Neuropsychiatric disorders are often associated with motor and coordination abnormalities that have important implications on the etiology, pathophysiology, and management of these disorders. Although the onset of many neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and attention-deficit hyperactivity disorder emerges mainly during infancy and adolescence, most of the behavioral studies in mice modeling neuropsychiatric phenotypes are performed in adult animals, possibly missing valuable phenotypic information related to the effect of synaptic maturation during development. Here, we examined which behavioral tests assessing both motor and coordination functions can be performed in mice at two different adolescent stages. As strain and sex affect mouse behavior, our experiments covered both male and female mice of three inbred wild-type strains, C57BL/6N, DBA/2, and FVB/N. Adolescent mice of both postnatal days (P)22–30 and P32–40 developmental stages were capable of mastering common motor and coordination tests. However, results differed significantly between strains and sexes. Moreover, the 10-day interval between the two tested cohorts uncovered a strong difference in the behavioral results, confirming the significant impact of maturation on behavioral patterns. Interestingly, the results of distinct behavioral experiments were directly correlated with the weight of mice, which may explain the lack of reproducibility of some behavioral results in genetically-modified mice. Our study paves the way for better reproducibility of behavioral tests by addressing the effect of the developmental stage, strain, sex, and weight of mice on achieving the face validity of neuropsychiatric disorder-associated motor dysfunctions. For instance, coordination and motor dysfunctions have been well documented in autism spectrum disorder (ASD)6, schizophrenia11, attention-deficit hyperactivity disorder (ADHD)14, and depression17 and have important implications for the etiology19, pathophysiology20, and management22 of these disorders. Moreover, they are considered established markers of illness episodes and severity19. These motor abnormalities vary greatly in different neuropsychiatric disorders. ASD patients are characterized by poor visual tracking, limited gross and fine motor functions when grasping or reaching for objects, inability to execute a sequence of actions (apraxia and difficulties in imitation), and postural balance difficulties24. Another class of motor-related alterations is observed in schizophrenia patients which include psychomotor slowing26, poor performance in complex motor tasks28, dystonia, akathisia, and hypokinesia29. On the other hand, 30% to 50% of children with ADHD show impaired gross motor and fine motor skills including difficulties in handwriting and clumsiness in performing motor skills, which can be attributed to their hyperactivity, impulsivity, and inattention31.For a long time, deficits in motor function in neuropsychiatric disorders have been traditionally neglected both in clinical practice and research despite their clinical and neurobiological relevance. Fortunately, they are now considered main diagnostic criteria of many neuropsychiatric disorders32. Thus, 50% of adults with neuropsychiatric disorders received a diagnosis before 15 years of age33. Motor abnormalities in ASD and ADHD specifically appear within the first year of life38, and it is known that neuromotor dysfunction in schizophrenia is even present before the onset of the disease and constitute an important indicator of it40, with poor coordination, clumsiness, and unfamiliar movements being the most common motor abnormalities among children later on developing schizophrenia41. However, behavioral studies modeling neuropsychiatric disorders in genetically-engineered mouse lines are mostly performed in adulthood taking the advantages of easier handling and the capabilities of mastering more complex behavioral and cognitive tasks. Unfortunately, these studies miss valuable information on the impact of synaptic maturation during development. Yet, our previous studies unraveled the capacity of adolescent mice in performing experiments assessing neuropsychiatric-like phenotypes including communication deficits42, anxiety, social impairment, and cognitive dysfunction43.The onset of several neuropsychiatric disorders emerges mainly during infancy and adolescence42 and home-cage activity43 are sensitive to a small developmental progress during adolescence, we performed our behavioral test battery on two cohorts of mice, one from P22 to P30 and the other from P32 to P40 in order to draw a full picture of the motor ability during adolescence. Because the genetic background and sexes are known to influence behavioral characteristics45, we performed our experiments on male and female mice of three different inbred strains, C57BL/6N, DBA/2, and FVB/N that are world-wide used in research and are the standard strains in neuroscience with their ability to discern the role of individual genes and the impact of allelic variation along with decreased variability49.In the present study, we aimed to investigate which behavioral tests recapitulating neuropsychiatric disorder-associated motor abnormalities can be performed in adolescent mice till P40. We have selected behavioral tests with high face validity that are commonly used to evaluate traits in sensory-motor functions. Our test battery included the grip strength, beam balance rod, inverted screen, cliff avoidance reaction, rotarod, and voluntary wheel running tests. As we have previously shown that behavioral results including ultrasonic vocalizations (USV)Our work revealed that the motor activity and coordination functions during adolescence differ among these mouse strains, highlighting the potential advantages and disadvantages of individual strains in behavioral studies.P = 0.007) and DBA/2 (P < 0.0001) mice  and FVB/N (P = 0.0003) mice  and the highest number of falls     with DBA/2 mice showing the least grip strength (P = 0.0003 vs. C57BL/6N) and C57BL/6N mice being intermediate  and DBA/2 (P = 0.021) mice     mice . These results indicate that the increased body weight of mice can increase the grip strength and thus, the latency to fall off a grid.To test whether the weight of mice can be a factor in determining the performance on various motor and coordination tests, we calculated the Pearson's correlation coefficient for each test in the behavioral test battery for the P32–40 cohort. Only the grip strength and inverted screen tests were positively correlated with the weight of mice , and cognitive tests . As motor abnormalities in neuropsychiatric disorders appear even before the onset of the core symptoms, it becomes mandatory to assess motor functions in young mice. Moreover, impaired motor functions can influence findings in other tasks, such as cognitive tests.Studying the behavior of genetically-modified rodents modeling neuropsychiatric disorders contributes to our understanding of the pathophysiology of these disorders and enhances the development of new therapeutics. Therefore, it is important to perform behavioral experiments during the developmental time window that matches the onset of symptoms in humans before or around puberty corresponding to P42 in miceWe here investigated which behavioral experiments assessing motor activity and coordination function can be performed during adolescence and established baseline levels of motor ability for three inbred wild-type mouse strains commonly used in biomedical and behavioral research. Our test battery covered the most frequently used assays including the grip strength, beam balance rod, inverted screen, cliff avoidance reaction, rotarod, and voluntary wheel running tests. To our knowledge, this is the first comprehensive study performed in young mice thoroughly exploring motor functions in different inbred strains. All three strains showed the ability to perform these tests in two developmental stages of adolescence. However, the three strains differed, with DBA/2 mice displaying weaker motor and coordination functions. These strain variations stress that the results of motor functions should be included to correctly interpret the outcome of other behavioral tests.53. Moreover, by performing several trials over multiple days, the rotarod test can unravel the motor learning ability that can indicate the degree of synaptic plasticity in various mouse strains during different developmental stages. Three studies comprehensively compared the performance of several inbred mouse strains on the rotarod. In the first study by Tarantino et al. (2000), four mouse strains, C57BL/6J, C3HeB/FeJ, DBA/2J, and 129/SvImJ, and aged around P60 were compared in an accelerating rotarod paradigm with three trials per day for two consecutive days54. Consistent with our finding, C57BL/6J mice stayed longer on the rotarod than DBA/2J mice. In the second study, the ability of eight inbred strains to perform on the rotarod was investigated with nine trials across three days55. In that study, C57BL/6J mice exhibited better performance on the rotarod compared to DBA/2J and FVB/NJ mice consistent with our findings55. When comparing the two previously mentioned studies together, DBA/2J mice performed better and showed increased motor learning in Tarantino et al.54 study than in McFadyen et al.55, which can be explained by the different protocols used in both studies. In the third study in young mice aged 5–6 weeks, again consistent with our results, C57BL/6J mice had the highest latencies for remaining on the rotarod compared to DBA/2J and FVB/NJ56. However, these results were in contrast to a previous study by the same group showing no difference between C57BL/6J, DBA/2J, and FVB/NJ mice57. On the other hand, motor and coordination functions differed between substrains. C57BL/6J mice exhibited significantly longer latencies than C57BL/6N and C57BL/6C mice58. Thus, care needs to be taken when comparing the results of studies in different mouse substrains. Notably, in our study, several mice made passive rotations i.e., mice held onto the bar and rotated for at least one revolution. If a mouse clings onto the rod in the rotarod test instead of running on top of it, no conclusions about its balance and coordination can be made60. Accordingly, we considered the first passive rotation of a mouse as the end of the trial since it can indicate the lost capability in the rotarod performance. As some laboratories consider the fall of the mouse off the rotarod as an endpoint, the different evaluation protocols will, at least partly, account for the lack of inter-laboratory reproducibility. Additionally, the apparatus itself including the diameter of the rod, material covering the rod, speed, and rate of acceleration of the rod can also impact the variability of behavioral results between laboratories62.The rotarod test is one of the oldest tests that has been extensively used to assess the innate motor ability in mice since 1950sSurprisingly, different coordination tests revealed intrastrain diverging results. For example, the P22–30 DBA/2 mice showed less coordination in the inverted screen and cliff avoidance tests but not the beam balance rod test. This implies that several behavioral tests assessing motor activity and coordination function need to be approached for a full characterization of genetically-engineered mice modeling neuropsychiatric disorders.66, 1.4:1 in schizophrenia68, 4:1 in attention-deficit hyperactivity disorder72 and 1:2 in depression78. Additionally, neuropsychiatric disorders exhibit sex differences in the age of onset, disease progression, and disease severity80. Females are known to reach puberty, the major feature of adolescence81, before males, which can have an impact on the behavior during this sensitive period. Various markers are used to determine puberty in mice including the vaginal opening, first vaginal cornification, onset of cyclicity in females and balanopreputial separation in males. These different markers make it difficult to determine the precise date of puberty in mice. Additionally, differences in the onset of puberty have been reported in different inbred strains, which add another layer of complexity in comparing the behavioral results of different strains. For instance, the vaginal opening in female DBA/2J and C57BL/6J mice occur, on average, at P25.78 and P26.45, respectively82. In contrast, the onset of cyclicity was earlier in female C57BL/6J (P48.67) than DBA/2J (P51.11) mice. For males, the occurrence of the balanopreputial separation was earlier in C57BL/6J (P29.71) than DBA/2J (P34) mice82. To this end, a rough dating of puberty in conventional preclinical models using laboratory wild-type and genetically modified mice is recommended for the accurate characterization of the behavioral outcome.Measuring motor function in both male and female mice is of particular relevance as the