O ácido úrico (AU), produto final do metabolismo dos nucleotídeos das purinas, participa dos processos de doenças metabólicas e cardiovasculares. Evidências experimentais sugerem que o ácido úrico é um mediador importante na resposta fisiológica ao aumento da pressão arterial.Avaliar a associação entre os níveis séricos de AU e pré-hipertensão e hipertensão em uma população chinesa.Conduziu-se um estudo transversal entre março e setembro de 2017, e 1.138 participantes com idades entre 35 e 75 anos foram incluídos neste estudo, onde 223 normotensos, 316 pré-hipertensos e 599 hipertensos foram selecionados para avaliar a associação entre níveis séricos de AU e hipertensão. Considerou-se um valor de p<0,05 estatisticamente significativo.odds ratios — ORs) e intervalos de confiança (IC) de 95% da pré-hipertensão, dos níveis séricos de AU mais baixos (referentes) aos mais altos foram 1,718 , 1,018  e 1,738 . Além disso, o segundo quartil dos níveis séricos de AU esteve significativamente associado à hipertensão, com uma OR (IC 95%) de 2,036 .Os níveis séricos de AU foram significativamente maiores no grupo pré-hipertensão e hipertensão em comparação com o grupo controle em toda a população . A análise quantitativa das características indicou níveis séricos de AU  linearmente aumentados em mulheres normotensas, pré-hipertensas e hipertensas, com um valor de p de 0,008. Os níveis séricos de AU nos quartis correlacionaram-se positivamente com a PAD , principalmente em mulheres. Após o ajuste para idade, sexo, índice de massa corporal (IMC), glicose (GLI), colesterol total (CT), triglicerídeos (TG), colesterol HDL , as razões de chances ( O presente estudo sugere que níveis séricos mais elevados de AU estão positivamente associados à pré-hipertensão e hipertensão entre adultos chineses. A taxa geral de prevalência padronizada por idade de doenças cardiovasculares aumentou significativamente de 1990 a 2016 — 14,7% — e o número anual de óbitos por DCV aumentou de 2,51 milhões para 3,97 milhões na China. 7 Um estudo de coorte mostrou que a hiperuricemia é preditor de hipertensão em homens e mulheres. 8 Pesquisas com animais revelaram que a hiperuricemia leve causa hipertensão e lesão renal em ratos por meio da estimulação do sistema renina-angiotensina e inibição da óxido nítrico-sintase neuronal (nNOS). 9 Como fator de relaxamento derivado do endotélio, o óxido nítrico é crucial para a manutenção da pressão arterial (PA). 10 Uma revisão sistemática e uma meta-análise verificaram que para um aumento de 60 umol/L nos níveis séricos de AU, o risco relativo de hipertensão aumentou em 13%, e esse risco parece mais pronunciado em indivíduos mais jovens e em mulheres. 11O AU é o produto final do metabolismo do nucleotídeo da purina, e o distúrbio do metabolismo da purina ou a excreção anormal do AU pode levar ao aumento dos níveis séricos de AU. Além disso, o aumento da concentração sérica de AU no corpo resulta em hiperuricemia, levando à ocorrência de gota. 12 O AU sérico também se mostrou um fator de risco independente para um padrão circadiano de hipertensão non-dipper.13 Quanto mais alto o nível de AU sérico, mais difícil é controlar a pressão arterial ambulatorial noturna, a pressão arterial diastólica noturna e o pico de pressão arterial matinal. 14 Em um estudo inicial, não se observou hiperuricemia em 25–40% dos hipertensos não tratados e 75% dos indivíduos com hipertensão maligna. 15 No entanto, não se encontrou nenhuma associação independente entre os níveis séricos de AU ou risco de hipertensão incidente entre homens mais velhos. 16A hiperuricemia está comumente associada à pré-hipertensão em adultos. Quando a hipertensão se complica com hiperuricemia, ambas causam e afetam uma à outra, o que agrava o desenvolvimento da doença. Portanto, apesar da associação entre AU sérico e hipertensão, seu mecanismo permanece obscuro. Assim, em nosso estudo, exploramos a associação entre altos níveis séricos de AU e hipertensão entre adultos chineses na província de Anhui, no norte da China.Este estudo foi realizado de março a setembro de 2017 no Centro de Exame Físico de um Hospital Popular na Província de Anhui, no norte da China. Um total de 1.191 participantes com idades entre 35 e 75 foram incluídos neste estudo, incluindo 643 casos de hipertensão e 548 indivíduos normotensos. Indivíduos sem valor sérico de AU (n=53) foram excluídos. Por fim, 1.138 adultos, incluindo 223 normotensos, 316 pré-hipertensos e 599 hipertensos, foram selecionados para avaliar a associação entre os níveis séricos de AU e hipertensão. O protocolo de estudo foi aprovado pelo Comitê de Ética da Wannan Medical College.2 ). Uma equipe de pesquisa bem treinada mediu a PA uma vez usando esfigmomanômetro eletrônico com o participante na posição sentada após pelo menos 5 minutos de repouso. Todos os indivíduos jejuaram durante a noite por pelo menos 10 horas antes da coleta de sangue. Amostras de sangue venoso de 5 ml foram coletadas para medir os níveis de colesterol total (CT), triglicerídeos (TG), colesterol HDL, colesterol LDL, glicose (GLI) e níveis séricos de AU. Os fumantes foram definidos como consumidores de cigarros que fumavam ao menos 20 cigarros por semana ou ao menos 3 meses por ano. Beber álcool no mínimo 2 vezes por semana ou no mínimo 6 meses por ano foi considerado consumo de álcool.Cada participante passou por entrevista presencial e preencheu um questionário padrão incluindo características demográficas, histórico clínico e características de estilo de vida. Todas as informações foram coletadas por uma equipe de pesquisa treinada. No exame físico, todos os indivíduos tiveram sua altura, peso e pressão arterial (PA) medidos. O índice de massa corporal (IMC) foi calculado como o peso corporal (kg) dividido pela altura ao quadrado  com distribuição normal; os dados qualitativos, como proporções. As diferenças de sexo nas características gerais foram analisadas usando o teste t pareado de Student para variáveis contínuas e o teste qui-quadrado (χ2) para variáveis categóricas. As diferenças para as variáveis entre os grupos foram determinadas por análise de variância (ANOVA) de um fator ou teste χ2, e as correções de Bonferroni foram usadas para comparações múltiplas. Além disso, aplicou-se a análise de regressão logística múltipla não condicional para estimar a relação entre AU e hipertensão. Realizou-se o teste do coeficiente de correlação de Pearson para avaliar as inter-relações entre as variáveis basais e os níveis séricos de AU. Utilizou-se o equipamento Epidata 3.1  para estabelecer bases de dados. Todas as análises estatísticas foram realizadas com o software SPSS 18.0 . Um p<0,05 bicaudal foi definido como estatisticamente significativo.Este estudo incluiu 1.138 indivíduos  com idades entre 35 e 75 anos. A p de 0,008 . Os níveis séricos de AU não mostraram diferença entre os grupos no sexo masculino. Outras análises quantitativas de características do AU sérico (mg/dL) indicaram que os níveis séricos de AU  aumentaram linearmente em normotensão, pré-hipertensão e hipertensão em mulheres, com valor de e 0,008   .A Os níveis séricos de AU estiveram positivamente correlacionados com IMC, pressão arterial diastólica (PAD), CT, TG, colesterol LDL e creatinina em ambos os sexos. Os níveis séricos de AU estiveram negativamente correlacionados com a idade e positivamente correlacionados com IMC, CT, TG, colesterol LDL e creatinina em homens. No sexo feminino, os níveis séricos de AU estiveram positivamente associados com IMC, PAD, TG, colesterol LDL e creatinina   .Na análise de regressão logística, a 20 O AU pode ser considerado um importante antioxidante, que não só estabiliza a atividade da óxido nítrico sintase endotelial (eNOS), mas também aumenta o armazenamento de gordura e triglicerídeos. 21 Estudos epidemiológicos têm demonstrado forte associação entre o AU e a doença arterial coronariana, aterosclerose e hipertensão. 22 Em nosso estudo, relatamos que níveis séricos de AU mais elevados estiveram positivamente associados com pré-hipertensão e hipertensão na população de meia-idade e idosos, e níveis elevados de AU sérico podem indicar um aumento correspondente na PAD. O risco geral de pré-hipertensão aumentou 73,8% para o quartil mais elevado vs. mais baixo dos níveis séricos de AU, mesmo após o ajuste para possíveis variáveis de confusão. Além disso, verificamos que uma associação mais robusta nas mulheres participantes.Alterações nos níveis de AU estão envolvidas na remodelação vascular e na disfunção endotelial, que pode ser a causa de distúrbios cardiovasculares. 23 revelaram que o aumento dos níveis séricos de AU foi preditor independente de desenvolvimento de hipertensão após um seguimento de curto prazo. Níveis elevados de colesterol LDL e AU sérico são fatores de risco para disfunção endotelial e envelhecimento vascular. A presença concomitante de valores subótimos de colesterol LDL e AU sérico está associada a risco aumentado de hipertensão em uma amostra populacional saudável. 24 Um estudo de coorte retrospectivo de 5 anos descobriu que o aumento de AU é um forte marcador de risco para hipertensão desenvolvida a partir da pré-hipertensão em adultos japoneses. 25 Além disso, estudos clínicos piloto sugerem que a redução dos níveis séricos de AU pode reduzir a pressão arterial em adolescentes pré-hipertensos. 26 Atualmente, a pré-hipertensão é comum na China. Aproximadamente 20–50% dos adultos são acometidos por pré-hipertensão em todo o mundo, e isso aumenta o risco de hipertensão incidente. 27 A prevalência de pré-hipertensão está aumentando rapidamente na China, mas suas causas e fatores associados não foram bem estudados.Estudos anteriores examinaram a associação entre os níveis séricos de AU e hipertensão, e os resultados estavam de acordo com nossos achados. Sundstrom et al., 28 também verificaram que a hiperuricemia estava associada à pré-hipertensão entre 1.773 mulheres chinesas com idade ≥30. Resultados semelhantes foram apresentados em um estudo de seguimento, no qual Strasak et al., 29 demonstraram que o AU sérico é um preditor independente de todas as principais formas de morte cardiovascular em mulheres idosas. A variação dos níveis de AU sérico em mulheres na menopausa sugere uma interação com os hormônios sexuais. 30 Pesquisas mostram que a diferença de sexo na pressão arterial começa a aparecer na adolescência e o surto de crescimento puberal ocorre mais cedo nas meninas do que nos meninos. 31 Há aumento mais significativo da PAS em meninos do que em meninas, enquanto há maior aumento da PAD em meninas do que em meninos. 32 Outras alterações fisiológicas e hormonais complexas podem contribuir para a hipertensão.Observamos que os níveis séricos de AU aumentaram linearmente na normotensão, pré-hipertensão e hipertensão em mulheres, e essa associação entre AU sérico e pressão arterial se mostrou mais forte entre as mulheres do que entre os homens. Além disso, os níveis séricos de AU estiveram associados positivamente à PAD, principalmente em mulheres. Alguns estudos anteriores demonstraram uma associação entre níveis séricos de AU e a hipertensão mais pronunciada em mulheres. Peng et al., Diversas limitações devem ser consideradas. Em primeiro lugar, o desenho transversal usado para avaliar a relação entre AU sérico e pré-hipertensão e hipertensão limita nossa capacidade de estabelecer uma relação causal. Este problema pode ser resolvido por estudos longitudinais no futuro. Em segundo lugar, o mecanismo de interação entre hipertensão e aumento de ácido úrico não foi explorado. Mais estudos ainda são necessários para examinar a possível diferença de sexo na associação entre os níveis séricos de AU e hipertensão em diferentes populações.Nossos achados sugerem que o AU sérico está significativamente associado à pré-hipertensão e hipertensão, e a associação se mostrou mais robusta nas participantes do sexo feminino. Portanto, o manejo precoce adequado dos níveis de AU em adultos pode ser importante para prevenir o desenvolvimento de hipertensão. 