Cases due to alcohol or substance abuse were excluded. Controls were selected on a randomized basis from the remaining patients (n = 128). All laboratory test parameters included in the routine blood and urine tests of the emergency department were collected. Results: The mean age of the patients was 81.24 ± 7.51 years, and 56.2% were males, while the mean age of the controls was 78.97 ± 7.99 years, and 45.3% were males. Significant differences were found between the cases and controls in relation to the following parameters: urea 43 (32–58) mg/dL versus 50 (37–66) mg/dL, respectively; neutrophils 69.6 (62.05–78.75)% versus 75.5 (65.1–83.2)%; monocytes 8.7 (7–10.4)% versus 7.6 (5.5–9.2)%; platelets 213 (159–266) × 109/L versus 224 (182–289) × 109/L; neutrophil–lymphocyte ratio 3.88 (2.45–7.07) versus 5 (2.75–8.83); platelet–lymphocyte ratio 281.4 (210–360) versus 357.1 (257.8–457.1); and mean platelet volume 10.6 (10–11.5) fl versus 10.4 (9.67–10.9) fl. Although the mean values were above desirable levels in both groups, they were higher for most parameters in the control group. No significant differences were observed in C-reactive protein concentration (9.99 (1.69–51) mg/L versus 12.3 (3.09–65.97) mg/L). Conclusions: The identification of delirium biomarkers poses difficulties due to the urgent nature of the disorders found in older people admitted to the emergency department. Research in this field is needed, since it would allow early identification and treatment of delirium.Background: The identification of biomarkers associated with delirium in the emergency department could contribute to the understanding, prediction and diagnosis of this disorder. The present study was carried out to identify biomarkers included in easily and quickly obtained standard blood examinations in older patients with delirium in the emergency department. Methods: A case–control study was carried out in the emergency department of Francesc de Borja Hospital . Older adults (≥65 years of age) diagnosed with delirium ( Delirium is a severe neuropsychiatric syndrome , definedDelirium in the emergency department (ED) is associated with a longer hospital stay, functional and cognitive impairment, an increased risk of falls and a greater likelihood of institutionalization, ultimately with an increase in patient morbidity and mortality ,5.In recent years, the detection of delirium in the ED has become a high-performance research objective, since it is an underdiagnosed disorder . StudiesIn addition to knowledge of the predisposing and triggering risk factors, comorbidities and drug therapies, alterations of certain blood parameters could contribute to the recognition of delirium . DiffereThere is little evidence in the literature on the clinical use of any delirium biomarkers, though some markers such as S-100 beta, insulin-like growth factor-1, cortisol and C-reactive protein (CRP) have shown some promising results ,3. The lThe lack of studies on the prevalence of delirium and its detection rates in the ED  make theA retrospective, unpaired case–control study was conducted to establish possible biomarkers for the detection of delirium in older persons (≥65 years of age) admitted to the ED of Francesc de Borja Hospital —a secondary, academic hospital serving a population of 188,000 inhabitants. The study has followed the STROBE guidelines for reporting in observational studies .The patients included in this study were aged 65 years or older and reported to the ED between 1 January and 31 December 2020. Cases were subjects coded according to the International Classification of Diseases—Ninth Revision (ICD-9) with delirium in the ED  as either a primary or secondary diagnosis recorded in the electronic medical record. The delirium indicator variable was obtained by the treating physician based on the DSM-V criteria . Cases o® tool.Once the cases were identified, the controls were randomly selected from the remaining patients meeting the inclusion criteria through computer-based block randomization using the XLstatA representative sample of 117 subjects per group was calculated for the unpaired case–control study, assuming an expected proportion of delirium in the patient group of 9% versus 1% among the cases, for an alpha error of 5% and a statistical power of 80%.All data were collected from the electronic medical records: sociodemographic data, age and sex, and the number of drugs prescribed together with the main pharmacotherapeutic groups related to delirium . The medAll laboratory test parameters included in the routine blood tests of the ED were collected. Biochemistry: glucose, urea, creatinine, sodium, potassium, chloride, total bilirubin, amylase, GOT/AST, GPT/ALT, cholinesterase and C-reactive protein (CRP). Blood count: erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), leukocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils , platelets, and neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR). Mean platelet volume (MPV), prothrombin time, Quick index, international normalized ratio (INR), activated partial thromboplastin time (aPTT), thromboplastin time ratio and fibrinogen, as variables related to coagulation, were also recorded. Finally, data related to urinalysis were included urine density, urine pH, urine protein, urine glucose, urine ketones, urine bilirubin, urobilinogen, urine erythrocytes and urine leukocytes. All blood and urine tests were performed during the course of the episode in the Emergency Department. The exact time of onset of delirium is unknown, but in most cases, subjects present within hours of symptoms onset, and at most within the first 12–24 h.This study was approved by the Ethics Committee of the Hospital. The data obtained were kept confidential in accordance with Spanish legislation on personal data protection . This study was conducted in accordance with the principles of the Declaration of Helsinki.t test) and non-parametric testing (Mann–Whitney U test) were used to compare quantitative variables, while the chi-square test was used for categorical variables. Two-factor between-subjects models were generated to analyze specific biomarker scores by groups (cases and controls), according to diagnostic groups. In order to adjust the p-value for multiple comparisons, Bonferroni corrections were used with a significance level of p < 0.003. All other statistical significance was reported at p < 0.05.Descriptive statistics were computed to characterize the sample and data distribution, and to check assumptions. The variables were reported as proportions and/or means and standard deviation (SD). The Kolmogorov–Smirnov test was used to assess normal data distribution. Parametric .A total of 128 patients and 128 controls were included in this study. The mean age was higher in the male-dominated patient group than in the control group. The median number of prescribed daily drugs was also higher among the patients . A signin = 67) and thus reflected probable organic causes of delirium. On the other hand, in 47.65% of the patients (n = 61), no cause of delirium was detected, and no secondary diagnoses were recorded in the medical history. In those cases where a secondary medical diagnosis was recorded, we observed a greater percentage of patients with neurological disorders (mainly dementia), and renal and cardiac problems  or anesthetics (p = 0.130)  .The increase in the number of hospital emergency department (ED) visits by older people is a worldwide phenomenon ,14,15. FThe detection of delirium is currently mainly based on the DSM-V diagnostic criteria, without the use of any specific biomarker. The scientific community is calling for the search for biomarkers that could help in the understanding and diagnosis of this syndrome . The patBased on this hypothesis, there are biomarkers in different fluids such as cerebrospinal fluid or blood that are useful for research, including cytokines, enzymes, growth factors and hormones, or which are useful in clinical practice, such as proteins, creatinine, NLR or PLR. Research in recent years has identified biomarkers of delirium in older people with acute and critical disease conditions, requiring differentiation among surgical, medical and critical patients; such biomarkers are difficult to apply in daily practice, and are not entirely consistent ,19.An ideal biomarker should be easily identifiable, cost-effective, reliable to allow targeted therapy, and at the same time should be highly sensitive and very specific. To our knowledge, no studies have evaluated biomarkers with these characteristics in the emergency care setting .Our results did not reveal any particular biomarker, and this may be due to several factors. A first factor is the nature of the ED itself, with the patients who attend being those requiring rapid care in the face of a recently established or aggravated disorder. A second factor is that a potential organic cause of delirium cannot be identified in the ED in all cases, and it is in later days during hospital admission or at home when the required information may be obtained—which also makes it difficult to interpret the analytical results. Lastly, the laboratory test data are obtained at the time of ED attendance, and at this point the new onset condition might not yet have modified the possible biomarkers in the biological samples. In this regard, postsurgical studies are characterized by serial pre- and postsurgical laboratory tests, while studies conducted in the medical setting typically analyze blood samples obtained from the first 24 h of admission onwards .In standard blood tests, the included biomarkers are albumin and creatinine, and may be useful in identifying an increased risk of delirium , since lIn contrast to the above, C-reactive protein (CRP) is considered to be a clinically useful biomarker of delirium in both medical and surgical patients ,20,24. SWe recorded no differences in cholinesterase values, in concordance with the findings of other authors ,30. ThisInterest in the neutrophil–lymphocyte ratio (NLR) has been on the rise ever since differences were found  