susceptibility to neuropsychiatric disorders differs between males and females with a ratio of 4:1 in ASD83. In P60–90 mice, female C57BL/6J mice had longer latency to fall off the rotarod than males55. In another study, female P30 C57BL/6J mice showed a trend towards increased duration on the rotarod, which reached significance at P15083, again confirming the effect of age on distinct behaviors. In contrast to C57BL/6N, DBA/2 mice in our study revealed no difference between males and females in both cohorts in all tests except the grip strength at P22. These results are consistent with a study showing no sex difference in DBA/2 mice in the rotarod test55 and with our previous study revealing no sex differences in DBA/2 mice in other behavioral tests related to neuropsychiatric disorders43. Furthermore, female FVB/N mice revealed more activity than males on the running wheel at P39, which is in line with our previous results showing a higher activity of P36 females in the home cage LABORAS test43. In summary, the sex-specific differences in motor activity and coordination function stress the necessity of testing both males and females in phenotyping mouse models of neuropsychiatric disorders.In our study, the C57BL/6N strain showed in the rotarod test the strongest difference between male and female mice with better performance by the latter. The difference being stronger in P26–27 than P36–37 mice may indicate an interrelation between age and sex. The better performance of female than male C57BL/6N mice is consistent with two previous findings in C57BL/6J mice tested at different ages84 but also reduced rotarod performance86, and altered locomotor activity87. Our results highlight the importance of small developmental windows and stress the importance of comparing mice of the same age since a few days’ differences can have a strong impact on the behavioral pattern.The 10-day interval between the two tested cohorts displayed strong differences in the behavioral outcome, mainly depending on the investigated strain and behavioral paradigm. Indeed, several studies revealed aging from young to middle age inducing not only increased body weight88. Consistent with a previous study performed on P30 and P150 C57BL/6J mice83, our analysis of male and female C57BL/6N mice revealed no statistically significant correlation between the body weight and rotarod performance. In contrast, a significant negative correlation was found in P60–90 C57BL/6J mice55. The discrepancy of the results in the aforementioned studies likely relies on age differences.The positive correlation between the weight of the mouse and performance on the grip strength and inverted screen tests needs to be taken into consideration when characterizing motor functions. Taking the weight into account can enhance the reproducibility of behavioral tests in genetically-modified mouse models, a main issue in the behavioral neuroscience field89, many automated methods with minimum handling of mice such as automated home-cage wheel-running90, CatWalk gait analysis91, and Erasmus ladder test92, are meanwhile replacing manual ones. These tests with a little interaction with mice will allow more precise characterization of motor function, coordination, and motor learning. However, careful attention to other confounding variables during testing, statistical analysis and interpretation remain important and require consideration. In addition to the experimenter and way of handling94, a large array of factors can impact the behavioral outcome of rodents. Some published examples are the cage type, density and its type of enrichment97, and even the cage position on the rack in the vivarium98 can significantly alter rodent behavior. Geographical location also can affected body weight, motor coordination, and motor learning capability of wild-type mice, which partially relied on the rodent diet and water quality83. Therefore, all these factors must be ruled out for approaching genuine differences in motor function and learning.As young mice are more active and hectic than adult mice, the animal-experimenter interaction can have an impact on the behavioral results. Because high standardization facilitates inter-laboratory reproducibility, increases efficiency, and reduces variabilityIn brief, differences in motor function as determined by performance in different motor and coordination tests uncovered the genetic difference as an important determinant in motor performance. Moreover, our study paves the way for better reproducibility of behavioral tests by addressing the effect of age during adolescence, sex, and weight on achieving the face validity of neuropsychiatric disorder-associated motor dysfunctions. Accordingly, behavioral characterization of knockout/knockin mice are recommended having littermates of the same delivery day, alike male/female ratios, and weight should be checked before and after every motor test since these three factors can strongly shift the results. Keeping these factors in mind will decrease variability, increase the face validity of the rodent model and concomitantly allow better defining “disease progression” and drug efficacy in motor abnormalities.43. All procedures were conducted in strict compliance with national and international guidelines for the Care and Use of Laboratory Animals. The experiments on mice were approved by the local government  and were carried out in compliance with the ARRIVE guidelines.Animals and housing conditions were similar to our previous studyAll behavioral tests were carried out during the daylight cycle. Mice were brought into the behavioral room half an hour before the behavioral testing. Behavioral tests started at 7 am. We analyzed the behavior in 2 cohorts of group-housed mice of both sexes with one cohort starting at P22 till P30 and the second starting at P32 till P40. The number of mice per cohort and the type of the behavioral experiments are listed Table . One malThe grip strength test was the first test to be applied to each cohort. One day later, we applied the beam balance rod test in the morning and the inverted screen test in the afternoon. Animals were maintained in the test room during this period. Then, we left the mice for two days without any experiments. At P26 and P36, we performed the rotarod test as one training trial permitting the mice to be habituated to the apparatus and to learn the task and two additional trials on the same day. On the following day, the data of three additional trials were collected. At P28 and P38, the cliff avoidance reaction test was performed. At P29 and P39, the voluntary wheel running test was performed for 24 h. The weights of male and female mice were measured before performing the tests and are shown in Supplementary Fig. After each trial, the apparatuses were carefully cleaned with 75% ethanol solution wetted tissue paper. All behavioral experiments were performed in a randomized manner with a separation of experiment conduction and data analysis.The strength of the forelimb, as well as the overall neuromuscular strength, were tested using a grip strength meter , which automatically measured the force (mN) needed for the mouse to release a grip after having to grasp and cling to it. During this task, the mouse was held by the tail. After an initial training session, the mice were tested for one session. A test session is consisting of three consecutive trials and the final result was calculated as the average of these three trials.The time during which the mouse was able to remain on a narrow, elevated rod without falling was measured. The apparatus is composed of 7 rods with diameters of 6, 8, 12, 15, 20, 25, and 32 mm, respectively. The length of each rod was 50 cm with a height of 20 cm above the ground. The mouse was put on each rod for 10 s starting from the widest to the narrowest. The score was calculated by the number of rods from which the tested mouse did not fall. If the mouse did not fall, the score was considered 8. The test was performed twice with 1 h interval.Muscle strength using all four limbs was tested in a modified version of the inverted screen test. The mouse was placed in the center of a metal grid screen. The grip screen is then carefully rotated to an inverted position and held steadily 45 cm above an empty cage containing bedding. The latency to fall off the grid was recorded with a maximum trial time of 5 min.The accelerated rotarod  test is a standard sensory-motor test to investigate animals’ motor coordination and learning skills by measuring the ability of the mouse to stay and run on the accelerated rod. The test was performed for a duration of 8 min with an acceleration of 4–40 rpm. As soon as a mouse fell off the rod or started to rotate with the rotarod without running, the time was stopped. After an initial training trial, mice were tested for five trials over two days. The recovery phase between trials was 10 min.99. The floor below the platform was carpeted with smooth tissue to prevent injury if the animal fell. The test was initiated by gently placing the animal on the middle of the platform. The latency from an initial placement on the platform until falling was recorded. Mice that fell off the platform were immediately put back on the platform and the total number of falls during the entire session (60 min) was counted.The cliff avoidance reaction assesses coordination and maladaptive impulsive behavior. It was assessed using a round plastic platform  supported by a rod (height 50 cm) similar to a bar stool100. Mice were placed individually in cages containing a running wheel and free access to food and water. The unrestricted voluntary wheel running activity started at 7–8 am and was digitally recorded for 24 h using the AWM counter  that uses an optical sensor to detect the total revolutions of the wheel and is connected to a USB Interface and PC running an AWM Software .The voluntary wheel running test was modified fromP value ≤ 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7 and Microsoft Office Excel software. The respective numbers of male and female mice are described in Table Two-way ANOVA was used with sex and genotype as the two factors. This was followed by Tukey’s post hoc test for multiple comparisons to determine differences between the three strains C57BL/6N, DBA/2, and FVB/N and Bonferroni correction to check differences between males and females within each strain. To compare the two developmental stages (P22–30) and (P32–40) within each strain, two-way ANOVA was used with sex and age as the two factors. Additionally, a correlation analysis between the weight of P32–40 mice and their performance on the tests was done and is discussed in the results section. All data were expressed as mean ± SEM. A Supplementary Figures and Tables


Few data were available on smoking and smokeless tobacco use in South Asian migrants in the United Arab Emirates (UAE). This study aimed to identify the prevalence and correlates of cigarette smoking and smokeless tobacco use in male South Asian migrants in the UAE.n = 433), Pakistani (n = 383) and Bangladeshi (n = 559) nationalities. We used multivariable logistic regression analysis to identify significant correlates of cigarettes smoking and smokeless tobacco use.We used a cross-sectional study to recruit a random representative sample of male South Asian migrants, including Indian  with a mean age of 34 years (SD ± 10). The overall prevalence of cigarette smoking was 28% (95%CI 25–30%) and smokeless tobacco use was 11% (95%CI 10–13%). The prevalence of current cigarette smoking was 21, 23, and 37% among participants from India, Pakistan and Bangladesh, respectively. The prevalence of current smokeless tobacco use was 6, 12, and 16% for Indian, Pakistani, and Bangladeshi participants, respectively. Among study participants, Bangladeshi nationality, hypertension, and alcohol use were significant correlates of current cigarette smoking. Significant correlates of smokeless tobacco use included increased age, less than college level education, alcohol use, and Pakistani or Bangladeshi nationality.Current smoking and smokeless tobacco use in South Asian migrants represent a significant public health burden in the UAE. Effective public health measures are needed to reduce tobacco use in this migrant population. Tobacco use is the single most preventable cause of death in the world today leading to over 6 million fatalities each year . More thTobacco use is a major risk factor for cardiovascular disease (CVD), cancer, and a wide range of chronic diseases especially chronic respiratory diseases , 3. ManuSouth Asians, estimated at 25% of the world’s population, represent one of the highest proportions of the global population, affected by current and future disease burden due to tobacco consumption. A high prevalence of tobacco use in this region has been reported, with 40% of males and 5% of females in India, 45% of males and 6% of females in Pakistan, and