1 High blood pressure (BP) has a major public health burden worldwide due to its high prevalence and it is a major risk factor for a series of CVD including stroke, myocardial infarction, heart failure and renal failure.2 According to the “Summary of report on cardiovascular diseases in China (2018)”, the number of hypertensive patients in China is about 245 million and the prevalence rate of males is higher than that of females.3 Hypertension, a highly heterogeneous disorder, is influenced by the interaction between many factors such as sodium intake, alcohol, smoking, overweight, and genetic factors.4 In recent years, many studies have shown that high serum uric acid (UA) levels are associated with increased incidence of hypertension.6The prevalence of cardiovascular diseases (CVD) is increasing rapidly in the world communities. The overall age-standardized prevalence rate of cardiovascular diseases increased significantly from 1990 to 2016 — by 14.7% — and the annual number of deaths from CVD increased from 2.51 million to 3.97 million in China.7 A screened cohort study has shown that hyperuricemia is a predictor of hypertension in both men and women.8 Animal research has revealed that mild hyperuricemia causes hypertension and renal injury in rats via stimulation of the renin-angiotensin system and inhibition of neuronal nitric oxide (NO) synthase.9 As an endothelial-derived relaxing factor, NO is crucial to the maintenance of blood pressure (BP).10 A systematic review and meta-analysis reported that for a 60 umol/L increase in serum UA levels, the relative risk of hypertension increased by 13%, and this risk appears more pronounced in younger individuals and women.11UA is the end product of purine nucleotide metabolism, and the disorder of purine metabolism or abnormal excretion of UA can lead to increased serum UA levels. Furthermore, increased serum UA concentration in the body results in hyperuricemia, ultimately leading to gout.12 Serum UA has also been shown to be an independent risk factor for a non-dipper circadian pattern of hypertension.13 The higher the level of serum UA, the more difficult it is to control nighttime ambulatory blood pressure, nighttime diastolic blood pressure and morning blood pressure peak.14 In an early study, hyperuricemia was reported in 25–40% of untreated hypertensive and 75% of malignant hypertensive subjects.15 However, no independent association between serum UA levels and risk of incident hypertension was found among older men.16Hyperuricemia is commonly associated with pre-hypertension in adults.When hypertension is complicated with hyperuricemia, both of them cause and affect each other, which aggravates the development of the disease. Therefore, despite an association between serum UA and hypertension, its mechanism remains unclear. Thus, in our study, we explored the association between high serum UA levels and hypertension among Chinese adults in Northern Anhui Province.This study was conducted from March to September 2017 at the Physical Examination Center of a People’s Hospital in Northern Anhui Province. A total of 1,191 participants aged 35 to 75 were enrolled in this study, including 643 hypertension cases and 548 normotensive subjects. Individuals with missing serum UA (n=53) value were excluded. Ultimately, 1,138 adults, including 223 normotensive, 316 pre-hypertensive, and 599 hypertensive subjects, were selected to evaluate the association between serum UA levels and hypertension. The study protocol was approved by the Ethics Committee of Wannan Medical College.2). A well-trained research staff measured BP once using electronic sphygmomanometer with the participant in the sitting position after at least 5 minutes of rest. All the subjects fasted overnight for at least 10 hours before blood sampling. Venous blood samples of 5 ml were taken for measuring plasma total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-C) levels, glucose (GLU), and serum UA levels. Smokers were defined as cigarette consumers who had smoked at least 20 cigarettes per week or at least 3 months per year. Drinking alcohol at least 2 times per week or at least 6 months per year was considered as alcohol consumption.Each participant completed a face-to-face interview and a standard questionnaire including demographic characteristics, medical history, and lifestyle characteristics. All information was collected by trained research staff. On physical examinations, all subjects were measured for height, weight, and blood pressure (BP). Body mass index (BMI) was calculated as body weight (kg) divided by height squared  with normal distribution; qualitative data as proportions. Gender differences in general characteristics were analyzed using Student’s unpaired t-test for continuous variables and the Chi-square (χ2) test for categorical variables. The differences for variables among the groups were determined by one-way analysis of variance (ANOVA) or χ2 test, and Bonferroni corrections were used for multiple comparisons. Additionally, multiple unconditional logistic regression analysis was applied to estimate the relationship between UA and hypertension. Pearson’s correlation coefficient test was performed to assess the interrelationships between baseline variables and serum UA levels. Epidata 3.1  was used to establish databases. All statistical analyses were performed with SPSS version 18.0 . A 2-tailed p<0.05 was defined as statistically significant.This study included 1,138 individuals  aged 35 to 75. The demographic and clinical characteristics of the participants are presented in By gender subgroup, of the 1,138 subjects, 568 were males, and 570 were females. The mean level of serum UA was 3.67 mg/dL in males and 3.11 mg/dL in females (p<0.05). Serum UA levels showed no difference between the groups in males. Further quantitative trait analysis of the serum UA (mg/dL) indicated that those for serum UA  increased linearly in normotension, pre-hypertension and hypertension in females, with a p value of 0.008   .Baseline information of the subjects in each serum UA quartile is presented in Serum UA levels were positively correlated with BMI, diastolic blood pressure (DBP), TC, TG, LDL-C, and creatinine in both genders. Serum UA levels were negatively correlated with age, and were positively correlated with BMI, TC, TG, LDL-C, and creatinine in males. In females, serum UA levels were positively associated with BMI, DBP, TG, LDL-C, and creatinine   .In logistic regression analysis, 20 UA can be regarded as an important antioxidant, which doe not only stabilize endothelial nitric oxide synthase (eNOS) activity but also increases fat storage and triglycerides.21 Epidemiological studies have demonstrated a strong association between UA and coronary artery disease, atherosclerosis and hypertension.22 In our study, we report that higher serum UA levels were found to be positively associated with pre-hypertension and hypertension in middle-aged and old-age population, and high serum UA levels causes a corresponding increase in DBP. The overall risk for pre-hypertension has increased by 73.8% for the highest vs. lowest quartile of serum UA levels, even after adjusting for potential confounding variables. Furthermore, we found that the association was more robust in the female participants.Abnormal UA levels have been involved in vascular remodeling and endothelial dysfunction, which may be the cause of cardiovascular disorders.23 revealed that increased serum UA levels were an independent predictor of hypertension development after a short-term follow-up. High LDL-c and serum UA levels are risk factors for endothelial dysfunction and vascular ageing. The contemporary presence of suboptimal LDL-c and serum UA values is associated with an increased risk of hypertension in an overall healthy population sample.24 A 5-year retrospective cohort study found that increased UA is a strong risk marker for hypertension developed from pre-hypertension in Japanese adults.25 Moreover, pilot clinical studies suggest lowering serum UA levels has been reported to lower blood pressure in pre-hypertensive adolescents.26 Currently, pre-hypertension is common in China. Approximately 20–50% of adults were affected by pre-hypertension worldwide, and this increases the risk of incident hypertension.27 The prevalence of pre-hypertension is rapidly increasing in China, but its causes and associated factors have not been well studied.Previous studies have examined the association between serum UA levels and hypertension, and the results were in agreement with our findings. Sundstrom et al.28 also found that hyperuricemia was associated with pre-hypertension among 1,773 Chinese women aged ≥30. Similar results were presented in a follow-up study, in which Strasak et al.29 demonstrated that serum UA is an independent predictor of all major forms of cardiovascular death in elderly women. The changing levels of serum UA in women at menopause suggests an interaction with sex hormones.30 Research has reported that the gender difference of blood pressure began to appear in adolescence, and pubertal growth spurt occurs earlier for girls than for boys.31 SBP increased significantly more in boys than in girls, while DBP increased more in girls than in boys.32 Other complex physiological and hormonal changes may contribute to hypertension.We observed that serum UA levels were increased linearly in normotension, pre-hypertension and hypertension in females, and this association between serum UA and blood pressure was stronger among females than in males. Besides, serum UA levels were positively associated with DBP, particularly in females. Some previous studies have demonstrated that the association between serum UA levels and hypertension was more pronounced in women. Peng et al.Several limitations must be considered. First, the cross-sectional design used to evaluate the relationship between serum UA and pre-hypertension and hypertension limits our ability to establish a causal relationship. This problem may be solved by longitudinal studies in the future. Secondly, the interaction mechanism between hypertension and increased uric acid has not been explored. Further studies are still needed to examine the potential gender difference of the association between serum UA levels and hypertension in different populations.Our findings suggest that serum UA is significantly associated with pre-hypertension and hypertension, and the association was more robust in the female participants. Therefore, proper early management of UA levels in adults may be important to prevent the development of hypertension.


Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs). The sustained functions of various tissues in the body are maintained by a population of cells with self-renewal ability called adult stem cells (ASCs) or tissue-resident stem cells. These cells are generally multipotent and differentiate into various effector cells, thus replenishing tissues like blood, skin, muscle, etc., throughout life. ASCs are usually more quiescent than their progenies, and they remain such unless the need arises for division or differentiation. Research over the years has identified tissue-resident cells with the varying capacity of self-renewal and differentiation. The identified ASCs so far include mesenchymal stem cells (MSCs), which give rise to osteogenic, adipogenic and chondrogenic lineages; hematopoietic stem cells (HSCs), which replenish cell types of the blood; germline stem cells giving rise to germ cells; epithelial stem cells of the skin, found on the bulge of hair follicles; neural stem cells in the central nervous system; muscle stem cells or satellite stem cells found usually in between the basal membrane and cell membrane of myofibers; stem/progenitor cells in the liver, lungs, cardiac tissue, pancreas etc. These identified adult stem cells could include “true” stem cells or a population of stem and progenitor cells. Research done by different groups has identified different subsets of such cells within the same tissue, making it difficult to give a consistent definition. When it comes to defining tissue-resident stem cells, the general consensus is their potential to self-renew and differentiate. Self-renewal may be defined as their potential to regenerate themselves during each division, and differentiation capacity is their ability to give rise to mature effector cells without losing the original stem cell pool. ASCs are classified as pluripotent, multipotent or unipotent based on their differentiation capacity. The self-renewal and multipotency of stem cells are found to diminish over time due to known and unknown genetic, epigenetic and environmental factors. Ageing is associated with this gradual reduction in the capacity of stem cells to replace old tissue. In the past couple of decades, there has been extensive research going on in the field of stem cell biology, primarily due to their potential in regenerative medicine and therapy. Even after decades of research, we still know very little about their true nature. This review aims to shed some light on adult stem cell dynamics based on In Vivo evidence from mainly four types of adult stem cells: intestinal stem cell (ISC), neural stem cells (NSC), hematopoietic stem cell (HSC) and epidermal stem cell (EpSC). The role of genetic factors is discussed here, emphasizing the insights gained from animal models.The self-renewal and differentiation of ASCs are strictly regulated by an intricate network of cell intrinsic and extrinsic factors. The intrinsic factors regulating stem cells include transcription factors, cell signaling pathways, cel -cycle control, and metabolic pathways . Some ofThe Wnts are a family of secreted glycoproteins that bind to a seven trans-membrane receptor called Frizzled, which may be coupled to a G-protein. The pathway downstream has been found to diverge into at least three different branches, viz. the canonical β-catenin pathway and two non-canonical β-catenin independent pathways: JNK  pathway and Wnt-Calcium pathway . StudiesISC: The Wnt signaling cascade is one of the major controllers of division and differentiation and studies using In Vivo models have shown the immense role they play in ISCs . The firDrosophila model system has also been used to study signaling cascades involved in ISC. Studies involving components of the Wnt signaling pathway like Wnt ligand Wingless (Wg), APC or shaggy  3-β) have reported that ISCs defective for these components show a higher proliferation rate with disrupted homeostasis, hyperplasia, and multilayering of the midgut epithelium. Similarly, reduction in the ligand Wg results in ISC quiescence and differentiation, whereas its overexpression caused an increase in the number of ISCs. It has also been reported that other components of the pathway, like Frizzled, Dishevelled and Armadillo, are necessary for ISC self-renewal in an autonomous manner. There are also suggestions that the Notch pathway operates downstream of Wg and is essential for maintaining the balance between self-renewal and differentiation. The effects of Wnt signaling in Drosophila ISCs are relatively mild compared to other signaling networks. For instance, APC is required for regulating stem cell proliferation but is not necessary for self-renewal or fate specification [fication ,9,10. NSC: Wnt signaling also plays a vital role in regulating neural stem cells (NSCs) . The modAdult neurogenesis occurs in two different niches viz subventricular zone (SVZ) and subgranular zone (SGZ). Wnt signaling plays a major role in regulating the proliferation of neural progenitors in the SVZ. Using the mice model, Adachi et al. have shown that increased β-catenin increases the proliferation of Mash1+ cells (marking type C and a subset of type B cells in SVZ). Pharmacological activation of β-catenin also increased the number of newly formed neurons in the olfactory bulb due to the increased number of neural progenitors in the SVZ . Later sOther In Vivo evidence for the role of Wnt in NSC comes from Zebrafish. Transcriptome analysis of Zebrafish embryonic NSCs to study glycine-mediated differentiation has identified major signaling pathways involved in NSC differentiation, one of which was Wnt signaling along with others like TGF-β, Shh (Sonic Hedgehog) and Calcium signaling . A studySmed-tcf1 has been reported to be primarily expressed in the brain and some stem and progenitor cells that may define the dorsal-lateral neuronal subtypes. Smed-tcf1 RNAi showed regeneration defects in forming proper dorsal-lateral neuronal subtypes, specifically GABAergic neurons. They also exhibited defective negative phototactic behavior and thinner and narrower brain lobes along the medial-lateral axis [Another model system in which the role of Wnt in NSC has been reported is the planarian system. Planarian LEF/TCF (lymphoid enhancer-binding factor/T-cell specific transcription factor) type transcription factor ral axis . Though ral axis . They arral axis . Woundinral axis . The rolral axis .HSC: The role of Wnt signaling has been extensively studied in hematopoietic development . ReportsWnt signaling is required for HSC maintenance in Zebrafish as well ,32. No rEpSC: Wnt signaling is also widely studied in epithelial tissue . One of Another major pathway involved in stem cell regulation is the Delta/Notch pathway. Notch signaling regulates proliferation, fate determination, cell death etc., in animals. Mammals possess four different single trans-membrane Notch receptors, namely Notch1-4. The ligands (Delta and Serrate/Jagged) for Notch are trans-membrane proteins, and thus Notch signaling acts to transduce a short-range paracrine signal. Ligand binding induces cleavage of Notch Intracellular Domain (NCID) which translocates to the nucleus where they regulate transcriptional complexes mainly containing transcription factor CSL . ISC: Notch signaling is essential to maintain tissue homeostasis and self-renewal of the intestine . InhibitDrosophila midgut development from larval to pupal and adult stages. The initial characterization in Drosophila showed that Notch signaling is essential for the differentiation of ISCs into enterocytes or enteroendocrine cells of the intestine. It was observed that the fate specification happens based on the levels of vesicular Delta. ISC having high Delta activates Notch which in turn downregulates Delta in the daughter cells that become enterocytes. ISCs have low levels of cytoplasmic vesicular Delta, which specifies the daughter cells to differentiate into enteroendocrine cells. Notch was also found to be necessary for the fate specifications of ISC daughter cells as well. It was also observed that a reduction in Notch leads to an increase in the midgut progenitors, whereas its activation leads to a decrease in proliferation [Notch pathway regulates feration ,50,51. NSC: Notch signaling plays an essential role in neural stem cells as well . The proDrosophila has shown that Notch signaling is vital in controlling apoptosis of neural progenitors. AbdA  homeobox protein, which provides special cues for cell death, is found to be regulated by Notch. This Notch signaling was activated by Delta present on the neighboring stem cell progenies [Drosophila. Inhibition of Notch in these cells causes a shift from symmetric to asymmetric division [Studies using Zebrafish report that the levels of Notch activity determine the shift between quiescence and dividing states in neural progenitors. Quiescence is promoted by inducing Notch, whereas inhibition of Notch signals causes progenitors to re-enter the dividing state and drives them towards neuronal commitment ,61. Thesrogenies . Furtherdivision . Drosophila and Zebrafish. Notch is necessary for early HSC specification in Zebrafish embryos [HSC: Earlier studies have shown that Notch is active in HSCs and as the cell differentiates, the levels of Notch decreases. Notch signaling is also required for the HSC emergence from hemogenic endothelium during embryonic development . Inducti embryos . Notch a embryos . Similar embryos . EHT is  embryos .EpSC: Notch pathway components are also widely expressed in epidermal stem cells and this pathway mainly regulates differentiation . Early rA recent study using the rabbit ear scar model reports that basic FGF (fibroblast growth factor) reduces scar formation by inhibiting EpSC differentiation towards myofibroblast lineage. This inhibition of differentiation was brought about by the activation of Notch/Jagged1 . This saThe Janus Kinase (JAK)/Signal transducers and activators of transcription (STAT) pathway is one of the conserved pleiotropic signaling cascades which transduces a variety of signals including cytokine signaling and growth factor signaling involved in development and stem cell regulation, normal tissue homeostasis, growth, immunity etc. Janus Kinases are the cell surface receptors which, upon activation, transduce the signal through STAT, which is activated, dimerized, and translocated to the nucleus for further transcriptional control. Drosophila strains to explore JAK/STAT signaling, it was observed that JAK/STAT pathway is normally active in ISCs and their immediate progenitors, but inactive in terminally differentiated cells. Researchers have also shown that lack of this signaling in individual ISC lineages results in failure of cell fate specifications. JAK/STAT signaling is an important regulator of proliferation, but not in self-renewal [ISC: In a study using -renewal   stem cells to neuroblasts (NB) during late fly development. Loss of this signaling causes differentiation in NE stem cells and leads to NE and NB depletion [In epletion ,88,89,90epletion . In anotepletion . There aepletion  and faciepletion .Drosophila definitive hematopoiesis in the lymph glands, JAK/STAT signaling is required for the maintenance of prohemocytes in its undifferentiated state. JAK/STAT signals maintain them in a primitive self-renewing state and differentiation is induced on inactivation of this signaling. JAK/STAT is also required for proliferation and production of lamellocytes, a specific type of blood cell other than the common hemocytes and crystal cells [V617F mice (gain of function mutation associated with neoplasm) show a reduction in the numbers of lineage− Sca-1+c-Kit+ (LSK) cells  cells . They weEpSC: One of the earliest In Vivo studies to investigate the role of JAK/STAT signaling in mice utilized a Cre Lox system to knock out STAT3 in keratinocytes. The report indicates that these mutants showed severe defects in skin remodeling after wound healing and impaired hair cycle. STAT3 disruption does not show any developmental skin defects, however, the second anagen phase was severely impaired with skin fibrosis and inflammatory cell infiltration. Wound healing was severely impaired in these mice with defects in re-epithelialization. This defect was due to defects in epidermal regeneration since granulation, inflammation, and neovascularization after wounding were found to be normal . JAK/STAHippo signaling is a highly conserved cascade generally involved in the control of organ size, cell proliferation, apoptosis, stem cell self-renewal, and cancer. Hippo  is a protein kinase that phosphorylates and activates Warts , which in turn inactivates transcriptional co-activators YAP (Yes-associated protein) and TAZ  by inhibitory phosphorylation. The hippo pathway is generally considered a tumor-suppressor pathway since mutations in this pathway result in an over-proliferation phenotype. Drosophila have established the role of Hippo signaling in regulating organ size and stem cell dynamics. Hippo signaling was initially identified as one of the signaling causing tumorigenesis in Drosophila intestines. The loss of Hippo signaling in precursor cells or epithelial cells stimulates ISC proliferation, suggesting Hippo signaling in adult cells is necessary to control ISC proliferation  in the mesenchyme, where usually undifferentiated progenies are observed. These animals also show an increase in the ζ neoblast  marker expression. Hippo knockdown is also associated with decreased apoptosis, cell cycle arrest, and dedifferentiation of post-mitotic cells . TGF-β family signaling is a major player in the regulation of cell growth, differentiation, and development. The TGF-β superfamily of molecules can be divided into the BMP group and TGF-β group. The binding of a suitable ligand initiates signaling that induces oligomerization of Ser/Thr kinase receptor. This leads to phosphorylation of SMAD2 and 3 (for TGF-β ligands) and SMAD1,5,9 (for BMP pathway). Phosphorylated SMAD proteins are associated with the common SMAD4 and are translocated to the nucleus, where they partner with various transcription factors and regulate gene expression. ISC: The TGF-β signaling pathways play a crucial role in ISCs . BMP sigDrosophila system as well. It was found that the two BMP ligands, Dpp and Gbb, were increased upon injury in Drosophila enterocytes, leading to an elevated BMP signaling in progenitor cells through an autocrine–paracrine mechanism which in turn causes an expansion of self-renewing ISCs. A negative feedback mechanism to maintain the number of ISCs was also observed where the BMP signaling in enterocytes inhibits the Dpp and Gbb production [BMP signaling has been studied in the oduction ,130. Stuoduction ,132.NSC: In the nervous system, TGF-beta signaling plays a variety of roles . It actsIn planarians, a recent study has demonstrated that inhibition of Wnt and TGF-β signaling during growth alters the patterning of mechanosensory neurons—a neural subpopulation that is distributed in accordance with worm size and modulates fission behavior . Hence, +/− or TGF-β type 2 receptor (Tgfbr2)-deficient mice also display a reduced potential for competitive repopulation [−/− mice show an increased number of immature neutrophils, monocytes, and platelets. It is also a necessary pathway for the maintenance of quiescence In Vivo [HSC: The role of TGF-β signaling in HSC has been studied extensively over the past few decades. BMP signaling is essential to maintain HSC niche size, and it also affects the reconstitution ability of HSCs. The role of BMP on HSC could also be indirect. BMP is known to influence the number of osteoblastic cells, which constitute the endosteal niche where more primitive LT-HSCs reside ,138. Usipulation ,142. TGF In Vivo . EpSC: BMP/TGF-β signaling plays an essential role in stem cell activation, differentiation, hair follicle morphogenesis etc. . While tThe zeta-neoblasts  in planarians express various genes depending on their dorsal–ventral position in the animals. The expression of PRDM1-1 is exclusive in dorsal zeta-neoblasts, whereas ventral zeta-neoblasts express Kal-1. It has been demonstrated that bmp4 expression from dorsal muscle cells regulates the polarization of the dorsal–ventral axis in planarians . KnockdoDrosophila and was later found to be important in the developmental context. Three types of Hedgehog proteins are present in mammalian cells, viz. Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh). The membrane receptor for hedgehog, called Patched, inhibits the downstream signaling in the absence of the hedgehog ligand. Binding of hedgehog to Patched lifts the inhibitory effect of Patched, leading to activation of transcription factor Gli.Hedgehog was initially discovered as a mutant involved in segment polarity in + ISC proliferation, disrupted mesenchymal architecture, reductions in numbers of crypt myofibroblast, and increased Wnt signaling [−/− mutants also show a substantial reduction in cell proliferation in the stem cell compartment [ISC: Studies in mice have shown that hedgehog signaling is essential in intestinal stem cell maintenance. Reports show the Indian Hedgehog (Ihh) expression in mice mature colonocytes, and they regulate colonic epithelial differentiation. Colonic epithelium of mice null for Indian hedgehog display multilayered cells which do not form crypts. They also show an increase in epithelial cell proliferation  Figure . Studiesignaling . Shh sigignaling ,152. Othignaling ,154. Thepartment . Here, tDrosophila also indicate that Hedgehog controls ISC proliferation. However, it is suggested that the pathway might not be necessary under normal homeostasis. Reports indicate that Hedgehog plays a major role during stress induced by damage or ageing [Studies in r ageing . An injur ageing . Schmidtea mediterranea causes reduced goblet cell differentiation in the developing intestinal regions of amputated head, tail, and trunk fragments. Reduction in goblet cell numbers was also observed upon Gli1 knockdown in uninjured animals. This effect was more profound in uninjured animals in the anterior, posterior, and lateral intestinal branches compared to the primary medial branch. Therefore, Gli transcription factor might be necessary for the normal homeostatic development of lateral intestinal branches. In contrast, it is essential to develop both lateral and medial branches in regenerating animals [Hedgehog transcription factor Gli1 also regulates the differentiation in planarians. RNAi-mediated knockdown of Gli1 in  animals .NSC: The role of Shh in Neural stem cells came to light after discovering Gli factors in the NSC niche, which are the effectors of Shh signaling. Several studies have identified the role of Shh signaling in maintaining NSC in sub-granular and subventricular zones of the brain. In Vivo experiments have demonstrated that the Shh pathway promotes proliferation, and Shh inhibition blocks proliferation ,161,162 HSC: In the hematopoietic system, hedgehog signaling has been implicated from the developmental stage itself. Studies investigating the signaling pathways involved in the induction of the HSC program in dorsal aorta (DA) in Zebrafish found that hedgehog signaling maintains the arterial program characteristic of the DA roof. Hedgehog signaling restricts the HSC formation to the DA ventral wall through transcriptional activation of tbx20. Transcription factor tbx20 and HSC emergence are mutually exclusive. Tbx20 is restricted to the dorsal side, whereas definitive hematopoiesis initiates at the ventral side. Hedgehog activates tbx20 on the dorsal side, and this distance from hedgehog signaling, along with BMP signaling, is thought to contribute towards the ventral localization of HSC . Similar+/− mice reported that Hedgehog activation expands primitive bone marrow LSK HSCs without affecting mature cells. However, this effect was at the expense of HSCs, resulting in an overall decline in the regenerative capacity due to lack of self-renewal and deregulation of cell-cycle related genes. Inhibition of this signaling was found to rescue the regeneration defect, suggesting hedgehog’s specificity in self-renewal In Vivo [Hedgehog signaling regulates the adult HSCs as well. The Hg regulates the HSC cell cycle during homeostatic as well as during acute regenerating conditions . A study In Vivo . There a In Vivo ,168. EpSc: Shh signaling is essential for the formation of hair follicles present in hair plaques during development . It is a) pathway plays a significant role in a wide variety of multiple and divergent cellular functions, including cell proliferation, self-renewal, differentiation, apoptosis, and survival. MAPK/ERK signaling occurs downstream of various growth factors, cytokines, and hormone signaling, including FGF, EGF , etc., and is well known for its role in cell proliferation. Three major groups of MAP kinases exist: the extracellular signal-regulated kinase (ERK) family, the p38 MAP kinase family, and the c-Jun NH2- terminal kinase (JNK) family.The MAP kinase or MEK (ERK kinase)/ERK  regulates intestinal progenitor cell differentiation. Silencing egfr1 and nrg1 impairs the differentiation of gamma neoblasts (gut lineage committed stem cells) into mature gastrodermal cells (phagocytes and goblet cells), without any effect on gamma neoblast specification. Silencing is also found to increase the number of gut progenitors as a consequence of differentiation failure ,182. NSC: Growth factor signaling through the MEK/ERK pathway influences the proliferation, differentiation, and migration of neural stem cells . A recenThe MEK/ERK pathway is also reported to play a role in regulating gliogenesis in mice. Deleting Mek1/2 in radial progenitors (capable of giving rise to neurons and glia) inhibits their transition to glial fate with reduced glial progenitors in the cortex. These mutants did not exhibit any defect in the formation of neurons. Conversely, overexpression of a constitutively active MEK1 induces precocious glial specification with increased astrocyte numbers, along with reduced neuronal numbers in the cortex . This reThe role of the EGFR signaling pathway in regulating stem cell dynamics in planarians has been recently thoroughly studied and reviewed . In planHSC: In hematopoietic stem cells, the MAP kinase pathway studies mostly concern the pathological conditions and malignancies and are performed in vitro, using isolated stem cells. Several studies indicate the role of MAP kinase signaling in controlling normal and malignant hematopoiesis ,200. SevEpSc: The classical MEK/ERK pathway plays an important role in controlling the proliferation and differentiation in the epidermis . ActivatASC dynamics need precise regulation to avoid any compromise with tissue health. Multiple extrinsic and intrinsic factors influence ASC behavior through genetic and epigenetic regulations. Detailed insights into these regulations would help us understand various pathophysiological processes such as regeneration, degenerative diseases, and ageing. It is well known that several microenvironmental factors influence ASC behavior. Therefore, In Vivo investigations are required to observe the multidimensional behaviors of ASCs in their natural microenvironment that permits their cross-talks with other cell types/ extracellular matrix. However, the accessibility of ASCs is a significant challenge for their In Vivo observations and manipulations. Very few animal models provide easy access to their ASCs for In Vivo investigations. One such model system is the planarians. Planarians possess a pool of ASCs distributed throughout their body and we can conveniently study their dynamics In Vivo inside the animals. Planarians would particularly be useful for studying dynamics of epidermal stem cells, intestinal stem cells, and neural stem cells but not for hair bulge stem cells or hematopoietic stem cells. Molecular techniques to study these animals have evolved significantly in the past two decades. This makes planarians a suitable model for studying ASC behaviors, and further investigations hold enormous promise for contributing novel insights into the current understanding of the regulation of ASC dynamics.


We present a data analysis and review of recent studies regarding the laboratory diagnosis of human T-lymphotropic virus 1 and 2 (HTLV-1/2) infections in Brazil.  Target populations, available diagnostic serological assays (screening and complementary tests), molecular assays (in-house), causes of false-positive and false-negative results, and flowcharts were analyzed.  A table presents the target populations, two diagnostic flowcharts (depending on laboratory infrastructure and study population), and recent research that may improve how HTLV-1/2 is diagnosed in Brazil.  Our results support the implementation of public policies to reduce HTLV-1/2 transmission and its associated diseases.  Retroviridae, followed by HTLV-2. HTLV-1 and -2 share molecular and biological characteristics, such as the integration of viral nucleic acids  into the cellular genome, establishing viral persistence, and maintaining and transmitting the virus. However, they differ in disease outcomes; HTLV-1 is responsible for at least two diseases with high morbidity and mortality, such as HTLV-1-associated myelopathy, and adult T-cell leukemia/lymphoma (ATL), whereas HTLV-2 seems to be less pathogenic. Thus, it is important to perform confirmatory and discriminatory diagnoses of HTLV-1/2 infections. Human T-lymphotropic virus 1 (HTLV-1) was the first human virus described in the family Information on the prevalence of HTLV-1/2 infections in Brazil was recently published in the Epidemiological Bulletin of the Health Surveillance Secretariat, Ministry of HealthAmong vulnerable populations such as intravenous drug users; men who have sex with men; male and female sex workers; and people infected with other sexually transmitted diseases, mostly with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV), high prevalence rates have been detected, varying from 0.7% to 5.3%Sistema Único de Saúde, SUS).Identifying PLHTLV is essential as it enables multidisciplinary follow-up of patients and asymptomatic carriers and allows for the implementation of measures to prevent transmission, thereby reducing the medium- and long-term impacts to the Brazilian Unified Health System . Routine screening assays detect antibodies against HTLV-1/2; however, in addition to the probability of false-positives, screening assays do not differentiate between the two infections, thereby requiring confirmation of the results by highly specific tests that can distinguish infection by each viral type. Confirmatory molecular assays have been designed to identify segments of the proviral genome of HTLV-1/2  in mononuclear cells present in blood and cerebrospinal fluid, among other specimens.SUS for individuals with ATLHTLV serology became mandatory in blood banks in Brazil in 1993Specific populations and contexts are associated with an increased risk of HTLV-1/2 infections. Therefore, testing these individuals is recommended . Notably,,SUS by more than 70%. In contrast, nonreactive results in routine screenings and true positives for HTLV-1/2 or for proteins produced by the HTLV genome may occur, although in small numbers, mostly when using reagents containing only recombinant proteins and/or synthetic peptides of the HTLV-1/2 viral envelope,Tax-only carriers, who are seronegative in an HTLV-1/2 CLIA screening assay, a particle agglutination assay, and an in-house immunofluorescence assay on the MT-2 cell lineCurrently, third-generation or sandwich-type immunoenzimatic screening tests (enzyme immunoassay [EIA] or enzyme-linked immunosorbent assay [ELISA] and chemiluminescence (chemiluminescent immunoassay [CLIA]) tests are available in the national market. They contain synthetic peptides and/or recombinant or chimeric proteins of HTLV-1/2 as antigens and conjugates, and the conjugate is labeled with enzymes (EIAs) or acridinium (CLIAs). These have high sensitivity (close to 100%) and specificity (ranging from 92.0% to 99.5%), depending on the population and the employed testenv [rgp46], gag [p19], and pX [Tax]) may increase the sensitivity of screening tests . In addition, chimeric antigen for each type of HTLV may reduce false-positive results due to the absence of p24 antigens in individuals who are truly infected with HTLV-1The use of a chimeric antigen containing recombinant proteins from three regions of the proviral genomes of HTLV-1/2 , and HBV and HCV have already been implemented by the Brazilian Ministry of HealthThe development of a point-of-care serological test with high sensitivity and specificity is of global urgency, as highlighted in the 2021 HTLV technical report issued by the World Health Organization,There are two types of complementary (confirmatory and discriminatory tests for HTLV-1/2 infections) tests: serological and molecular. Serological complementary tests examine the presence of antibodies against specific HTLV-1/2 antigens. The tests available in the national market include the WB assay, which uses nitrocellulose strips containing HTLV-1 whole viral lysate and recombinant HTLV-1 (rgp 46-I), HTLV-2 (rgp 46-II) envelope proteins, and the transmembrane glycoprotein common to HTLV-1/2 (GD 21), and the LIA, which identifies synthetic peptides and recombinant proteins common to both viruses and specific to each virus adsorbed on nylon strips. These complementary tests have different sensitivities and can generate inconclusive results (untyped HTLV and indeterminate), especially when applied to samples obtained from individuals with HTLV-2 infections who are coinfected with HIV, HBV, or HCV. In these populations, the LIA sensitivity varied from 94.8% to 97.2% and the specificity was approximately 82.0%, while the WB sensitivity varied from 82.4% to 82.8% with a specificity of approximately 60.0%; thus, in these cases, LIA is the test of choice,,-Inconclusive results in complementary serological tests may be associated with different factors, including (1) the period of seroconversion, (2) stringent manufacturer positivity criteria, (3) HTLV strains distinct from those used in the tests, (4) the presence of defective viral particles, and (5) point mutations in the long terminal repeat viral promoter region and regions that encode structural and regulatory proteins, mainly in the HTLV-2 viral envelope, among othersComplementary tests using molecular techniques can identify the proviral DNA of HTLV-1/2 because HTLV maintains infection mainly by clonal expansion and the presence of viral RNA is extremely rare in the plasma. These tests are based on polymerase chain reaction (PCR) in various formats: nested PCR, PCR followed by a restriction enzyme site search resulting in fragments of different sizes , real-time or quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP).pol and tax segments) were used. In Brazil, commercial assays are not available, only in-house assays, many of which have not been validated. There is no consensus on which test has the best performance, especially for HTLV-2 strains that circulate in Brazil. Moreover, depending on the study population, positivity percentages differ from more than 95% for individuals with HTLV-1 monoinfection and close to 60% for the HIV-coinfected population,-SUS ,In all molecular assays, oligonucleotides (primers and/or probes) specific to conserved regions of the proviral genome of HTLV-1/2 . This approach differs from that described previously in Brazil, which was standardized using only one master mix reagent and one specific type of equipment,,qPCR assays can be used for both the diagnosis and monitoring of proviral loads in patients with symptoms related mainly to HTLV-1 infectionRegarding an HTLV diagnostic flowchart applicable to Brazil, laboratories without a molecular biology infrastructure can confirm results using the WB or LIA serological tests, preferably LIA due to the higher sensitivity shown for HTLV-1 monoinfection and HTLV-1/2 coinfections with other viruses,,When samples have inconclusive results in complementary serological tests and the laboratory is not able to perform molecular assays, it is recommended to retest the patient after three months, as the first test may have been conducted during the period of seroconversion . If the These flowcharts apply to the general population, pregnant women, and children over 18 months of age if they are not breastfeeding. Pregnancy does not interfere with the detection of anti-HTLV antibodies or with the detection of HTLV-1 proviral genome segmentsAlthough there are gaps, the current Brazilian experience with HTLV diagnosis can provide support for health authorities and managers in the implementation of reliable tests for the surveillance and diagnosis of HTLV-1/2. This may help to reduce transmission, provide adequate treatment and care for PLHTLV, and implement compulsory notification of HTLV-1/2 infections. A nationwide multicenter validation study of in-house molecular tests is important and should be considered a priority for improving the laboratory diagnosis of HTLV in Brazil.