in olderPlatelets count and PLR looks like it could also be a marker of delirium . Our resSignificant diversity is currently found in the literature regarding the type of study design, the biomarkers examined, the patient populations and the settings in which research is conducted . It can be affirmed that until properly designed studies become available, it is unlikely for altered biomarker levels to be of help in clinical practice .To our knowledge, this is the first article to provide all the clinical analytical data obtained in the ED. Despite not finding a possible biomarker, our results will allow researchers to compare different cohorts and analyze according to pathologies. The limitations of the present study include those inherent to its retrospective design, which precludes the analysis of other possible blood biomarkers in the sample analyzed. Likewise, it was not possible to monitor laboratory test parameters beyond the first 24 h of admission, when greater biomarker alterations could perhaps be observed—though the values then might also be biased as a result of the treatments administered. The race of the participants in this study was also not analyzed. Although the majority were Caucasian, these data are not available. Lastly, there is little literature on biomarkers of delirium in older people in the ED, which makes it difficult to compare results for patients of this kind.Older patients with delirium admitted to the hospital emergency department showed out-of-range mean values in urea, neutrophils%, monocytes%, platelets, NLR, PLR, and mean platelet volume. Despite this, no biomarker was identified, due to the high values also found in the control group. There is difficulty in identifying inflammatory biomarkers of delirium in the ED, due to the urgent nature of the disorders mainly seen in the hospital ED setting.


To explore the clinical effect of different delivery methods and the safety of vaginal delivery of second pregnancy after cesarean section and analyze the related factors.  A total of 738 eligible pregnant women who underwent cesarean section from September 2018 to August 2020 were randomly selected from our hospital. Among them, 527 pregnant women successfully delivered vaginally were selected as the observation group, and 211 pregnant women who failed vaginal delivery were selected as the control group. To analyze the factors that influence the success of vaginal delivery of second pregnancy after cesarean section and compare the outcomes of mother and infant in two groups. P > 0.05). There were significant differences in fetal head orientation, fetal abdominal circumference, fetal biparietal diameter, uterine height, premature rupture of membranes, Bishop score, and epidural anesthesia during labor between the two groups (P < 0.05). Multivariate logistic regression analysis showed that fetal abdominal circumference, fetal head orientation, Bishop score, and epidural anesthesia during labor were independent factors affecting the success of VBAC (P < 0.05). There was no significant difference in the incidence of uterine rupture between the two groups (P > 0.05). The amount of postpartum hemorrhage in the observation group was significantly lower than that in the control group (P < 0.05). There was no significant difference in Apgar score, asphyxia rate, and hospitalization rate between the two groups (P > 0.05). There was no significant difference in the incidence of complications between the two groups (P > 0.05).  There was no significant difference in age, prenatal body mass index (BMI), and thickness of lower uterine segment between the two groups ( There are many factors that influence the success of vaginal delivery after cesarean section. Through prenatal comprehensive evaluation of vaginal delivery conditions, we can guide the parturient to choose a reasonable mode of delivery, reduce the incidence of complications, and improve the outcome of mother and baby. In recent years, with the continuous maturity and development of cesarean section, the pain experienced by mothers during delivery has been significantly reduced, and the success rate and safety of cesarean section have been significantly improved , 2. HoweTherefore, this study selected 738 women who were pregnant again after cesarean section in our hospital as the research object, aimed at exploring the clinical effects of different delivery methods of pregnant again after cesarean section and the safety of vaginal delivery and analyze the relevant influencing factors.From September 2018 to August 2020, 527 pregnant women undergoing vaginal delivery (VBAC) after cesarean section in our hospital were randomly selected as the VBAC group. Among them, the age of the enrolled was 18-42 years, the average age was 31.55 ± 3.86 years, the gestation was 31-41 weeks, the median value was 39.29 weeks , and the interval between the last cesarean section was 1-16 years, including 7 cases in 1-2 years, 198 cases in 2-5 years, 258 cases in 5-10 years, and 64 cases in more than 10 years.A total of 211 