an overall tobacco use prevalence of 21%. Bangladesh has one of the highest reported prevalence of tobacco use with 58% among males and 29% in females, with an overall tobacco use of 43% .Cigarette smoking is also associated with a significant loss of work productivity. Prospective cohort studies have shown that compared with non-smokers, current smokers had a 33% increase in the risk of absenteeism and were absent for an average of 2.7 more days per year compared to non-smokers . The totThe United Arab Emirates (UAE) has one of the largest proportions of a culturally diverse migrant population. Expatriate workers account for almost 80% of the UAE’s total population and about two-thirds are South Asian migrant workers from India, Pakistan, and Bangladesh . ResearcThis study aims to estimate the prevalence and correlates of cigarette smoking and smokeless tobacco use among South Asian migrants in the UAE who originated from India, Pakistan, and Bangladesh.The study used a cross-sectional design. Ethical approval was obtained from the Al Ain Medical District Human Research Ethics Committee (AAMDHREC 10/21), and the Abu Dhabi Health Services Company’s Research Committee (08 February 2012). Study participants signed a written informed consent.zzα2 p (1- p)/d2] to estimate the sample size for our study, where n is the sample size; zα is the normal deviate (1.96) at 5% level of significance. The estimated prevalence (P) of cigarette smoking , the precision of estimate (5%), and decided to recruit 1800 participants.The target study population consisted of all male South Asian migrant workers aged 18 years and older from India, Pakistan, and Bangladesh. We used the formula for binomial distribution [n = The Abu Dhabi Health Services Company or SEHA  provides health care in the Emirate of Abu Dhabi, through hospitals, clinics, and ambulatory health care centers. Ten of the ambulatory health care centers are disease prevention and screening centers, primarily for infectious disease screening required for residency visas. All expatriate workers seeking employment or renewing their visa in the UAE are required by law to undergo health and communicable disease screening at one of the screening centers. The sampling frame in our study was a list of all expatriate workers from India, Pakistan, and Bangladesh who were enrolled for medical examination at the only visa screening center in the city of Al Ain, Abu Dhabi during the process of obtaining a new or renewing their visa in 2012 –17.The sampling frame included male adult (age ≥ 18 years) South Asian migrant workers from India, Pakistan and Bangladesh, attending visa screening in Al Ain, Abu Dhabi. Briefly, study participants had to self-identify with a South Asian ethnicity of India, Pakistan, or Bangladesh, be an adult aged ≥18 years, and be able to speak and /or read Urdu, Hindi or Bengali, and had to provide a written informed consent to be eligible to participate in the study. Due to the low literacy rate among the South Asian expatriate population in the UAE, the study questionnaire was interviewer-administered and all interviews were conducted in Urdu, Hindi, or Bengali, led by a native Urdu, Hindi, or Bengali speaking research assistant who had received appropriate training. Every third person on the list of eligible participants registered for the visa screening was invited to participant in the study and it took six months to recruit the study sample.“have you ever smoked cigarettes, cigars /biddies/or shisha?”We used an adapted version of the World Health Organization (WHO) global standard “STEPS” survey questionnaire titled “Chronic Diseases Prevention in Immigrants: putting CVD risk factors on surveillance screen” for population-based assessment of the prevalence of noncommunicable diseases (NCDs) risk factors including cigarette smoking and use of smokeless tobacco . Moreovep = 0.135) [We used an interviewer-administered survey because of the low literacy in South Asian migrants in the UAE. Studies have shown a higher sensitivity (5.2%) with interviewer administered self-reports of tobacco smoking than that of self-administered survey reports, but this difference was not significantly different from zero (= 0.135) .We collected information on demographics lifestyle factors, family and personal disease history, home country residence setting , occupation, monthly salary , years lived in the UAE, current type of accommodation, history of current and past cigarette smoking and tobacco chewing, history of exposure to second-hand tobacco smoke, and alcohol consumption.’ weight and height measurements were performed using standard weight and height scales  and procedures. Body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters. We used BMI categories based on World Health Organization recommendations, being overweight (BMI 25.00 to 29.99 kg/m2), obesity class I (BMI 30.00–34.99 kg/m2), obesity class II (BMI 35.00–39.99 kg/m2), and obesity class III (≥ 40.00 kg/m2) [Participants0 kg/m2) . In the 0 kg/m2) .p-value ≤0.05  and the corresponding 95% confidence interval (CI).Data were entered into Microsoft Access and then imported into Stata version 11.0  for analysis. Demographic characteristics and CVD risk factors were evaluated in association with both smoking cigarettes and/or smokeless tobacco use. Continuous and categorical variables were assessed using t-test and/or chi-square tests, respectively. Univariate and multi-variate adjusted logistic regression models were used to examine the association of different demographic and clinical parameters with cigarette smoking and/or smokeless tobacco use. Being a current cigarette smoker and/or being a current smokeless tobacco user were the outcomes of interest in our logistic regression models. Statistical significance was assessed based on a Out of 1800 eligible males, 1375 (76%) participated in our study . In Table The overall prevalence of cigarette smoking was 28% (95%CI 25–30%) and overall smokeless tobacco use was 11% (95%CI 10–13%) among South Asian migrants. The prevalence of cigarettes smoking was 21% (95%CI 17–25%), 23% (95%CI 19–27%), and 37% (95%CI 33–41%) for participants from India, Pakistan, and Bangladesh, while the prevalence of smokeless tobacco use was 6% (95%CI 4–9%), 12% (95%CI 9–15%), and 16% (95%CI 13–19%) for the three nationalities, respectively. Of the total current smokeless tobacco users, the majority were chewing tobacco including naswar , or using paan masala (65%), followed by zarda (14%), betel nut (12%), mawa (7%), and Gutka (3%). Combined tobacco use was relatively low and was found to be 2, 3, and 8% for India, Pakistani, and Bangladeshi participants, respectively. The mean (±SD) age of smoking initiation in our population was 21 (±7) years of age. A total of 4% of South Asian migrants initiated smoking below the age of 10 years, while 8, 20, and 68% initiated smoking between the ages of 11–14, 15–18, and 18+ years, respectively. Migrants smoked an average of 9 (±6) cigarettes per day. A total of 44% of migrants smoked fewer than 6 cigarettes per day, while 32% of migrants reported smoking between 6 and 10 cigarettes and 24% smoked 11 or more cigarettes per day.In Table Odds ratios and 95% confidence intervals for the association of different parameters with cigarette smoking are presented in Table To our knowledge, this is the first study to examine the prevalence of tobacco use amongst South Asian migrants living in the UAE; and report migrant smoking prevalence in the Gulf. We found a relatively high prevalence of cigarette smoking among Indian and Pakistani migrants and an even higher prevalence among Bangladeshi migrants. We found strong and significant associations between alcohol consumption and cigarette smoking and smokeless tobacco use in this migrant population. Furthermore, despite this population being relatively young, we found a clustering of CVD risk factors among smokers albeit not statistically significant.A study from India based on the Global Adult Tobacco Survey (GATS) reported a prevalence of cigarette smoking of 15% and smokeless tobacco use of 28% among males aged 25–44 years . AnotherSouth Asian migrants are also living in many other countries of Europe and in the United States. The United Kingdom, home to a large South Asian migrant population from India, Pakistan, and Bangladesh, has reported figures for smoking by country of origin over time. Cigarette smoking prevalence among Indians, Pakistanis, and Bangladeshis living in the UK was reported as 20, 29, and 40%, respectively. Smokeless tobacco use among the same migrant population in the UK was 4, 2, and 9%, respectively . TobaccoThe prevalence of cigarette smoking in our study showed an inverse association with age. On the contrary, a positive association with age was observed for smokeless tobacco use. The association between smoking prevalence and age is usually attributed to a “cohort effect”; namely, the higher prevalence of cigarette smoking in younger age groups may be associated with current societal trends in the country of residence (UAE) as opposed to smokeless tobacco use, which may represent a stronger habitual effect among older migrants being influenced in a more significant fashion from their country of origin. We also found an inverse association between educational level and tobacco use both for cigarette smoking and smokeless tobacco. This observation may be associated with a lack of awareness about health risks among those with lower educational attainment although this explanation has recently been questioned .The average age of smoking initiation was 21 years in our study, which may suggest that migrants, especially from rural areas in their home countries, might have initiated smoking during migration at an older age, given the different age of smoking initiation seen among different ethnic groups. Other studies have also reported on the different age of smoking initiation among migrant populations . AggressIt is well known that migrants are “selected” and therefore differ from their home population . UsuallyStrengths of this study include the large representative sample and the use of standardized questions to classify respondents as cigarette smokers or users of smokeless tobacco. There are some limitations in the estimates presented in this study. First, from the adapted WHO STEPS questionnaire used in this study, we only had estimates of current smoking and average number of cigarettes smoked during the last 24 h. Secondly, we were not able to assess the use of tobacco products prior and after migration, which would have provided useful information on the impact of migration. This could be important since a high proportion of migrants live in shared accommodation and therefore, may be influenced by peer pressure and behavior. Our study is also limited to male migrants. Because this was a cross-sectional study, we could not draw any conclusions on potential causal associations between cigarette smoking and the demographic, socio-economic, and CVD risk factor variables examined. Finally, it is possible that tobacco use based on self-reported questionnaires may be under-estimated and we did not validate self-reports with other objective measures of smoking such as cotinine levels. Studies have shown trends of underestimation when smoking is based on self-report and there have been varying sensitivity levels depending on the population studied. On average, the sensitivity was 88% and specificity was 89% . SensitiAlthough the overall prevalence of tobacco smoking in the UAE is lower than many other countries in the Middle East, this hides important variations – especially the very high rates seen in South Asian migrants, and particularly in Bangladeshis. Concerted public health action is required to develop tobacco prevention and control strategies for this specific population group, which accounts for the largest proportion of the expatriate population. Being the first study of tobacco use amongst South Asian migrants in the Gulf, it provides important population-based data on the prevalence of tobacco use. There is an alarmingly high level of cigarette smoking and relatively large burden of smokeless tobacco use in these migrants originating from India, Pakistan, and Bangladesh. Given the large number of South Asian migrants in the UAE and Gulf countries, smoking cessation and prevention programs should be a public health priority for the Ministries of Health in the region. In addition, the strong association of cigarette smoking and smokeless tobacco use with alcohol drinking calls for concerted efforts in addressing multiple unhealthful behaviors with comprehensive preventive programs among migrants in this region.Additional file 1. Our supplementary file is our study questionnaire titled “Chronic Diseases Prevention in Immigrants: putting CVD risk factors on surveillance screen” as has been cited in page 5, under the sub-heading, Measures of the manuscript.