More specifically, a very strong linear relationship for the peak area for the co-existing I− and IO3− ions was reached, and a high resolution value between two peaks was observed, which proves the effectiveness of our combined IC-ICP-OES method at analyzing iodine species. We observed lower accessible porosity for the diffusion of both I− and IO3− in samples of bentonite clay using IC-ICP-OES detection methods, where the effective diffusion coefficient varied based on the anion exclusion effect and the size of the diffusing molecules. In fact, the distribution coefficients (dK) of both I− and IO3− were close to 0, which indicates that there was no adsorption on bentonite clay. This finding can be explained by the fact that no change in speciation took place during the diffusion of I− and IO3− ions in bentonite clay. Our IC-ICP-OES method can be used to estimate the diffusion coefficients of various iodine species in natural environments.An accurate and effective method combining ion chromatography (IC) and inductively coupled plasma optical emission spectrometry (ICP-OES) was applied in this work to qualitatively and quantitatively analyze individual and co-existing iodide (I The design of a deep geological repository for spent fuel or high-level radioactive waste (HLW) generally adopts a “multi-barrier system”. Generally, the underground facilities, waste containers, and buffer/backfill materials  are known as engineering barriers, and the surrounding geological formations are known as natural barriers. In such a system, the engineering barriers and the geological medium serve a dual purpose: they ensure both the containment of the source term and the protection of the biosphere. In order to retard radionuclide release, the materials used in the construction of an HLW repository must have specific properties that help to prevent the dispersion of radioactivity. Such design parameters make it possible both to delay the time required for the radionuclides to reach the biosphere and to reduce their concentration by means of retarding mechanisms, including sorption, diffusion, and dispersion, so as to ensure the safety of the final disposal of radioactive waste ,2.Bentonite clay is just such an engineering barrier that has often been used as a buffer/backfill material because of its retention effect on the migration of radionuclides to the biosphere ,3. Condu129I, 99Tc, and 79Se [129I is characterized by a long half-life (t1/2 = 1.57 × 107 years), a high fission yield, easy volatilization, easy migration, a high radioactive toxicity, and a high bioavailability. Furthermore, the pH of and the redox potential in aqueous solutions determines the form that iodine takes in the environment, including iodide (I−), iodate (IO3−), I2, and organic iodine. Thus, 129I is the main source of potential risk posed, not only by HLW repositories, but also by nuclear accidents such as the 2011 Fukushima Daiichi nuclear disaster. This threat has recently been made obvious when 129I was observed in the Pacific Ocean around Japan, where fast dispersion by the movement of the seawater and deposition caused it to migrate into the marine sediments [In addition to transuranic elements (TRU), the possible environmental impact of long-lived radionuclides that exhibit a relatively high mobility under certain geochemical conditions must be taken into consideration when designing an HLW repository. In particular, weakly adsorbed radionuclide anions are the most important, for example and 79Se ,11,12. Aediments .129I, which can exist in various forms, of which I− and IO3−are the main anions found in groundwater [− and IO3− in the bentonite clay buffer is considered a major issue for the assessment of the possibility of the release of radionuclides into the environment from an HLW repository.While carrying out a safety assessment (SA) for the disposal of HLW, numerical models should be used in order to consider and evaluate a variety of scenarios, including the possibility of radionuclides being released into the environment and thus exposing people to radiation, as in the scenario in which radionuclides migrate into groundwater following the failure of the multi-barrier system after 10,000 years . In factundwater ,16. TherdK values, and diffusion coefficients under various conditions. For example, some research has recorded very low dK values (~0) [125I−: 5.1 ± 0.8 × 10−11 m2 s−1 [− and IO3−), on the one hand, because of the difficulty of separating the iodine from the environmental matrix and, on the other, because of the high cost of achieving accurate detection with a combination of advanced instrumental methods such as accelerator mass spectrometry (AMS), nanoscale secondary ion mass spectrometry (NanoSIMS), and inductively coupled plasma mass spectrometry (ICP-MS) [Several previous studies on fission products have been performed to calculate sorption and diffusion parameters, ues (~0) ,18. Anot1 m2 s−1 . Moreove1 m2 s−1 ,20. Fina(ICP-MS) ,16,21,22− and IO3− by combining IC and ICP-OES to create IC-ICP-OES. More specifically, we investigated the variability of the concentrations of I− and IO3−, both as individual ions and as co-existing ions, separating and analyzing them by calibrating the IC-ICP-OES to be able to carry out both qualitative and quantitative analyses. We also performed through-diffusion experiments to examine the diffusion behavior of I− and IO3− in compacted bentonite clay based on their diffusion coefficients. Our experiments with the IC-ICP-OES can serve as an important reference case while carrying out future SAs of engineering barriers used in HLW repositories in China.In recent years, ion chromatography (IC) has become a popular and effective method applied to the analysis of anion speciation in environmental samples . AnotherThe material tested in this work was a sample of Wyoming Na-bentonite clay purchased from American Colloid Company. It had a montmorillonite content of approximately 85–90 wt.%. Previous studies have investigated its mineralogical and chemical properties using X-ray diffraction analysis techniques to identify several minor mineral components such as quartz and feldspar ,24. In t® ultrapure water has a resistivity of 18.2 MΩ.cm . A standardized potassium iodide (KI) solution containing 1.20 ± 0.05 g of KI and a potassium iodate (KIO3) solution containing 1.30 ± 0.05 g of KIO3 were prepared in deionized water and 3–5% of nitric acid (HNO3). Finally, these solutions were diluted 100 times with deionized water to 1 L in a volumetric flask. The concentrations of I− and IO3− in the diluted solutions were measured and analyzed by means of IC and ICP-OES  to the point at which the variation of element concentrations was within 5% of the corresponding liquid phase concentrations.All chemical reagents used in our experiments were of analytical grade of purity, and the Milli-Q− and IO3− anions could be effectively detected and separated through the combination of IC and ICP-OES techniques. In addition, an ICS600 system  was equipped with a gradient pump, an injection valve with a 10 μL sample loop, and an anion-exchange column (250 mm × 4.6 mm inner diameter). Moreover, the eluent solution used in the IC procedure consisted of 1.0 mM NaHCO3 and 3.2 mM Na2CO3, with degassing performed with a DG-2410 degasser  before the injection of individual and co-existing I− and IO3− ions. Finally, the resolution (R) was calculated based on the peak areas and retention times:1 and M2 represent the peak areas of I− and IO3−, respectively; a is the peak width at 5% of the peak height (for I− and IO3−); and s is the time difference between the peak heights of I− and IO3−. The results showed that two peaks could be effectively separated and identified at R > 0.5.We used an IC instrument equipped with a conductivity detector to analyze the different iodine species, and then we quantitatively measured them with ICP-OES. By deriving an analytical solution, the I2, respectively. The sample slab was inserted and fixed in a holder and was sandwiched between two glass microfiber filters (0.4 mm thickness) with pore size of 0.7 um , a Teflon O-ring, and constrainers. 125I− and 134Cs+ [We developed and verified a precise and reliable apparatus with sandwich-like columns to conduct through-diffusion (TD) experiments, as shown in d 134Cs+ .