Intervertebral disc degeneration.In order to obtain the proteomic and transcriptomic data, we performed label-free proteome analysis on freshly isolated nucleus pulposus cells and obtained transcriptome profiling data from the Gene Expression Omnibus repository. To identify the key regulatory network of intervertebral disc degeneration in nucleus pulposus cells, we performed bioinformatic analyses and established a protein-RNA interacting network. To validate the candidate genes, we performed in vitro experimentation and immunochemistry labeling to identify their potential function during nucleus pulposus degeneration.The label-free proteome analysis identified altogether 656 proteins, and 503 of which were differentially expressed between nucleus pulposus cells from degenerated or normal disc cells. Using the existing nucleus pulposus transcriptomic profiling data, we integrated the proteomic and transcriptomic data of nucleus pulposus cells, and established a protein-RNA interacting network to show the combined regulatory network of intervertebral disc degeneration. In the network, we found 9 genes showed significant changes, and 6 of which  showed concordant changes in both protein and mRNA level. Further functional analysis showed these candidates can significantly affect the degeneration of the nucleus pulposus cell when altering their expression.This study is the first to use combined analysis of proteomic and transcriptomic profiling data to identify novel regulatory network of nucleus pulposus cells in intervertebral disc degeneration. Our established protein-RNA interacting network demonstrated novel regulatory mechanisms and key genes that may play vital roles in the pathogenesis of intervertebral disc degeneration. Lower back pain and neck pain are common musculoskeletal disorders that could result in severe social and economic burdens on patients . CurrentSince the initial degeneration change appears in the nucleus pulposus (NP) region that compromises the function of imbibing water, leading to a decrease in the intradiscal pressure . SubsequStudies showed multiple factors could affect the degeneration process, such as increased activities and expressions of extracellular matrix metabolic enzymes such as matrix metalloproteinases (MMPs), TIMPs, inflammatory molecules  . On the mRNA translation involves mechanism such as post-transcriptional or translational regulation. Disturbances of the expressions of these mechanism related factors may cause uneven translation of mRNAs, thus causing global expression change . Taking Informed consent was obtained and signed by both the patient and their relatives before acquiring the intervertebral disc tissue during surgery as requested by the ethics committee in our institute. The experimental protocol was also authorized by the ethics committee of Naval Medical University. Magnetic resonance Imaging (MRI) T-2 weighted images were used to evaluate and diagnose IDD according to the modified Pfirrmann grading system . RelativFor primary cell extraction, disc tissue specimens were washed twice with PBS, the NP region of the disc was isolated under a microscopy and then minced and digested with 2 U/mL protease in DMEM/F12  for 30 min at 37 °C [For gene knockdown, we used synthesized siRNAs  to perform siRNA mediated knockdown on the candidate genes . The traTrizol  was used to extract the total RNA from human nucleus pulposus cell samples according to the manufacturer’s instructions as previously reported . Total R–∆∆CT method [P < 0.05 was assumed for significance in differences, and all experiments were repeated at least for three times independently.Real-time polymerase chain reaction (RT-PCR) analysis of CHI3L1, KRT19, CLIP, COL6A2, DPT and COL11A2 were performed using SYBR premix Ex Taq™  in a Step One Plus real-time PCR system  according to the manufacturer’s instructions as previously reported [g for 30 min at 4 °C, and 100 μL lysis buffer  were added into each sample followed by ultrasonication to extract the total proteins of NP cells [g for 30 min. The concentration was measured according to the manufacturer’s instructions using Qubit quantification kit  to determine the concentration [NP cell samples were centrifuged at 10,000×NP cells . Trichlontration .p < 0.05 and signal more > 1.5-fold were considered as differentially expressed proteins.Label-free proteomic profiling of the protein extracts were done by Beijing BangFei Bioscience Co., Ltd. In brief, 60 μg protein samples were used and addition of 100 mmol/L DTT (DL-Dithiothreitol) to its final concentration of 10 mmol/L was performed and mixed at 37 ℃ for 60 min. The mix was then diluted with 250 mmol/L IAM (Iodoacetamide) and kept in dark for 60 min . 