Radiofrequency ablation (RFA) is recommended to prevent potential neurological injury or intrauterine foetal death (IUFD) of the co-twin(s) in complicated monochorionic (MC) pregnancies. However, the impacts of various indications on the pregnancy outcome following RFA remain unclear. This study aimed to determine how the indications influence the perinatal outcomes in complicated MC pregnancies undergoing radiofrequency ablation.This was a retrospective cohort study performed in a single centre. All consecutive MC pregnancies treated with RFA between July 2011 and July 2019 were included. The adverse perinatal outcomes and the survival rate were analysed based on various indications. The continuous variables with and without normal distribution were compared between the groups using Student’s t-test and Mann–Whitney U test, respectively, and for categorical variables, Chi-square and Fisher’s exact tests were used. P < 0.05 indicated a significant difference.We performed 272 RFA procedures in 268 complicated MC pregnancies, including 60 selective intrauterine growth restriction (sIUGR), 64 twin–twin transfusion syndrome (TTTS), 12 twin reversed arterial perfusion sequence (TRAPs), 66 foetal anomaly and 66 elective foetal reduction (EFR) cases. The overall survival rate of the co-twin was 201/272 (73.9%). The overall technical successful rate was determined at 201/263 (76.7%). The IUFD rate in the co-twin was 20/272 (7.4%). The TTTS group had recorded the lowest survival rate , and the survival rate was significantly correlated with Quintero stages (P = 0.029). Moreover, the sIUGR III subgroup had a lower survival rate compared with sIUGR II . The subgroup of foetal anomaly of gastroschisis or exomphalos had the highest IUFD rate , followed by sIUGR III  and dichorionic triamniotic (DCTA) subgroup . In EFR group, eight IUFD cases were all coming from the DCTA subgroup and received RFA before 17 weeks.The perinatal outcome of RFA was correlated with the indications, with the lowest survival rate in TTTS IV and the highest IUFD incidence in abdominal wall defect followed by sIUGR III. Elective RFA after 17 weeks may prevent IUFD in DCTA pregnancies. Around one-third of the monochorionic (MC) multiple pregnancies suffer from specific complications such as twin–twin transfusion syndrome (TTTS), selective intrauterine growth restriction (sIUGR), twin reversed arterial perfusion sequence (TRAPs) and twin anaemia polycythaemia sequence (TAPS) , 2. For UCO techniques include laser cord coagulation , cord liThe primary indications for RFA are TRAPs and other lethal foetal anomalies . After RHowever, the effects of these indications on the pregnancy outcome following RFA remain undefined. Therefore, by examining a large cohort of complicated MC pregnancies undergoing RFA, we determined how the various indications influenced the perinatal outcome after RFA treatment.This retrospective cohort study enrolled all cases of consecutive MC pregnancies treated with RFA in a single tertiary centre between July 2011 and July 2019. The indications for RFA were as follows: sIUGR II and III ; TTTS stPatients underwent a comprehensive ultrasound examination to determine the chorionicity status, placental position, malformations, amniotic fluid’s deepest vertical pocket, cervical length, umbilical artery blood flow and the middle cerebral artery peak systolic velocity (MCA–PSV). Patients and their families received detailed counselling regarding the risks of RFA, including co-twin demise, miscarriage, preterm labour and neurological or thermal injury to the surviving foetus. Written consent forms were signed by patients.All RFA procedures were performed under local anaesthesia, and either supine or lateral position was used depending on the convenience of access to the targeted vessel. Under ultrasound guidance, a 17-gauge Sarburst radiofrequency needle  was inserted to get near to the abdominal segment of the foetal umbilical vessel, trying to avoid the placenta and amniotic sac of the retained foetus(es). The accuracy of the ablation area is essential for the success of the RFA procedure. If the umbilical vessels could not be surrounded by the tines, the position of the needle was adjusted.Three electrodes were ejected to surround the cord vessels after the tine position was confirmed. Radiofrequency energy was applied for 3 min at 150 watts to generate the target temperature of 100 °C–110 °C. If the three electrode tines did not exhibit a synchronous temperature increase (difference: >30 °C), they were retracted, and the position was then adjusted. During and after each ablation cycle, the blood flow in the targeted vessels was observed using the colour Doppler until the blood flow signal was completely absent. The heart rate of the targeted foetus was then monitored until it decreased to < 60 bpm. However, the disappearance of the blood flow signal in the umbilical vessels does not always indicate a complete blockage; it may be merely too weak to be detected after a reflective decrease of the heart rate and output or spasm of the vessel. If the heartbeat stopped earlier or the decrease of the heartbeat was slower than expected, another ablation cycle was planned.An ultrasound examination was performed 24 h after the procedure to evaluate the surviving foetus, the systolic/diastolic pressure of the umbilical artery and MCA–PSV and cervical length. Thereafter, further ultrasound scan was performed every 2 weeks. MRI was also performed at 24–28 weeks to detect any foetal brain damage. Subsequently, the patients were allowed to consult and deliver in local hospitals.The following data of all the consecutive patients who underwent RFA were retrieved from our database: maternal age, indications for foetal reduction, gestational age at procedure, postprocedural complications, subsequent ultrasound scan and foetal brain MRI results. Furthermore, the following perinatal outcomes were also recorded: IUFD of the co-twin, PPROM within 2 weeks after the RFA, termination of pregnancy (TOP) and preterm delivery < 28 weeks, gestational age at delivery and birth weight and perinatal mortality. In cases delivered elsewhere, we gathered the delivery and neonatal information, and a neonatologist evaluated the condition of the newborns according to the Gessel Developmental Schedule , both viPatients were divided into five groups based on their conditions, namely, sIUGR, TTTS, TRAPs, MTFDA and EFR. They were further split into subgroups according to the type of sIUGR, Quintero stage of TTTS, position of foetal anomaly and the chorionicity of EFR. The incidence rate was calculated per procedure because some patients had undergone two procedures. The survival rate was calculated when the co-twin(s) were delivered after 28 weeks and the newborn was alive at 28 days. Technical successful rate was calculated to exclude those TOP cases after RFA because of foetal anomaly or other reasons and those who gave up further treatment on the premature newborns. Take-baby-home rate per patient was also calculated because most triplet pregnancies receiving RFA were successful despite the co-twin demise.t-test and Mann–Whitney U test, respectively; for categorical variables, we used the chi-square and Fisher’s exact tests. Differences were considered significant when P < 0.05.Data were analysed using the SPSS version 25.0 software (IBM SPSS Statistics). Continuous variables are presented as mean and standard deviation, whereas categorical variables are expressed as absolute numbers and percentages. The continuous variables with and without normal distribution were compared between the groups by Student’s P < 0.001). The EFR group achieved the best pregnancy outcome followed by the sIUGR group, with the same co-twin survival rate (80.0%) and a higher take-baby-home rate in the EFR group (85.1%). The overall survival rate of the co-twin was determined to be 201/272 (73.9%). The median gestational age at delivery was 36.3 ± 2.9 weeks, with an average birth weight of 2662.5 ± 707.8 g. After the exclusion of those who opted for TOP (n = 7) and who gave up on postnatal treatment (n = 2), the overall technical successful rate was 201/263 (76.4%).Table n = 3) and tricuspid insufficiency (n = 2) in the TTTS group and cleft palate in the MTDFA group (n = 1) and of maternal complication (n = 1) in the TRAPs group. The highest IUFD rate was found in the EFR group , followed by the MTDFA group , whereas the lowest IUFD rate was found in the TRAPs group , followed by the sIUGR group . Meanwhile, the lowest survival rate of 37/64 (57.8%) was recorded in the TTTS group, considering the three IUFD (4.7%) and five PROM (7.8%) cases within 2 weeks after the procedure, five TOP cases for tricuspid insufficiency and severe hydrops foetalis and the highest miscarriage rate.IUFD of the co-twin occurred in 20/272 (7.4%) procedures of the total population and 17/20 (85.0%) procedures within 2 days after RFA. The incidence of premature rupture of membranes (PROM) within 2 weeks after RFA was at 14/272 (5.1%). Elective TOP was preferred by parents for dysplasia in the co-twin including severe oedema (P = 0.029). The technical successful rate in sIUGR III  was lower than that in sIUGR II , but the difference was not significant, probably owing to the small sample size of the sIUGR III subgroup (P = 0.069). The highest IUFD rate  was found in the subgroup of foetal anomaly in the abdominal wall , followed by the subgroups of sIUGR III  and dichorionic triamniotic (DCTA) for EFR .The pregnancy outcomes stratified by the types of sIUGR, Quintero stages of TTTS, position of foetal anomaly in the MTDFA group and chorionicity of EFR are presented in Table P = 0.607). The eight IUFD cases were all from the DCTA subgroup and received RFA before 17 weeks; for those who received RFA after 17 weeks (n = 19), they all ended with desirable outcomes regardless of the chorionicity , and 5 MCDA twin pregnancies choosing foetal reduction. With no IUFD after RFA, the pregnancy outcome in the MCTA subgroup was found to be slightly better than that in the DCTA subgroup . Treatment of the two preterm newborns delivered at 27This study investigated the perinatal outcome of 268 MC pregnancies undergoing 272 RFA procedures, and the overall survival rate was 73.9% in the co-twins. Postprocedural complications of RFA include thermal injuries , 8, IUFDThe perinatal outcome was found to be significantly different with respect to various indications. Regarding the survival rate, the lowest was recorded in the TTTS group, whereas the highest was in the EFR and sIUGR groups, conforming to previous studies , 19, 25.The perinatal outcomes were found to be significantly correlated with the Quintero stages of TTTS, which was worst in TTTS IV, having higher incidences of polyhydramnios and miscarriage, and selective termination for severe tricuspid insufficiency or oedema in the retained foetus. The sIUGR II subgroup was able to obtain a better survival rate than the sIUGR III subgroup, probably contributed by the higher incidence of IUFD in the subgroup sIUGR III, which involves larger artery–artery anastomosis , 25, allAdditionally, two subgroups had high IUFD rates. As for the high rate of 40% in the subgroup of foetal anomaly in the anterior abdominal wall , with the proximity of the target vessel and the heart of the targeted twin, we speculate that the disappearance of the cord blood flow during RFA might be the consequence of the damage to the heart caused by ablation energy rather than a complete blockage of the blood flow, and subsequent exsanguination resulted in the co-twin demise. This finding provided an evidence to consider other UCO techniques in similar situations. Furthermore, an unexpected higher rate of 8/46 was found in the DCTA subgroup, with all IUFD cases occurred in those performed before 17 weeks, wherein technical difficulty was worsened by the small size of the targeted foetus. Meanwhile, IUFD did not occur in the MCTA subgroup with the same gestational age, probably because of the flexible option in any of the three foetuses, making the process technically simpler than in DCTA cases wherein only two of the three foetuses could be targeted. Technical difficulty and subsequent IUFD in the DCTA subgroup could be partially avoided by performing the procedure after 17 weeks.The accurate mechanism of the co-twin demise remains unclear. Subsequent exsanguination caused by an incomplete blockage in the targeted vessel after ablation might be a reason . In thisThe incidence of brain damage and neurodevelopmental impairment in the reserved co-twin was reported to be 2/103 in a systematic review  and 5/74The limitation of this study lies in its retrospective nature. The initial case selection such as selective avoidance of sIUGR III and TTTS IV, leading to the small sample size in certain subgroups, may result in statistical bias. Moreover, some surgical details, such as placental penetration, entry of a twin sac and whether the reduced twin was the presenting twin, were missed. Another shortcoming is owing to the distance from the patients’ home to our hospital, most patients were followed up and delivered at local hospitals where viable preterm newborns had to be given up and most of the neurodevelopmental follow-up was merely assessed via telephone interview. Long-term follow-up with a standard cognitive and developmental scale is necessary.Compared to previous publications, this is the largest cohort study regarding the perinatal outcomes of RFA stratified by all possible indications. Furthermore, all of the procedures were conducted by one experienced operator and the same sonographer, thereby maximally decreasing the influence of the technique and measurement bias. In conclusion, the pregnancy outcome after RFA was found to be correlated to the indication, with the lowest survival rate in TTTS IV cases and the highest IUFD incidence in foetal anomaly cases affecting the anterior abdominal wall, followed by the sIUGR III cases. Selective RFA after 17 weeks may help prevent IUFD in DCTA pregnancies.