pregnant women with failed vaginal delivery (VBNC) after cesarean section at the same period were selected as the VBNC group. They were 21-48 years old, with an average age of 31.36 + 4.56 years, 32 + 2 − 41 + 3 weeks pregnant, with a median value of 39.36 weeks . The time from the last vaginal delivery was 1-16 years, including 38 cases in 1-2 years, 103 cases in 2-5 years, 54 cases in 5-10 years, and 16 cases in more than 10 years. This study was approved by the Ethics Committee of Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine.Inclusion criteria: (1) the pregnant women who became pregnant again after cesarean section met the relevant diagnostic criteria after cesarean section, and all had one cesarean section experience; (2) the time from the second pregnancy to the previous cesarean section was >2 years, and all of them were singleton cephalic; (3) patients and their families should be informed and sign informed consent.Exclusion criteria: (1) prenatal B-ultrasound showed pelvic abnormalities, scar position, and uterine thickness <0.2 cm; (2) there were complications such as incision infection and postpartum hemorrhage after the previous cesarean section; (3) complicated with pregnancy diabetes, pregnancy induced hypertension, and other diseases; (4) complicated with severe cognitive impairment and unable to cooperate with treatment.TOLAC pregnant women receive the whole process management of pregnancy, nutrition, and exercise guidance during pregnancy, and they control their body mass during pregnancy. In the third trimester of pregnancy, the thickness and continuity of the myometrium at the incision of the uterus were evaluated by ultrasound, the weight of the fetus was evaluated, and the pelvic condition was measured. At least two doctors with the title of deputy director were evaluated to make a delivery plan.If there was no indication of induction of labor, pregnant women expect natural labor; those who met the indications for induction of labor were admitted to the hospital for induction of labor. According to the cervical score, small doses of oxytocin were selected for intravenous drip induction of labor, or water capsules were used to promote cervical maturation. During VBAC, continuous fetal heart rate monitoring should be carried out, and preparations for cesarean section should be made. In case of maternal and infant abnormalities, the surgical indications should be relaxed and emergency cesarean section should be carried out.The possible factors affecting the success of VBAC were recorded, including age, prenatal BMI, fetal abdominal circumference, fetal biparietal diameter, premature rupture of membranes, Bishop score, uterine height, fetal head orientation, thickness of lower uterine segment, and epidural anesthesia during labor.Bishop scoring standard  includinApgar score, neonatal asphyxia rate, and neonatal hospitalization rate of the two groups were statistically analyzed. The Apgar score is based on a total score of 1 to 10. The higher the score, the better the baby was doing after birth. A score of 7, 8, or 9 was normal and was a sign that the newborn was in good healthThe incidence of uterine rupture in the two groups was counted, and the bleeding volume at 2 and 24 hours after delivery was recordedThe incidence of pregnancy complications in two groups were recorded, including GDM, hypoactive hypothyroidism, thyroid hyperactivity, syphilis, gestational hypertension, and liver injuryt-test was performed. The count data in the study were expressed by percentage [n (%)]. X2 test was used. Set the statistical test level as: α = 0.05, P < 0.05 was statistically significant. The relative risk or value and 95% confidence interval (95% CI) were calculated for various factors. After multivariate logistic analysis, P < 0.05 was considered as statistically significant.The data of this study were processed and analyzed in the software SPSS 20.0. The measurement data generated in the study were described in the form of mean ± mean and parallel A total of 738 women who became pregnant again after cesarean section underwent vaginal trial delivery, and 527 women delivered vaginally, accounting for 71.41%. 211 cases of failed trial delivery were changed to cesarean section, 47 cases of parturients gave up continuing trial delivery, 32 cases of continuous pain in the lower abdomen, 63 cases of fetal head descent stagnation, 53 cases of active early stagnation, and 16 cases of cervical edema.P > 0.05); There were significant differences in fetal head orientation, fetal abdominal circumference, fetal biparietal diameter, uterine height, premature rupture of membranes, Bishop score, and epidural anesthesia during labor between the two groups (P < 0.05), as shown in There was no significant difference in age, prenatal body mass index (BMI), and thickness of lower uterine segment between the two groups (P < 0.05), as shown in The results of multivariate logistic regression analysis showed that fetal abdominal circumference, fetal head orientation, Bishop score, and epidural