100 μL 2-transformed and quantile-normalized signal data were used, and genes with p < 0.05, signal fold-change ≥  ± 1.5 were filtered and considered as differentially expressed genes. Unsupervised hierarchical clustering was performed to detect the expression pattern change using the profiling data by average linkage and median centering, the processed data was visualized with TreeView [P value were used to selected candidate genes to form the network, and the presented network is further drawn according to the KCore value and the calculated Degree value. Other analysis like Gene Ontology (GO) analysis was carried out using annotations in PANTHER database v 6.1 (www.pantherdb.org) [http://www.genome.jp/kegg/pathway.html) respectively [We downloaded the processed data of GSE70362  from GenTreeView . To consrdb.org) , and patectively .To localize the expression of DPT, COL6A2, KRT19, CLIP, COL11A2 and CHI3L1 in NP tissue samples, we made use of three normal  and three degenerated NP tissue samples  for the immunohistochemistry analysis as previously reported [P value < 0.05, Additional file To gain initial knowledge of the global protein and RNA expression pattern of primary NP cells during intervertebral disc degeneration (IDD), we first performed label-free proteomic analysis of IDD and normal NP cells. Three normal NP  and three IDD NP  tissue samples were collected from male patients of similar ages, The NP cells were initially isolated to exclude further influences of protein degradation, and cells were extracted for proteomic profiling. In the data, we identified 656 proteins, and 503 of which were identified as differentially expressed proteins , while mRNA changes mainly focused on steroid and cholesterol metabolism , extracellular matrix related metabolism , stress and inflammatory response  in Fig. Here, we construct the co-expression network to clarify the key component and potential relationship between these differentially expressed mRNAs and/or proteins. By comparing the normal and degenerated NP data, we constructed a co-expression network according to the mRNA or protein expression trend (fold change). From the network we can found that the degenerated co-expression network is rather focused, with each key node (gene) inter-connected with another, forming one big network loop Fig. a. HoweveFrom the degenerated NP co-expression network, we found only 9 genes showed significant correlation with other genes both in mRNA and protein forms , and only 6 of them  showed concordant changes between mRNA and protein level during degeneration. This finding indicated the important functional roles of these candidate genes during IDD. Among which, KRT19 and COL6A2 were considered as important degeneration markers in the previous studies , 21, whiTo further validate the interacting network, we first examined the expression of a few candidates. Here, we selected TNFAIP6, CHI3L1, KRT19, DPT, COL6A2 and COL11A2 as candidate genes for further validation for they showed concordant changes in both RNA and protein level in NP degeneration, which may potentially exert significant function in IDD process. By immunohistochemistry, we found that the expression levels of the candidates were all consistent with the transcriptomic and proteomic sequencing data Fig. a, b. UsiTo further show their function in IDD, we designed small interfering RNAs targeting these candidates to perform knockdown in NP cells. The efficiency of the knockdown siRNAs was tested prior to functional examination Fig. a. By usiThe degeneration of intervertebral disc is a complex biological process that the mechanism of which is less known . Many reRecently, post-transcriptional regulation mechanism has been identified as important molecular mechanism that affect the pathological events in intervertebral disc degeneration process . Post-trIn order to testify this hypothesis, we those high through-put profiling data to gain insight into the regulatory network of intervertebral disc degeneration. In our constructed network, we found many unreported genes that had the potential to act as important regulators during the IDD process. Looking into the degenerated network we constructed, we can find the network is more condensed and interconnected with genes of many different functions, which indicated that the degeneration process may act in cascades. This means that all the included genes could affect each other and take a role in the pathogenesis of IDD in an uncovered mechanism. During which, we can find genes such as COL6A2, COL11A2, KRT19, COL1A2, TIMP1, and ITGBL1 are known extracellular matrix related factors in the regulation of IDD , 37, 38.Among the network, we found enzymes that related to chondroitin sulfate (CS) biosynthesis and catalysis  showed significant changes during NP degeneration, which further support our previous findings  in decipBesides, we observed that osteogenesis related genes like IBSP, RSPO3, SOX4 and OGN are also potential regulators that were included in the degeneration related network, which indicated a vital role of ossification or osteogenesis related genes during the IDD pathogenesis. It has been reported that Runx2, a master transcriptional factor of chondrocyte hypertrophy and ossification, is the abundantly expressed in the nucleus pulposus cells during its degeneration . The resHowever, our study had limitations. First of all, the samples were collected from surgical patients, which would inevitably cause damage to the nucleus pulposus tissue, which would have effects on the gene expression of the NP cells . So, the tissue samples, especially the normal NP samples, would have some differences to its physiological state. Moreover, the sample size for proteome study is rather small (only 3 control and 3 degenerated samples were used), which needs further validation for each candidate gene we reported in this study. Secondly, our