Oral Tongue Squamous Cell Carcinoma (OTSCC), a distinct sub-group of head and neck cancers, is characteristically aggressive in nature with a higher incidence of recurrence and metastasis. Recent advances in therapeutics have not improved patient survival. The phenomenon of occult node metastasis, even among the purportedly good prognosis group of early-stage and node-negative tongue tumors, leads to a high incidence of locoregional failure in OTSCC which needs to be addressed. In the current study, transcriptome analysis of OTSCC patients identified the key genes and deregulated pathways. A panel of 26 marker genes was shortlisted and validated using real-time PCR in a prospective cohort of 100 patients. The gene expression was correlated with clinicopathological features including occult node metastasis, survival, and therapeutic outcome. The up-regulation of a panel of 6 genes namely, matrix metalloproteinase 9 (MMP9), Laminin subunit Gamma 2 (LAMC2), Desmoglein 2 (DSG2), Plasminogen Activator Urokinase (PLAU), Forkhead Box M1 (FOXM1), and Myosin 1B (MYO1B) was associated with failure of treatment in the early stage . Up-regulation of Tenacin C (TNC) and Podoplanin (PDPN) was significantly correlated with occult node positivity. Immunohistochemical analysis of LAMC2, MMP9, and E-Cadherin (ECAD) confirmed these markers to be indicators of poor prognosis. We propose this panel of valuable prognostic markers can be clinically useful to identify poor prognosis and occult node metastasis in OTSCC patients. There is a sharp increase in the incidence of tongue cancers not only in India1 but also in young white women2 and in Nordic countries3. Striking aspect of OTSCC epidemiology is the increase in incidence of OTSCC among young patients5 and women below 44 years of age7.Oral Tongue Squamous Cell carcinoma (OTSCC) represents a major portion of oral cavity cancers, especially in India. It is now evident that OTSCC needs to be studied as a separate entity and not be included in the broad scheme of oral cancers, as often reported—an approach that increases the power of the study but diminishes the credibility of the inference made from such studies. As per the data from National Cancer Registry Program  was done to identify the differentially expressed genes (DEG), in a set of OTSCC samples with validations by qPCR and Immunohistochemistry to derive salient markers for prognostication of OTSCC patients in a prospective study cohort.Transcriptome sequencing (RNA-seq) as a platform of analysis not only has a better sensitivity and dynamic range than traditional microarrays, but also enables identification of novel transcripts and more exhaustive study of biological pathwaysP value < 0.05) in tongue carcinoma samples. The quality of the results depends on the number of genes analyzed, which in turn is based on cut-offs and statistical significance. Hence for further functional analyses, we filtered only highly significant 2073 DEGs  with P value < 0.01 and Fold Change > 10. The heatmap representing the expression of top 100 DEG in OTSCC was derived from results obtained by transcriptome sequencing  studied by transcriptome sequencing is shown in Supplementary Table The top 15 over expressed and under expressed genes are listed in Table To understand the biological roles of the DEG from OTSCC, we performed a gene set enrichment analysis on the DEG derived by transcriptome sequencing. We found GO terms for biological process enriched for Inflammatory response, Extracellular Matrix organization, Cell adhesion, Collagen Catabolic Process, Immune response and Angiogenesis. The top most significant enriched GO term for Cellular components were related to extracellular matrix remodeling. The top most significant enriched GO term for Molecular Function were Metalloendopeptidase activity, Heparin binding, Extracellular matrix structural constituent, Collagen binding and Receptor activity. The chief deregulated KEGG pathways included Cytokine-cytokine receptor interaction, Focal adhesion, ECM-receptor interaction and PI3K-Akt signaling pathways. The GO enrichment and KEGG pathway for differentially expressed genes (DEG) is shown in Supplementary Figure P value < 1.0e−16. The top 10 nodes are presented in Supplementary Table The PPI network generated in STRING using the 1698 DEGs included 1392 nodes and 2516 edges with a PPI enrichment Biomarkers for a qPCR-based validation in prospective OTSCC patients n = 100) were chosen based on interrelationships assessed by Pearson correlation with respect to the genes involved in ECM remodeling and EMT. The markers chosen were LAMC2, TNC, PDPN, SPP1, PLAU, PLAUR, MMP9, FOXM1, VIM, CTNNB1, GLUT1, HIF1A, SOX2, OCT 4, VEGF, MYO1B, RBP1, SVN, CA9, DSG2, SURVIVIN, S100, TWIST2, CCND1, ECAD, POSTN. Figure 00 were cP value = 3.16E−08), PLAU , PDPN , SPP1  and PLAU  while SPP1 correlated strongly to UPAR  and PLAU. MMP9 expression was positively related to FOXM1  and TNC . VIM expression was strongly associated with CTNNB1  while DSG2 was similarly correlated to TNC  and S100 to GLUT1 . TNC was positively related to PDPN , UPAR , HIF1A , PLAU  and MYO1B; PDPN to PLAU , UPAR and MYO1B. The hypoxia marker HIF1A was correlated to the expression of SOX2, PLAU , FOXM1 , MYO1B , SVN  and GLUT1. TWIST2 was positively linked to SOX2 , OCT4  and MYO1B ; SOX2 strongly linked to OCT4  and OCT4 to VEGF . Similarly, CA9 correlated with RBP1 , PLAU  and GLUT1 , OCT4 to VEGF  and SVN; RBP1 to PLAU , FOXM1 , SVN  and GLUT1 ; PLAU to FOXM1 , MYO1B, SVN  and GLUT1  and FOXM1 correlated to SVN  and GLUT1 . Moderately strong to weak significant correlations was observed among the biomarker expression in the tongue tumors and listed in Table The interrelationships derived is shown as follows. LAMC2 expression was most strongly correlated to TNC  is shown in Supplementary Table P value = 0.02), LAMC2 , DSG2 , PLAU , FOXM1  and MYO1B  to be associated with failure of treatment in the early stage patients  cases and 77.5% (31) cases showed NED. Among the panel of 19 biomarkers studied, we identified a panel of 6 genes showing overexpression namely, MMP9  and PDPN  significantly indicated occult node positive status while the overexpression of LAMC2 , VIM , TWIST2  and SOX2  were the significantly associated with the cervical lymph node involvement.Overexpression of TNC , VIM , HIF1A , TWIST2  and SOX2  were found to be significantly differentially expressed among the early and advanced stage tumors. The differential expression of potential markers between early versus advanced staged OTSCC has been shown , MMP9  and ECAD expression at ITF  had a significant correlation with the treatment outcome. LAMC2  and MMP9 expression , also correlated with the pattern of failure. Altered ECAD expression at ITF and MMP9 overexpression denoted nodal positivity and poor outcome in OTSCC. The pattern of ECAD expression at ITF  and over-expression of MMP9  correlated with the cervical node status. MMP9 also correlated with the locally advanced stage of tumor  with higher MMP9 expression seen in 84.4% of all locally advanced tumors.As shown by the qPCR-based mRNA expression studies, the immunohistochemical analysis also confirmed that LAMC2 , alcohol (P = 0.04), clinical factors like node status (P = 0.003), stage (P = 0.01) and occult node status (P = 0.005) were found to be significant predictors of disease recurrence in OTSCC. Tobacco chewing, positive nodal status and advanced stage of tumor increased the hazard of tumor recurrence by more than two-fold while a positive occult node increased the risk of recurrence by 4.27-fold. The same clinico-pathological parameters were also significant predictors of death. Tobacco chewing (P = 0.02) and alcohol (P = 0.01) were associated with an increased hazard of death as seen by the HR of more than 2. Similarly, positive cervical node status (P = 0.008), advanced clinical stage (P = 0.01) and grade (P = 0.04) of the tumor were also significant predictors of death. Occult node positivity increased the risk of death by 5.19-fold and was the most significant clinic-pathological parameter among all factors.  Kaplan–Meier survival analysis also confirmed the parameters clinical stage, node, presence of perineural invasion and occult node status to be predictors of disease recurrence and death  and an increased risk of death (P = 0.006). Loss of membrane positivity of E-cadherin was associated with an increased hazard of disease-recurrence as seen by the increased HR of 3.19 for DFS (P = 0.001), when compared to no E-cadherin expression. It was also a significant predictor of death (P = 0.003) with a HR of 3.11. High MMP9 expression was associated with an increased risk of both disease recurrence (P = 0.002) and death (P = 0.004). These results were also mirrored by the Kaplan–Meier survival analysis of factors predicting DFS and OS  and death (P = 0.03) with an increased HR of 25.39 and 10.81 respectively. These results were confirmed by the Kaplan–Meier survival analysis  components playing a crucial role in patient prognosis. Based on the data obtained from RNA-seq, the top up regulated genes included members of Matrix metalloproteinases and Melanoma associated gene families. The Gene Ontology terms enriched among the DEGs included inflammatory response, ECM organization, cell adhesion, collagen catabolic process and metalloendopeptidase activity. Studies have shown that biomarkers of stromal microenvironment might have a greater impact on prognosis compared to biomarkers related to tumor cells23. We found ECM receptor interaction and Focal adhesion as the most significant deregulated pathways in OTSCC. However, biomarkers related to tumor microenvironment have not been widely studied except activin A24. Degradation of the ECM that expediates the movement of tumor cells to surrounding tissue, is one of the important hallmarks of cancer progression and is facilitated by collagenases. Focal adhesion kinase is a tyrosine kinase that mediates signaling by the integrin family of cell surface receptors for ECM, leading to tumor cell migration and progression25. The module analysis additionally identified Neuroactive ligand–receptor interaction pathway, which is implicated in OTSCC26 and other types of cancers, like breast27 and bladder28 to be dysregulated in OTSCC. Collectively, these results complement each other and reinforce the pivotal role played by ECM remodeling and stromal microenvironment in OTSCC which in turn clarifies the aggressive nature of the disease and its propensity to metastasize.The findings of our current transcriptomic sequencing study were in complete agreement to our published meta-analysis study on OTSCC29. APP was identified as the most significant hub gene in both transcriptomic and meta-analysis studies23. APP, a type-I integral membrane protein, has shown higher expression levels in OSCC and is known to be associated with poor prognosis30. Secretory proteins assume a lot of importance in the context of a high-effect local disease like oral cancer owing to its potency to be detected in saliva. With more than 500 of the DEGs in the current study being secreted proteins, patient-based saliva proteomics could be an engaging