− (C0 = 1000 ± 50 ppm) and IO3− (C0 = 1000 ± 50 ppm) ions, and various Teflon® (PTFE) units and connectors. The PP columns were made of pressure-resistant polypropylene (<10 MPa) and had a length of 13.6 cm and an inner diameter of 5 cm. They were filled with bentonite clay having a total porosity of 0.26 and a bulk density of 2.0 g/cm3.The setup consisted mainly of a highly precise multichannel isocratic peristaltic pump  for use with two polypropylene (PP) columns , an initial tracer reservoir containing individual ID) for I− and IO3− in compacted bentonite clay. The one-dimensional diffusion equation for the radionuclides can be written as follows:eD and aD are the effective and apparent diffusion coefficients, respectively; α (capacity factor) is a function of the bulk density of the dry material (bρ), the total porosity of compacted samples (θ), and the distribution coefficient dK; and C is the concentration of the solute in the liquid phase. The rate at which molecules spread determines the diffusion coefficient (D), which is the proportionality constant between the mass flux and the concentration gradient of the solute. Both boundary and initial conditions limit the through-diffusion experimental method and can be expressed as follows:C0); the ion concentration in the opposing reservoir is kept close to zero; and L is the overall duration of the compaction of the clay samples.In this work, Fick’s second law was applied to estimate the diffusion coefficients  into consideration and V and S are the diffusion volume and the cross-section area of the compacted samples, respectively. When sufficient time is allowed to carry out the TD test, the diffusion process reaches a steady state. Consequently, the exponential term in Equation (3) tends to zero, and the profile curve illustrating the distribution of the CR(t) shows a linear relationship with time (t). Moreover, using a method for statistical data analysis that had previously been developed [− and IO3− and evaluated them in terms of the steady-state diffusion condition and its relation to time. The combination of the IC and ICP-OES techniques makes it possible to determine whether or not the speciation of I− and IO3− changes during the diffusion process. Therefore, using both techniques while concurrently performing experimental and numerical analyses of I− and IO3− provides an effective and important tool for carrying out future safety assessments of HLW repositories.As defined by Crank , the cumeveloped , we comp− and IO3− in bentonite clay. More specifically, the standard ASTM batch method [− and IO3− in the bentonite was observed by measuring the pH, Eh, and the final ion concentrations. In order to introduce stable isotope tracers, an individual I− and IO3− stock solution was added to the initial bentonite solution prior to the batch testing. The I− and IO3− stock solution was prepared at initial concentrations (C0) of 5, 10, and 20 ppm. All batch tests were conducted on solutions with a solid–liquid ratio of 1 g per 30 mL. The centrifuge tubes were placed in an oscillating thermostatic shaker, and the solutions were uniformly mixed at 200 rpm for 7 days. The solid and liquid phases were separated by being put in a high-speed centrifuge  for 30 min. Approximately 10 mL of the supernatant was reserved to analyze the final concentrations of the I− and IO3− by means of a combination of the IC and off-line ICP-OES techniques, at which time changes in pH and Eh were also recorded. As a result, the distribution coefficient (dK) for I− and IO3− can be written as follows:C0 is the initial ion concentration of I− (or IO3−); C is the final ion concentration; and V and m are the volume (mL) and mass (g) of the samples, respectively.We performed a simulation of batch sorption experiments with Ih method  was appl− and IO3− ions were injected into an IC system, with measurements simultaneously taken by a conductivity detector and ICP-OES equipment and elution performed with a solution of 1.0 mM NaHCO3 and 3.2 mM Na2CO3  containing various concentrations of I ppm See . This fi− and IO3− ions (− and IO3− ions was reached (0.99), and a high resolution value (R: 52.30~79.36) between two peaks was observed, which proves that IC provided a very effective qualitative and quantitative analysis of the two forms of iodine investigated in this study.IC was also used to analyze solutions with different concentrations of co-existing IO3− ions . The res− and IO3− ions to diffuse out of the bentonite clay was approximately 24 h, with the steady-state condition reached in 7 to 10 days as a result of a constant diffusion flux. The effective diffusion coefficient (eD) and the capacity factor (α) for the I− and IO3− ions in compacted bentonite were estimated on the basis of numerical analyses, including calculating graphical asymptotes, finding analytical solutions with Lsqcurvefit, and modeling with dual period diffusion model (DPDM) [− and IO3− ions in bentonite clay could be suitable fitting, which can be explained by the fact that no change in speciation was detected by means of the IC-ICP-OES technique.The results of the TD experiments with our IC-ICP-OES method indicate that the time required for the Il (DPDM) ,28 (See − and IO3− in three samples of bentonite clay reached the steady-state condition in approximately 7 days. dK) for the I− and IO3− ions in compacted bentonite. Our findings indicate that no sorption of I− and IO3− has taken place in the bentonite clay samples, which can be explained by the fact that no change in speciation was detected by means of the IC-ICP-OES technique.The batch experiments conducted with the IC-ICP-OES method indicated that the adsorption of I3− ions were consistently detected first, after which individual I− was extracted by elution from the solution containing co-existing I− and IO3− ions. Generally, the affinity of the anion exchange resin is quite different for I− and IO3−, with I− being subject to a higher adsorption affinity and anion exchange and showing a slower retention time (~20 min) than IO3− (~4 min) during the elution process. In fact, it has been shown that some ions interfere with the IC-based detection of I− and IO3− ions in complex and natural environments [− and IO3− ions in solutions at various concentrations.In this work, individual IOronments ,29, a pr3H2O), a non-reactive radiotracer, was used to estimate the total porosity  to both I− and IO3− ions was lower than that accessible to HTO, which was determined on the basis of the anion exclusion effect (dK~0) [− and IO3− ions have a similar mobility as does 3H2O when the effective diffusion coefficients (De) are compared. Moreover, there were significant differences between the anions present in bentonite clay with regards to the exclusion effect, which has been associated with the size or hydrated radius of I− (~2.16 Å), IO3− (~3.30 Å), and 99TcO4− (~3.50 Å) [acc) to SeO32− (IV) might be higher than that accessible to 3H2O as a result of the anion exclusion effect and the weak adsorption effect (dK > 0) [− and IO3− anions were investigated in this paper and the 99TcO4− and SeO32− (IV) anions were examined in previous research [In this study, tritiated water (t (Kd~0) ,25 and tt (Kd~0) ,30,31. T~3.50 Å) ,33. On t(Kd > 0) . The parresearch ,25.− and IO3− ions through a combination of ion chromatography (IC) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques, which we call IC-ICP-OES. The anion exchange resin exhibited a different affinity for I− and IO3−, with I− being subject to a higher adsorption affinity and anion exchange. This finding demonstrates that ICP-OES was able to identify the I− and IO3− ions as forms of iodine after linear calibration had been performed. In fact, there was obvious interference with the performance of the conductivity detector during the IC-based I− and IO3− analysis procedure, which suggested the possibility of combining IC with an offline ICP-OES detection system to obtain accurate measurements of I− and IO3− chromatographic peaks at different retention times. Therefore, an analysis of the diffusion of I− and IO3− ions in bentonite clay was performed, with the results showing that lower accessible porosity was related to the anion exclusion effect and the size or hydrated radius of the ions. The effective diffusion coefficient (eD) and the capacity factor (α) obtained in this study could be used as input data for future safety assessments of radioactive waste disposal facilities. In conclusion, 129I anions, in the form of I− and IO3−, are radionuclides that are critical factors to take into account when carrying out safety assessments of deep geological repositories for HLW, which should be a focus of future R&D studies. In addition, our results confirm that Wyoming bentonite clay shows potential for use as a buffer/backfill material to counter the diffusion of radionuclides from containers used for storing HLW.In this paper, we developed a relatively simple, effective, and reliable method to detect individual and co-existing IWe have been relying on nuclear power for decades to meet our electricity needs. Today, governments around the word continue to invest in nuclear technology as part of their strategies to combat climate change and transition to low-carbon economies. Our role is to ensure there is a safe, long-term solution to the obstacles facing the containment and isolation of any spent nuclear fuel created by nuclear power generation, including what already exists today and any produced in the future.