study mainly focused on deciphering the interaction network of coding genes using RNA sequencing and protein sequencing data from NP cells, and due to the limitation of the techniques we used, some type of the proteins (like proteoglycans) and other functional RNAs like microRNAs and lncRNA (long non-coding RNAs) were not or could not be considered in this study. However, since the coding genes were of strong function in disease pathogenesis, we think that our data would throw light on further in depth studies based on our findings. Thirdly, due to the nature of the NP tissue, it is hard to collect large number of NP cells using one patient’s sample , so the Taken together, our study provides new evidence and insights into the regulatory network of IDD. By combining the mRNA and protein profiling data, we purpose new IDD related interacting RNA–protein network and validated key regulators  with potential functional roles in the IDD process, but the detailed mechanism is up to further investigation and validation.Additional file: 1. Oligonucleotide Sequences used in this study. The sequence of oligonucleotides used in this study.Additional file: 2. Differentially expressed proteins in IDD. Contains the processed data of label-free proteomic profiling of normal (IVD) and degenerated (IDD) nucleus pulposus cells.Additional file 3. Differentially expressed mRNAs in IDD. Contains the processed data of transcriptome profiling of normal (IVD) and degenerated (IDD) nucleus pulposus cells.Additional file 4. Comparison of differentially expressed mRNA and Protein expressions. Annotation and comparison of processed protein and mRNA expression of normal and degenerated nucleus pulposus cells.Additional file 5. GO and pathway analysis of differentially expressed protein and mRNA in IDD. Contains four sheet detailing the pathway and gene ontology analysis results of differentially expressed Protein and mRNA in IDD.Additional file: 6. Degree value for network construction. The calculated value of gene degree for construction of protein-mRNA interacting network.


It remains unclear if, and to what extent, current estimations of ITV are biased. The most common method used to quantify ITV is the coefficient of variation (CV), which is dimensionless and can therefore be compared across traits, species, and studies. Here, we asked which CV estimator and data normalization method are optimal for quantifying ITV, and further identified the minimum sample size required for ±5% accuracy assuming a completely random sample scheme. To these ends, we compared the performance of four existing CV estimators, together with new simple composite estimators, across different data normalizations, and sample sizes using both a simulated and empirical trait datasets from local to regional scales. Our results consistently showed that the most commonly used ITV estimator (CV1= sampleσ/sampleμ), often underestimated ITV—in some cases by nearly 50%—and that underestimation varies largely among traits and species. The extent of this bias depends on the sample size, skewness and kurtosis of the trait value distribution. The bias in ITV can be substantially reduced by using log-transforming trait data and alternative CV estimators that take into consideration the above dependencies. We find that the 4CV estimator, also known as Bao's CV estimator, combined with log data normalization, exhibits the lowest bias and can reach ±5% accuracy with sample sizes greater than 20 for almost all examined traits and species. These results demonstrated that many previous ITV measurements may be substantially underestimated and, further, that these underestimations are not equal among species and traits even using the same sample size. These problems can be largely solved by log-transforming trait data first and then using the Bao's CV to quantify ITV. Together, our findings facilitate a more accurate understanding of ITV in community structures and dynamics, and may also benefit studies in other research areas that depend on accurate estimation of CV.Intraspecific trait variation (ITV) is common feature of natural communities and has gained increasing attention due to its significant ecological effects on community dynamics and ecosystem functioning. However, the estimation of ITV  Intraspecific trait variation (ITV) is the overall difference in traits values among conspecific individuals in one or more traits, such as height, specific leaf area, and wood density . Such vaAccurate estimation of ITV is essential for fully understanding species distributions and abundances from a trait-based perspective because the absolute extent of ITV is thought to be closely linked with species tolerances to the abiotic environment and responses to neighborhood interactions . Specifiad hoc assumptions of the underlying probability distributions for each trait. Importantly, CV is unitless, and thus offers a convenient way to directly compare variation  among species with different abundances under various environments (Most current studies use the coefficient of variation (CV = σ/μ) to quantify the absolute extent of ITV and evaluating whether ITV varies among species or traits, and ultimately in thinking about the response of populations or communities to environmental change (see our literature review in ronments . For exaronments : speciesronments .CV1= sampleσ/sampleμ) is biased . Despite the potential importance of ITV in many ecological disciplines, no study has yet examined the effect of sample size. Moreover, because traits have various distributions and often take extreme values, whether there exist bounds for the minimum sample size of ITV estimation remains largely unknown.In addition to choosing a suitable CV estimator and data normalization method, few studies quantify the minimum sample size needed for accurate estimation of ITV. As reviewed by ple size . This reHere, we attempt to address the above knowledge gaps and improve the estimation of ITV by answering the following three questions: (i) To what extent the current estimations of ITV are biased? (ii) Are there more accurate estimators and data transformations for estimating ITV? (iii) Are there prescriptive rules for identifying the minimum sample size needed for a given level of accuracy (here ±5%)? To answer these questions, we reviewed the previous literature and evaluated the performance of CV estimators using simulated and empirical trait datasets. Finally, we evaluated the accuracy of the best performing ITV estimator across various sample sizes.http://thomsonreuters.com/web-of-knowledge/) in Sep. of 2019 for research articles containing the topic “intraspecific variation”, or “intraspecific variability”, or “individual variation”  in the past 19 years, from 2000 to 2019. Studies that only used quantitative statistical tests such as ANOVA, Levene's test, and linear mixed-effects models to analyze or account for ITV were omitted, because comparing ITV among populations, species, studies, and regions requires knowledge about the absolute extent of ITV. We downloaded the full text of each remaining article and checked if and how ITV was calculated. Specifically, we summarized the absolute and relative number of studies that used each ITV estimation method, data normalization scheme, and the minimum sample sizes for each species in our literature survey (1CV (see below) as their estimator of ITV.To find the most commonly used methods for the quantification of ITV in recent literature, we searched the Web of Knowledge   , where N2CV is derived under the assumption of a normal distribution, it may underperform when trait values are not normally distributed. 1CV also depends on the skewness and kurtosis of the distribution of trait values. Therefore, 3CV and 4CV , respectively, which do not assume any specific trait distribution:Because N is the sample size, and γ1 and γ2 are the Pearson's measures of skewness and kurtosis of the trait sample distribution, respectively.where 3CV and 4CV—which are only approximate estimators of CV—often underestimate or overestimate the population CV in our simulation and empirical data , we found that data see S4E. The7CV and 8CV have almost identical performance compared to 5CV and 6CV, respectively, so we will only present the results of 5CV and 6CV in the remainder of the text. Note that 3CV and 4CV were derived without any assumption of trait value distribution, thereby their composite estimators, 5CV and 7CV, also make no assumptions of the true trait value distribution.We found i,mink for estimator i as the minimum integer k that reaches ±5% accuracy of the ITV estimation. Because different traits of species may have different distributions, we calculated i,mink for each trait of each selected species separately.Accurate estimation of ITV not only depends on the choice CV estimator and data transformation, but also on sample size. Generally, the accuracy of any estimator increases with the sample size. Therefore, it is crucial to know how many samples are sufficient to reach a specific accuracy requirement in the estimation of ITV. Here, we defined the minimum sample size 1β and a scale parameter 2β, which were two independent random variables following a uniform distribution from 1 to 10 and from 5 to 30, respectively. The ranges of 1β and 2β were determined by fitting empirical trait value distributions using a gamma distribution for each trait of each species with abundance ≥100 in our trait plot (see details of the trait plot below). We chose the gamma distribution here because its domain, like empirical functional trait value, is always positive and its shape is flexible enough to model various distributions of traits. Additionally, empirical trait data often have a few (4.8% on average) extreme large values, which are defined here as trait values greater than three standard deviations from the mean. These extreme values may have large impacts on the accuracy of CV estimators. Therefore, we added 480 extreme large values to each simulated trait pool. The extent of the extreme value follows a truncated exponential distribution from mean+3*standard deviation of the trait pool to infinity and parameter λ equals 1. Note that we recognize that we cannot simulate all possible trait distributions observed in natural communities; here, we tried as best as possible to cover a large portion of the ranges of empirical trait distributions.To find the optimal CV estimator and data transformation, we evaluated the performance of the above estimators using simulated trait data. First, 200 trait pools were generated by simulation. Specifically, for each trait