approach to biomarker discovery in oral cancer31. SPOCK1 was found to be up-regulated in OTSCC but its mode of action and its role in OSCC has not been explored and merits an exhaustive analysis. SPOCK1 has the potential to act as a prognostic marker and the added advantage of it being a secretory protein, offers the possibility of non-invasive detection.The hub genes in a network are expected to be key drivers influencing the normal functioning of a cell, owing to the central role played by them in the network with multiple interactions35. However there has been no single, decisive prognostic/predictive factor for a positive occult node32 and clinical staging is insufficient and often underestimates the extent of occult node positivity36. These shortcomings have called for an exigent need to identify molecular markers to predict occult tumor burden and our study has attempted to answer it.While surgery is the accepted first line of treatment in OTSCC, there exists several views over the clinical management of node negative patients with suggestions ranging from observation to elective neck dissectionOur cohort comprised of stage I to stage IV cancers and was representative of the entire spectrum of OTSCC. Out of the 26 markers evaluated, 19 of the tested biomarkers were able to significantly differentiate tumor from normal samples. We report 12 genes namely, LAMC2, VIM, HIF1A, TWIST2, SOX2, TNC, PDPN, MMP9, DSG2, PLAU, FOXM1 and MYO1B found to be relevant clinical markers that can be easily tested in OTSCC tumors to indicate prognosis, cervical and occult node positivity and outcome. We have derived a panel of molecular markers significantly predicting recurrence of the early stage OTSCC based on the gene expression levels of 6 genes namely, MMP9, LAMC2, DSG2, PLAU, FOXM1 and MYO1B.39. Additional studies by Zhou et al., showed up-regulation of the MMP9 gene to be associated with advanced OTSCC and having a predictive value for the identification of lymph node metastasis40. These evidences along with our findings emphasize the importance of evaluating MMP9 in OTSCC.Several studies including our previous study have shown that members of MMP family are involved in OTSCC with very significant upregulation42 with its overexpression indicating an advanced stage and positive cervical node status. Our results are in confirmation of earlier studies identifying LAMC2 as a prognostic factor in early stage and advanced stage OTSCC tumors43. Interestingly, in our cohort, the tumor area facing the stroma had a higher expression of LAMC2. Most of the extracellular spaces of the tumor areas showed accumulation of LAMC2 and expression of LAMC2 at tumor stroma interfaces indicates invasion and cancer progression44.LAMC2 was identified as a relevant gene and an important indicator of poor prognosis and cancer progression in OTSCC45 and gastric carcinoma46. Our observation of PLAU as a marker of relapse in OTSCC is in line with prior study in OSCC demonstrating the role of PLAU as a strong independent prognostic factor of DFS47. Baker et al., have previously reported the over-expression of PLAU and its correlation to aggression of tumors in OSCC48.Other relevant genes were DSG2, and PLAU. DSG2 is an adhesion molecule and a known epithelial EMT marker overexpressed and is also known for bad prognosis marker in hepatocellular carcinoma50, glioblastoma51, non-small-cell lung cancer52 and most recently, also in oral cancer where it is proven to predict poor patient survival53. This is similar to our findings where FOXM1 overexpression is associated with relapse in OTSCC patients. MYO1B, a motor protein involve in cellular motility54, is known to be overexpressed in HNSCC55 and is found to play a pivotal role in lymph node metastasis by way of augmenting cancer cell motility56. SOX2 is a transcription factor and stem cell marker, known to be expressed in several cancers, playing a major role in cell proliferation, metastasis57, tumor drug resistance58 and is hence a valuable therapeutic target59. SOX2 maintains the stemness of CSC, ultimately resulting in cancer relapse and resistance to treatment60. Liu et al. demonstrated the involvement of SOX2 in promoting tumor aggressiveness and EMT in OTSCC61. In this scenario, our finding of SOX2 to be a marker of failure in OTSCC cases treated by radiotherapy is significant.Several studies have implicated FOXM1 to play a major role in chemo-resistance in gastric cancer62, has been established as a diagnostic marker and potential cancer-associated fibroblast marker in breast ductal cancer63 and cervical cancer64 and a prognostic marker in early stage OTSCC65. The functions of TNC and its role as a cancer stem cell (CSC) marker66 makes it an ideal marker of occult node positivity as found in our study. Another important marker of occult node positivity and poor outcome identified in radiation-treated locally advanced OTSCC was PDPN. PDPN is a trans-membrane receptor glycoprotein that increases clonal cell capacity, EMT, invasion and metastasis67, playing a pivotal role in several cancers. Preclinical studies have identified PDPN as a therapeutic target to combat several cancers including oral cancer71 and nasopharyngeal cancer72. These findings corroborate our identification of PDPN as an occult node and poor prognosis predictor.We identified TNC and PDPN to be significant markers of occult node metastasis. TNC, a protein of the extracellular matrix with important functions in angiogenesis and tumor cell proliferation34. In another study on OTSCCs from our centre, the incidence of occult metastasis was 32.69%73. Currently, sentinel Lymph node biopsy has been suggested as a minimally invasive technique for nodal staging to reduce the needless morbidity of nearly 70–75% of pathologically node negative patients undergoing elective neck dissection. Since, sentinel node is generally believed to be the first lymph node or lymph nodes group receiving lymphatic drainage from the primary tumor, if this node is metastasis negative, the non-sentinel nodes in the neighboring regional basins are also deemed to be negative of metastases. Currently, there are no molecular markers that can identify metastasis in lymphatic basins, and we suggest that TNC and PDPN expression can be evaluated in a larger series of cases to identify patients at risk of occult micrometastasis.Our cohort had about 26.9% of occult positive cases that falls within the range of previous studies reported75. Our validations based on protein expression predicting risk of disease relapse and death in OTSCC patients confirmed the expression of LAMC2, MMP9 and ECAD at ITF as the important prognostic indicators. Thus, among our panel of markers, MMP9, LAMC2, VIM, DSG2 and TNC are well-known EMT markers, emphasizing the importance of EMT in OTSCC.Survival analyses of the clinicopathological factors predicting recurrence and death identified clinical stage, cervical node, occult node and tobacco habits as the classic prognostic factors in tongue cancer77. Loss of membrane ECAD at ITF predicted both risk of recurrence and death. Among the locally advanced cases treated by radiation, LAMC2, MMP9 and POSTN were the predictors of disease relapse while node, LAMC2 and POSTN were the prognostic factors predicting death.In the exclusive cohort of early stage patients treated by surgery, cervical node and peri-neural invasion significantly predicted failure in early stage OTSCC as reported earlier78. Our locally advanced tumors were treated by radiation and POSTN turned out to be an important prognostic factor in these tumors. High levels of POSTN are found to be associated with aggressive tumor behaviour, tumor progression, resistance to treatment and hence, bad prognosis81. Although Choi et al. have identified POSTN to be a diagnostic marker in OSCC by tissue microarray82 and its role in OSCC tumorigenesis is well established84, its prognostic role in OTSCC has not been reported earlier.Murthy et al. reported node to be an important prognostic factor in oral cancer patients treated by radiotherapy and went on to state that among the different oral subsites, OTSCC patients treated by radiotherapy perform poorly85, very few have derived meaningful mRNA-based prognostic markers86, more so in OTSCC. Using a panel of markers has superior discriminatory power over using individual candidate genes and hence, our panel of biomarkers with further validations in a larger study, may have better utility in the clinical setting to indicate the risk of recurrencee in OTSCC.Although many earlier studies have reported diagnostic biomarkers based on mRNA expression in OSCCOTSCC transcriptome sequencing upholds prior findings on the pivotal role of ECM degradation in OTSCC tumorigenesis and progression. Our cohort study addressed the most pressing issue of OTSCC management, namely, occult node metastasis prediction with 2 molecular markers and identified a panel of 6 molecular markers that can be used to differentiate the early stage tumors at higher risk of recurrence. Identification of novel biomarkers for early detection, prediction of response, and for use as treatment targets is of utmost importance to increase survival in OTSCC and the current study has fulfilled that. With further validation in a larger cohort, preferably, a multi-centric one, the panel of prognostic markers ascertained in our study can be used routinely to make important clinical decisions with respect to treatment modalities.later and stored in − 80 °C till further use. The patients were treated as per the NCCN guidelines. Variables recorded and evaluated for the study included age, sex, site, size of the tumor, clinical stage, histological grade, tobacco habits, alcohol habits, comorbids like Diabetes mellitus and Hypertension, node status, perineural invasion and occult node status.The Ethical approval number of the study was CIWIA/Protocol 1/HNCOG 2014, namely Cancer Institute Womens India Association/ Protocol 1 /Head and Neck Oncology Group 2014). All research involving human participants had been approved by the authors’ Institutional Review Board (IRB) and all clinical investigations had been conducted according to the principles expressed in the Declaration of Helsinki. A written informed consent was obtained from all the participants and the content of the informed consent was approved by the Institutional Research Board.Prospective primary tongue cancer samples (n = 100), both punch biopsy and tissue samples from surgical specimens with corresponding apparently uninvolved adjacent tissue were collected from the patients presenting with OTSCC between the years 2014 and 2016 to the Head and Neck Oncology Clinic, Cancer Institute (WIA), Chennai, India. Collected samples were immediately immersed in RNA87. Briefly, the libraries were multiplexed and sequenced on HiSeq 2500 to obtain on average ~ 108 million single end 95 bp reads per sample. RNA-seq reads were mapped to the human genome version NCBI GRCh37 using GSNAP. Only uniquely mapped reads were considered for downstream analysis. Gene models were based on RefSeq transcripts, NCBI and ENSEMBL gene annotations. Differential gene expression was performed with the R/Bioconductor package