There were no significant differences in the time intervals between the discontinuation of anesthetics, first response, and extubation. Although flumazenil did not reduce the incidence of emergence agitation in patients undergoing orthognathic surgery with sevoflurane anesthesia, it can be considered as an option for awakening patients in terms of improving emergence profiles.Flumazenil, a gamma-aminobutyric acid receptor antagonist, can promote arousal even under general anesthesia without the use of benzodiazepines. We hypothesized that flumazenil could promote arousal and reduce emergence agitation in patients undergoing orthognathic surgery with sevoflurane anesthesia. One hundred and two patients were randomly allocated to the control or flumazenil group. Saline or flumazenil was administered at the end of the surgery. The incidence of emergence agitation was measured by using Aono’s four-point scale, with scores of 3 and 4 indicating emergence agitation. The primary outcome was the incidence of emergence agitation. Secondary outcomes included duration of emergence agitation and time intervals between the discontinuation of anesthetics, first response, extubation, and post-anesthesia care-unit discharge readiness. The incidence of emergence agitation was 58.3% and 38.9% in the control and flumazenil groups, respectively, but it was not statistically significant. However, the duration of emergence agitation was shorter in the flumazenil group ( Emergence agitation is characterized by confusion, restlessness, and hyperactive physical behavior resulting from a temporary loss of consciousness during recovery from general anesthesia. It has an incidence of 0.25% to 90.5% and is considered a very important dilemma in postoperative care because it increases the risk of self-extubation, unintended removal of catheters, surgical site injury, and postoperative bleeding ,2. The rIn a previous study, when desflurane was used during orthognathic surgery, rapid awakening resulted in early situational awareness; therefore, emergence agitation was reduced compared to sevoflurane . HoweverMeanwhile, flumazenil is a competitive inhibitor of γ-aminobutyric acid (GABA) receptors and reverses the hypnotic and sedative actions of benzodiazepines acting on GABA receptors. Recent studies have reported that arousal is promoted when flumazenil is administered, even when general anesthesia is administered through inhalation anesthetics or propofol and remifentanil ,9,10,11.This study was approved by the Institutional Review Board of Chung-Ang University Hospital, Seoul, Korea  and was registered with Clinical Research Information Service (#KCT0006209). The American Society of Anesthesiologists physical status I or II patients aged 18–65 years who were scheduled for elective orthognathic surgery under general anesthesia were recruited. Patients weighing less than 45 kg or more than 100 kg, and those who took medications for neurological/psychological disorder, cardiopulmonary disease, or a history of these were excluded. Written informed consent was obtained from all participants. We fully explained the fact that all subjects may feel a sense of suffocation due to orofacial edema and the presence of a nasotracheal tube during arousal from anesthesia after surgery, and that severe discomfort may be experienced. Participants were blinded to their allocation group.According to a computer-generated table of random numbers, using randomization block size 6, participants were allocated to either the control (group C) or the flumazenil (group F) group randomly. Group allocations were sealed in sequentially numbered opaque envelopes by an investigator who was not involved in the perioperative management and outcome assessment. Another investigator who did not assess the outcome opened the envelope and prepared the experimental drug in a 5 cc syringe by the group allocation, which was either normal saline (3 mL) or flumazenil .2O in oxygen was used to maintain anesthesia. The respiratory rate was regulated to maintain ETCO2 between 30 and 35 mmHg, and mechanical ventilation was controlled with a tidal volume of 6 mL/kg. Age-corrected minimal alveolar concentration (MAC) was monitored continuously by using a Carestation 650 A1 ventilator . Sevoflurane with oxygen/nitrous oxide in 0.5 FiO2 was administered at a 1.5–2.0 MAC to maintain anesthesia. Orthognathic surgery was performed by an experienced surgical team who was blinded to the group assignments. Before incision, a combination of 2% lidocaine and 1:200,000 epinephrine was administered to the oral submucosa. For bimaxillary orthognathic surgery, Le Fort I osteotomy was performed first, followed by short lingual osteotomy of the mandible. If tachycardia (>110 beats/min) and elevated blood pressure (systolic blood pressure > 120 mm/Hg) persisted even after increasing sevoflurane up to 2.0 MAC during the operation, additional fentanyl (25 µg) was administered intravenously twice, followed by labetaolol at 5 mg. Phenylephrine (50 mg) for hypotension , esmolol 20 mg for tachycardia (>110 beats/min), and glycopyrrolate (0.2 mg) for bradycardia (<40 beats/min) were intravenously administered as required. The standard monitoring methods, which included electrocardiography, non-invasive arterial pressure, pulse oximetry, and neuromuscular transmission, were applied after the patient entered the operating room. Propofol (2 mg/kg), fentanyl (2 µg/kg), and rocuronium (0.6 mg/kg) were administered intravenously to induce general anesthesia. Nasotracheal intubation was performed by using a 6.0 mm or 7.0 mm tracheal tube for women and men each. Then, with a steady fresh gas flow, sevoflurane with 50% N2 of 40 to 45 mmHg. Before the end of the operation, a syringe with the experimental drug was delivered to an anesthesia provider who was blinded to the group. At the completion of surgery, all anesthetics were discontinued with a fresh gas flow of 100% O2. Normal saline or flumazenil, sugammadex (2 mg/kg), and ramosetron (0.3 mg) were injected intravenously. During emergence, patients were not disturbed, except for gentle oral suction and repeated verbal requests to open their eyes. Once patients responded to verbal commands by opening their eyes or nodding their heads, and sufficient spontaneous breathing had been confirmed, extubation was performed. Subsequently, they were transferred to the PACU.Approximately 30 min before the end of surgery, fentanyl 1 µg/kg was intravenously administered, and sevoflurane was reduced to decrease the concentration to 1.0 MAC until the end of surgery. After confirmation that the train-of-four count was greater than 2, ventilation was controlled so that the patients could breathe spontaneously while maintaining an ETCOAn outcome assessor who was blinded to the patient group allocation continuously evaluated the emergence state, using a four-point categorical scale based on Aono’s scale: 1 = calm; 2 = not calm, but can be easily calmed by verbal instructions and able to tolerate ordinary fixation straps for both arms and legs; 3 = not calm despite frequent verbal instructions, moderately agitated or restless, and requires physical restraint; 4 = combative, excited, disoriented, and strongly requires physical restraint. Scores of 3 and 4 were regarded as emergence agitation ,12. OnceIn the PACU, an 11-point numeric scale, ranging from 0 (no pain) to 10 (worst pain imaginable), was used to evaluate postoperative pain. When a patient in the PACU complained of an NRS score of 5 or higher, 20 mg of nefopam was mixed with 100 mL of normal saline and administered intravenously. In the case of postoperative nausea or vomiting, 10 mg of metoclopramide was administered. The time when the modified Aldrete score exceeded 9 was recorded as PACU discharge readiness time, and all participants were questioned if they remembered their status during the recovery phase. The primary outcome measure was used to calculate sample size. In a previous study, the incidence of emergence agitation in the sevoflurane group was 70.8% . Assumint-test, and non-normally distributed data were analyzed by using non-parametric tests. Descriptive variables were subjected to χ2 analysis or Fisher’s exact test, and statistical significance was set at p < 0.05. Data are presented as mean ± standard deviation (SD) or absolute number (%).Statistical analyses were conducted by using the SPSS ver. 23.0 . For intergroup comparisons, the distribution of data was first evaluated for normality, using the Shapiro–Wilk test. Normally distributed data were compared by using Student’s p = 0.012) (p = 0.04)  . There w = 0.04) . Pain scIn this study, the incidence of emergence agitation showed a lower tendency when flumazenil was administered (58.3% vs. 38.9%), without reaching statistical significance. Instead, the duration of emergence agitation was shorter in the flumazenil group (2.5 ± 1.6 min) than in the control group (1.5 ± 0.8 min). There were no significant differences in the time to first response, extubation, and time interval between the first response and extubation, but they were slightly shorter in the flumazenil group. The time to PACU discharge was significantly shorter in the flumazenil group. This is thought to be caused by the antagonistic effect of flumazenil in patients under general anesthesia. In fact, several studies revealed that patients who were anesthetized with intravenous or inhalation anesthetics had faster recovery and reversal of amnesia when flumazenil was administered ,9,10. In Kim’s study, patients were anesthetized with sevoflurane/fentanyl, and 0.3 mg of flumazenil was administered in the same way as in this study. Spontaneous breathing, eye opening, and hand squeezing on verbal command, extubation, and date-of-birth recollection were significantly faster when flumazenil was administered . The timThere are three possible mechanisms to explain the action of flumazenil in patients under general anesthesia without benzodiazepine administration. First, it is related to the GABA type A receptor, which is part of an important cellular mechanism involved in the unconsciousness, and anesthetics target this part of the central nervous system (CNS) . It is nMeanwhile, the characteristics and mechanisms of emergence agitation in adults are considered different from those in children. Emergence agitation in children is defined as a disturbance in a child’s awareness of attention to his or her environment, with disorientation and perceptual alteration, including hypersensitivity to stimuli and hyperactive motor behavior in the immediate post-anesthesia period, and it occurs frequently in the PACU ,22. SevoChoi et al. concluded that, in contrast to children, adults with faster recovery of consciousness experience less agitation because they were given a detailed explanation of emergence prior to anesthesia and their rapid recognition of the post-operative environment. This might have led to a significantly lower incidence in the desflurane group than in the sevoflurane group . In ChoiEmergence agitation is a hyperactive state that is related to the light plane of anesthetic depth. Therefore, we analyzed the time of arousal divided into time to first reaction and first reaction to extubation in order to differentiate the emergence phase into the phase of the light and deep planes. Both times were slightly shorter in the flumazenil group, but the difference was not significant. The time to PACU discharge readiness was reduced in the flumazenil group. It can be supposed that flumazenil accelerates awakening in patients throughout the overall phase, but the effect is weak and not sufficient to shorten each phase of arousal. In addition to not reducing the time to extubation sufficiently, flumazenil’s failure to shorten the phase of the light plane could be another reason for not reducing the incidence. Besides the reasons mentioned above, the reason why the incidence did not decrease is that flumazenil also has a partial benzodiazepine agonist-like effect. Several studies have shown that flumazenil has agonist activity against the GABA type A receptor and can potentiate the hypnotic activity of propofol ,26,27,28However, when flumazenil was administered, the duration of emergence was significantly reduced. If emergence agitation is interpreted as a patient expressing discomfort in the light plane, the stimulus due to discomfort itself can promote awakening of the patient. In this dynamic situation, although the effect of flumazenil was insufficient to reduce the incidence or time to extubation, it is thought that the arousal effect of flumazenil was accentuated and caused a decrease in duration. 2O. The effect of flumazenil on emergence agitation in various situations may differ depending on the anesthetic method and the type of surgery. Further studies are needed.There were some limitations to this study. Emergence agitation, according to Aono’s scale, refers to a state in which physical restraint is required and cannot be pacified with words, but scoring may vary depending on the evaluator, as it is a subjective evaluation. This may have led to the different agitation incidence of sevoflurane from the previous study. In a previous study by Choi et al., the incidence was 71%, whereas it was 58.3% in the control group in this study [Although flumazenil did not reduce the incidence of emergence agitation in patients undergoing orthognathic surgery with sevoflurane anesthesia, it can be considered an option for awakening patients in terms of improving emergence profiles.