pool, 9,520 trait values were drawn from a gamma distribution with a shape parameter k, k trait values were sampled from each simulated trait pool and i,kCV was calculated for each CV estimator i. The mean CV, k. Then the bias of the ith estimator under sample size k was defined as trueCV is the population CV that is known in our simulated data. Note that trueCV is only required in our performance evaluation, and is not demanded in the estimation of ITV. The sign of B indicates whether i,kCV under- or overestimates ITV. To facilitate estimator comparison, we calculated the proportion of bias  = B /trueCV) for each sample size and estimator combination, and total absolute bias For each sample size x-minx)/maxx, where x is the raw trait value and minx and maxx are the minimum and maximum values of all sampled trait values, respectively. The second method is log-transformation with the natural logarithm base. For trait with values smaller than 1, 1 was added to all values of that trait before log-transformation. The last is cube-root transformation. To compare the effect of data normalizations on ITV estimation, PB and TPB(i) were calculated for the raw trait data and three types of data normalizations for each CV estimator.Our literature review further found that three types of data normalization methods are used in the estimation of ITV in conventional studies Table 1.k from the observed trait values for each trait of each species, then PB and TPB(i) were calculated for all i,kCV. Here, trueCV was estimated by the value of 1CV based on all individuals for each trait and each selected species.The performance of all the above estimators and data normalizations were also evaluated using three individual-based trait datasets, each representing a different geographic scale. For each dataset, we used the same sampling scheme as in the simulated data: 9,999 replicates were drawn with replacement for each sample size PB and TPB, which depend on the trueCV, can be estimated within ±0.55% accuracy by any of the above estimators when the sample size is larger than 400. The second trait dataset contained two traits (SLA and LDMC) from four tree species from 72 small (20 m × 20 m) plots in the whole Ningbo region , Zhejiang Province, China. It distributed along a 30 km gradient from seaside to inland—and is thus representative of regional scale data. The number of trait measurements for each trait in the Ningbo tree dataset was larger than 170. The third trait dataset contained five traits, head length (HL), interorbital distance (IOD), tympanum diameter (TYD), outer metacarpal tubercle width (OPTW), and tibia width (TW), of a mountain frog species (Feirana quadrana) from the mountain region covering Longmen-Qinling-Daba Mountains , Central China. A total of 545 individuals were measured for each trait.The first dataset contained four traits: mean leaf area (MLA), specific leaf area (SLA), leaf dry mass content (LDMC) and individual height (Height). The dataset included 20,248 individuals (DBH≥1 cm or Height >1.3 m) belonging to 108 tree species in a 5ha subtropical forest plot, Tiantong , Zhejiang Province, China . This dahttps://www.github.com/guochunshen/CV). All other performance tests and sample size analyses were performed in R environment , Ningbo tree data , and Mountain frog data . 4CV was again found to be the most accurate estimator with log-transformed data, with one exception: 6CV was the most accurate in the Mountain frog data. The cube-root transformation produced a similar, but relatively weaker reduction of ITV bias, and min-max normalization had no significant effects  still required more than 100 samples. Finally, combined use of 4CV with log-transformed trait data reduced the minimum sample size need to reach ±5% accuracy to just 20 samples for almost all examined traits and species.The accuracy S2. For 1CV is often biased under sample sizes that are commonly applied . When the sample size is around 10, the underestimation of ITV can exceed 48.9%. This pervasive underestimation of ITV by 1CV has largely been ignored in previous studies and has therefore potentially misled subsequent ecological inferences when comparing ITVs among species. The bias of 1CV was different among traits and species and these differences cannot be completely removed by using the same sample sizes, because the source of bias in 1CV includes the skewness and kurtosis of the underlying trait distributions . The higher accuracy of 4CV results from its consideration the skewness and kurtosis of the trait distributions. However, 4CV may overestimate ITV when the distribution of raw or log-transformed trait data is close to the normal distribution. 4CV is an approximate form of CV, and therefore can only be truly unbiased when the underlying trait distribution is known exactly. Like 1CV, 2CV is derived under the assumption that trait values follow a normal distribution, which has no skewness and kurtosis. The contrasting behaviors of 4CV and 2CV allowed us to construct a simple composite estimator 6CV, which was more accurate when the distribution of trait values is close to the normal distribution  and the National Natural Science Foundation of China , as well as the ECNU Multifunctional Platform for Innovation (008).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.