DESeq2. For clustering, count data were transformed by variance stabilization and genes were centered to have mean zero. Clustering was performed using 1-Pearson correlation as distance metric and average linkage. Heat maps were generated using the R/Bioconductor package NMF. Fold change was calculated based on comparing the average of tumor samples with the average of normal samples. The relative abundance metric parameter FPKM (Fragments Per Kilobase of exon per Million reads sequenced) was used to represent the value of gene expression. To detect and filter DEGs, we applied the Student’s t test , and the FDR correction .Transcriptome libraries for sequencing were constructed using 0.5 μg of total RNA as per manufacturer’s instructions  and was analysed as described previously88, a web-based tool for Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses89. A Benjamini-corrected P value less than 0.05 was used to identify a statistically significant analysis. The hub proteins were identified using the Cytoscape v3.6.0. plugin called ‘Network Analyzer’90. The hub proteins were evaluated by analyzing the highest closeness centrality (CC), betweenness centrality (BC), and the node degree. Genes with degree ≥ 10 were defined as hub genes in the present study. In addition, the Molecular Complex Detection (MCODE) plugin of Cytoscape software was also employed to identify functionally related and highly interconnected clusters from the PPI network with a degree cutoff of 2, node score cutoff of 0.3, k-core of 4, and maximum depth of 100 (http://baderlab.org/Software/MCODE)91. Significant modules were identified with MCODE score ≥ 4 and nodes ≥ 6. Subsequently, based on modules selected from the PPI network, functional enrichment analysis was performed using DAVID with the criterion of P < 0.05. The protein–protein interactions PPI was visualised using Search Tool for the Retrieval of Interacting Genes (STRING) online database (v10.5) (www.string-db.org) for network construction92.Functional interpretation of the DEGs was performed with Database for Annotation, Visualization and Integrated Discovery 23. Triplicates were performed for each gene and average expression value was computed for subsequent analysis. The relative expression level of the genes was calculated using the 2−∆∆Ct method. We defined the cut-off value for over-expression of genes based on the median log RQ values for each of the marker. Patients with pathological stages I and II disease were grouped as ‘early stage’ tumors and patients with pathological stages III and IV were grouped as ‘advanced stage’ tumors. We used t test to identify the markers whose expression varied most among the staging, node positive and outcome subgroups.Total RNA was extracted from the tissue samples, homogenising them in liquid nitrogen, using RNeasy mini kit  using standard manufacturer protocol. Total RNA (2 µg) was converted to cDNA using High capacity cDNA reverse transcription kit  as per instructions of the manufacturer and stored at − 40 °C.Primers used for the study are listed in Supplementary Table 23. The IHC detection of MMP9, LAMC2, ECAD, was performed on five-micron sections of FFPE tissues. The sections were deparaffinised in xylene, rehydrated in absolute ethanol and endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in Phosphate-buffered Saline (PBS) for 30 min. The sections were then subjected to heat-induced epitope retrieval according to the conditions listed in Supplementary Table The IHC methodology is mentioned previouslyOverall Survival (OS) was calculated as time in months from the date of primary treatment to the date of death due to the disease. Disease free survival (DFS) was calculated as time in months from the date of primary treatment to the date of objective tumor recurrence . The samples were blinded with respect to clinical data during the molecular testing. All statistical analysis was done using SPSS, version 16 (IBM).Supplementary Information 1.Supplementary Information 2.


No significant differences were found in questionnaire scores. The correlations of weekly exercise behavior scores with exercise motivation and self-efficacy scores were stronger in the KD group. Conclusions: adolescents with KD history had significantly lower aerobic metabolism capacity and peak exercise load capacity than controls. The correlations of amount of weekly exercise with exercise motivation and self-efficacy were stronger in the KD group.Introduction: Kawasaki disease (KD) is a childhood illness causing blood vessel inflammation. Children with KD have similar cardiopulmonary function to healthy children, but lower moderate-to-vigorous activity and exercise self-efficacy—possibly harming their cardiopulmonary function in adolescence. The purpose of this study is to investigate the cardiopulmonary function, exercise behaviors, exercise motivations, and self-efficacy of adolescents who once had KD. Methods: adolescents who once had KD and adolescents matched to the KD group in age and sex were enrolled. The cardiopulmonary exercise test was used to assess cardiopulmonary function. Weekly exercise behavior, exercise motivation, and self-efficacy were assessed with questionnaires. Results: this study recruited 50 and 30 participants, respectively, to the KD and control groups. The KD group had a lower ratio of VO Kawasaki disease (KD) is an illness in children that causes inflammation of blood vessels throughout the body and primarily affects children younger than 5 years old. Approximately 1000 new cases of KD occur every year in Taiwan, placing Taiwan third worldwide, behind South Korea, for the number of individual cases. Japan has the highest incidence of 10,000–20,000 new cases each year. KD can cause inflammation of the coronary arteries, leading to coronary artery aneurysm (CAA), and is, therefore, the most common cause of acquired heart disease in children. Patients with coronary artery disease may need to take aspirin, a blood thinner, for the rest of their lives, as such disease may lead to heart failure and can thus be fatal. Furthermore, failure to treat child patients during the golden treatment period may result in severe heart disease sequelae.Scholars have discovered that although children with KD have lower rates of myocardial perfusion while they are exercising, their cardiopulmonary function and exercise load capacities do not significantly differ from those of healthy children ,2. HowevThe experiment was conducted through a cross-sectional, observational study.This study was conducted at a medical center in Southern Taiwan. The research team contacted patients listed in the medical center’s KD case management database, which had been established by the children’s rheumatology and immunology department. Contact was achieved through clinical recruitment, posters, and cold calling. The patients who agreed to participate in the study were assigned to the KD group. Age- and sex-matched healthy volunteers were recruited through posters advertising the study and assigned to the control group (hereafter referred to as the C group).The inclusion criterion for the study was age 13–19 years. The exclusion criteria were (1) having had KD but the onset date could not be confirmed; (2) having a heart disease that can affect cardiopulmonary functions ; (3) having a pulmonary disease that can affect pulmonary functions (such as asthma or chronic obstructive pulmonary disease); and (4) other factors that would affect their results or that prevented the patient from accepting cardiopulmonary exercise tests or surveys.Demographics: sex, age, height, weight, and body mass index (BMI).The recorded KD onset date for the KD group participants and number of years since the onset.In accordance with the KD group participants’ most recent echocardiography test results, Z score was calculated using the Taiwan Society of Pediatric Cardiology’s online calculator and the widest diameters of the left coronary artery (LCA) and right coronary artery (RCA) ,5. CAA wWhether the KD group participants took aspirin as a blood thinner.After recording the participants’ personal information, the two groups underwent the cardiopulmonary exercise test (CPET) and survey assessments:2) plateaued within 2 min; (4) any myocardial ischemia should be observed  or arrhythmia occurred; and (5) other circumstances that may affect the safety of the CPET. Throughout the test, the participants’ blood pressure and heart rate were measured, and the gas analyzer measured the participants’ VO2, VCO2, and minute ventilation (VE) throughout the test. The participants’ respiratory exchange ratio (RER) was derived from the VCO2/VO2 ratio; RER ≥ 1.10 indicates that a patient has reached sufficient exercise intensity [2 and VE/VCO2 values were employed to calculate the participants’ anaerobic threshold (AT)9. The AT is a significant increase in anaerobic glycolysis to provide energy when the oxygen supply in the circulatory system is insufficient to fulfill the current oxygen consumption levels at the exercise intensity [2/kg at the AT and the predicted VO2/kg at peak (hereafter referred to as AT%) were used to assess the aerobic metabolism capacity; a higher ratio indicates that a person’s aerobic metabolism and circulatory system oxygen supply capacity during exercise can withstand a high exercise load. VO2/kg at peak exercise intensity was used to reflect the participants’ peak exercise capacity [2/kg at peak (this ratio is hereafter referred to as Peak%) being 85% or higher is taken as the benchmark for “normal” test results [2 pulse indicates oxygen intake and heart rate ratio and can be used to assess stroke volume [2/kg at peak, and O2 pulse, which were compared with the participants’ test data.Cardiopulmonary exercise test: the CPET was used to assess the participants’ cardiopulmonary functions and exercise load capacity performance. This test was conducted using the MasterScreen CPX . First, pulmonary function was evaluated in a resting state. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and the FEV1/FVC ratio were recorded. Next, the participants were asked to put on masks connected to a gas analyzer and were connected to an electrocardiogram monitor and underwent treadmill exercise tests, according to the Bruce protocol . The conntensity ,8. The V results . O2 pulsThe participants’ exercise behaviors were assessed using a Chinese version of the Godin Leisure-Time Exercise Questionnaire. This questionnaire was designed by G. Godin and R.J. Shephard ,12 and aThe participants’ exercise motivation and behavioral regulation were assessed using the Behavioral Regulation in Exercise Questionnaire 2nd edition (BREQ-2) in Chinese ,14. The not confident at all and 10 for very confident). This revealed the participants’ assessment of their own confidence in all dimensions.Self-efficacy for exercise was assessed using the Multidimensional Self-Efficacy for Exercise Scale in Chinese. This scale comprises nine statements that begin “How confident are you that you can…” and can be sorted into three efficacies relating to exercise: task efficacy , coping efficacy , and scheduling efficacy  . The parFirst, the following information about the participants who met the inclusion and exclusion criteria and were willing to participate was recorded:t-test. One way analysis of variance (ANOVA) was used to compare the CPET results of the KD group—participants with and without CAA—and C group, the CPET results between the KD group—taking and not taking aspirin—and C group, and CPET results within the KD group among participants with different degrees of CAA. Post hoc verification was conducted using the Scheffé test. Pearson’s correlation coefficients were calculated to determine the correlations between the Z score and CPET results, between the survey scores and CPET results, between the survey scores and body weight/BMI, and between several survey results. Correlation coefficients (CCs) were calculated, and the results were divided into four ranges: strong correlation (0.70–0.99), moderate correlation (0.40–0.69), weak correlation (0.10–0.39), and almost no correlation (0.01–0.09). The survey results of the five groups—group of participants with KD and CAA, group of participants with KD but without CAA, group of participants with KD who were taking aspirin, group of participants with KD who were not taking aspirin, and the C group—were compared using the Kruskal–Wallis test, and the post hoc test was conducted according to the method of Dunn [p < 0.05 indicated a statistically significant difference.Statistical analysis of the data was conducted using MedCalc version 18 . The Kolmogorov–Smirnov test was employed to test the normality of the data distribution. The chi-squared test was used to compare the KD and C groups in terms of sex, distributions in age ranges, reasons for terminating the CPET, RER ≥ 1.10, and predicted number of participants with Peak% ≥ 85%. The CPET and survey results were verified using the independent-samples  of Dunn ,17. In tThe study was conducted in accordance with the Declaration of Helsinki. The proposal for this study was reviewed and approved by the Chang Gung Medical Foundation Institutional Review Board (case no. 201601136B0C502). All participants were informed of the research process by the chief investigation prior to signing a consent form.From January 2017 to December 2018, 88 people who met the inclusion criteria  were recruited. Eight subjects refused to participate in the study . Finally, 80 participants were enrolled in the study .p = 0.909).Because physical development is rapid during adolescence, we classified all participants into three age ranges  and analyzed the age distributions in both groups. In the KD group, 16 participants were aged 13–15 years (32%), 32 participants were aged 16–18 years (64%), and 2 participants were aged 19 years (4%); in the C group, 11 participants were aged 13–15 years (37%), 18 participants were aged 16–18 years (60%), and 1 participant was aged 19 years (3%). No significant difference existed between the age distributions in the two groups . In the remaining two cases, one participant’s chief complaint was foot pain, and the other participant asked to stop the test because of sweat in the eyes. In the C group, 14 participants stopped due to soreness in their legs (46.7%) and 10 from shortness of breath (33.3%). In six cases, the test was terminated because the participant’s VO2 plateaued (20%). Comparison of the reasons for test termination in the two groups yielded no significant difference (p = 0.534). Whether the participants achieved sufficient exercise intensity (RER ≥ 1.10) was analyzed; 42 participants in the KD group achieved sufficient exercise intensity (84%), and 8 participants did not; 7 of those 8 participants stopped the test due to soreness in their legs, and the 8th stopped the test due to foot pain. In the C group, 27 participants achieved sufficient exercise intensity (90%), and 3 did not; 2 of the 3 stopped the test due to soreness in their legs and the other due to shortness of breath. No significant intergroup difference was determined regarding whether sufficient exercise intensity was achieved (p = 0.453).CPETs were successfully conducted for all the participants, and the electrocardiograms obtained in the testing process did not exhibit any abnormalities. Regarding the reasons the KD-group participants terminated their CPET, 27 participants ended their test due to soreness in their legs (54%) and 17 due to shortness of breath (34%). In four cases, the test was terminated because the participant’s VOp = 0.061). The other data did not exhibit any significant intergroup differences.p = 0.036), and using the Scheffé test in post hoc pairwise comparisons indicated no significant differences between the three groups. No significant intergroup differences were observed in other data . Correlation analysis of Z scores and CPET results for the participants with CAA revealed that although the RCA Z score and CPET results were not significantly correlated, there was a trend showing their negative correlation with VO2/kg at peak . Peak Z score was not significantly correlated with LCA Z score or CPET results.The participants without CAA (Z score < 2.5), with small aneurysms (Z score ≥2.5 to <5.0), and with large aneurysms (Z score ≥5.0 to <10.0) within the KD group were compared, and no significant differences were discovered between these groups for VOp = 0.038), and using the Scheffé test in post hoc pairwise comparisons revealed no significant differences between the three groups. No significant differences were observed in other data between the three groups  or total score .n = 19), with CAA and not taking aspirin (n = 13), without CAA and taking aspirin (n = 8), and with CAA and taking aspirin (n = 10) groups. These four groups were then compared with the C group; however, these comparisons revealed no significant differences.The KD group was divided into four groups based on whether the participant had CAA and whether they regularly took aspirin; they formed the without CAA and not taking aspirin (p = 0.025) and external regulation , weakly positively correlated with identified regulation  and intrinsic regulation , and moderately positively correlated with RAI score , weakly positively correlated with corresponding efficacies  and moderately positively correlated with planning efficacy  and total score in the Multidimensional Self-Efficacy for Exercise Scale . For the C group, a moderately positive correlation was discovered between the Godin Leisure-Time Exercise Questionnaire score and intrinsic regulation in BREQ-2 , but no significant correlations between other BREQ-2 items and multidimensional self-efficacy for exercise items were found.p = 0.012) and a moderate positive correlation between Peak% and BREQ-2 external regulation score in the C group . Neither the Godin Leisure-Time Exercise Questionnaire scores nor Multidimensional Self-Efficacy for Exercise Scale scores were significantly correlated with the CPET results. Correlations between body weight, BMI, and the survey results were analyzed, but no significant correlation was found.Correlation analysis of the survey and CPET results revealed a weak negative correlation between Peak% and BREQ-2 external regulation score in the KD group  and the C group (53%), suggesting that adolescents in both groups engage in insufficient physical activity. According to the latest survey by the WHO, the global prevalence of insufficient physical activity in adolescents is 81.0%. In Taiwan, where this study was conducted, 84.4% of adolescents engage in insufficient physical activity, a worse rate than the global average [Although the results of this study indicate that adolescents who had once had KD had lower aerobic metabolism capacity and peak exercise load capacity, this does not mean they are unable to engage in exercise normally. The World Health Organization (WHO), recommends 75 min of vigorous aerobic exercise and 150 min of moderate aerobic exercise per week . The Ame average . Thus, iIn addition to investigating cardiopulmonary functions, this was the first study to evaluate the exercise behaviors, exercise motivations, and self-efficacy of adolescents who had once had KD. This study discovered that compared with peers in the same age group, adolescents who had once had KD did not have a significantly different self-assessed amount of weekly exercise, exercise motivation of any type, or task efficacy, coping efficacy, or scheduling efficacy. Moreover, division of the KD group on the basis of whether the participants had CAA or regularly used blood thinners and subsequent analysis revealed no significant differences in self-assessed amount of weekly exercise, exercise motivation, and self-efficacy, indicating that CAA and use of blood thinners do not appear to affect the amount of exercise performed by adolescents who had once had KD or their motivations and confidence regarding exercise. Correlation analysis demonstrated that in the KD group, the participants’ amount of weekly exercise was moderately positively correlated with RAI—which reflects overall exercise motivation—and overall self-efficacy score; conversely, in the control group, the amount of weekly exercise was moderately positively correlated with only intrinsic regulation among all the types of exercise motivation; no significant correlations were discovered with other exercise motivations or self-efficacies. This indicates that the exercise behaviors of adolescents who had once had KD were more likely to be affected by exercise motivation and self-efficacy, compared with the behaviors of typical adolescents. Therefore, how the exercise motivation and confidence of this group, and consequently also their health and cardiopulmonary fitness, can be improved through approaches such as health education campaigns and an upgrade to exercise environments to improve is worthy of research. The positive effects of improving the exercise motivation and confidence of this population may be even more significant than for typical adolescents.This study had several limitations. First, the sample was relatively small, and second, the participants were recruited from one medical center and thus their circumstances may not reflect the circumstances of all adolescents who had once had KD. Third, in this study, Z scores were calculated using the online calculator provided by the Taiwan Society of Pediatric Cardiology, because this is the only algorithm constructed to be applicable to Taiwanese people. However, the algorithm was developed using data only from subjects aged 6 years and under ; therefoCompared with control adolescents, adolescents who had KD history had lower aerobic metabolism capacity and peak exercise load capacity, which should not affect their ability to engage in daily leisure exercise. CAA and regular use of blood-thinning medication did not significantly affect aerobic metabolism capacity and peak exercise load capacity, nor did it have significant effects on daily exercise, exercise motivation, and self-efficacy. Furthermore, for the adolescents who once had KD, significant positive correlations were discovered between amount of weekly exercise and exercise motivation and between amount of weekly exercise and self-efficacy; these correlations were stronger than in peers of the same age.