anesthesia during labor were the independent factors affecting the success of VBAC (P > 0.05). The amount of postpartum hemorrhage in the VBAC group was significantly lower than that in the VBNC group at 2, 12, and 24 hours (P < 0.05), as shown in There was no significant difference in the incidence of uterine rupture between the two groups (P > 0.05), as shown in There was no significant difference in Apgar score, asphyxia rate, and hospitalization rate between the two groups (P > 0.05). In the absence of obvious contraindications to delivery, vaginal delivery could be performed under close monitoring. As shown in By comparing the complications of pregnant women during pregnancy, it was found that there were more pregnant women with gestational diabetes mellitus (GDM) in the two groups. The data showed that the highest incidence of pregnancy complications was pregnancy diabetes, and the incidence of VBAC group (22 cases) was higher than that of VBNC group (14 cases). But there was no significant difference in the incidence of complications between the two groups . It was speculated that there were more cases with good lifestyle and moderate body weight. In addition, the overall childbearing age of maternal was small, which may also be related to the small number of included samples. Fetal abdominal circumference, fetal biparietal diameter, and uterine height are common indicators to determine fetal body mass. The heavier the fetus, the lower the success rate of vaginal delivery [The results of this study showed that 738 pregnant women who were pregnant again after cesarean section performed vaginal trial delivery, and the natural delivery rate was 71.41% (527/738), which was equivalent to 60%~80% reported at home and abroad , 12, whidelivery . A studydelivery , uterinedelivery , fetal fP < 0.05). Bishop score is an important indicator to evaluate cervical maturity, including cervical orifice position, cervical tube regression, cervical hardness, cervical orifice expansion degree, and the position of presentation. The higher the Bishop score, the higher the degree of cervix, and the higher the success rate of induction of labor [P > 0.05), which was presumably related to the strict screening of the included samples. Studies have shown that [This study showed that there was a significant difference of Bishop scores between the two groups , indicating that vaginal delivery of pregnancy after cesarean section was safe and reliable for both mothers and infants when conditions permit. Some studies believe that pregnant women who become pregnant again after cesarean section should make prenatal preparations and strictly grasp the indications of vaginal delivery, which can improve the rate of vaginal delivery [In addition to the amount of postpartum hemorrhage, there was no significant difference between the two groups in maternal and infant outcomes, especially in the rate of uterine rupture, Apgar score of newborns, asphyxia rate, and hospitalization rate (delivery . Most stdelivery , 26.There are some limitations in our study. First, the sample of our study was relatively small. Second, our study had regionalism, which could not represent the whole pregnant women. Therefore, further relative studies should be carried to in the future.To sum up, there are many factors that affect the success of vaginal delivery after cesarean section. Prenatal comprehensive evaluation of vaginal delivery conditions can guide mothers to choose a reasonable mode of delivery, reduce the incidence of complications, and improve the outcome of mother and baby.


Platichthys stellatus). Platichthys stellatus  was fed with dietary cadmium-ascorbic acid (Cd-AsA) composed of C0A0, C0A500, C0A1000, C40A0, C40A500, C40A1000, C80A0, C80A500, and C80A1000 mg of Cd-AsA per kg diet for four weeks. Our results showed that Cd accumulation significantly increased in proportion to the Cd concentration, where the highest levels were observed in the intestine, followed by the kidney, liver, and gills. Dietary AsA significantly mitigated the Cd accumulation in all tissues, and the reduction in Cd accumulation was proportional to the increase in AsA concentration. Dietary Cd has adverse effects on growth performance  and can alter the hematological parameters , plasma components , and antioxidant responses . Dietary AsA restored the decreased growth performance parameters and the altered hematological parameters, plasma components, and antioxidant responses caused by the dietary Cd exposure. The results of this study showed that dietary Cd is toxic to P. stellatus, while dietary AsA is effective in mitigating the toxic effects of Cd.This study evaluates the toxic effects of dietary Cd and mitigative effects of AsA supplementation by measuring the growth performance, bioaccumulation, hematological parameters, plasma components, and antioxidant responses of Starry flounder ( Additiombalance .L-ascorbic acid (AsA) is a vital nutrient in fish diets since they lack L-gulonolactone oxidase activity that catalyzes the conversion of L-gulonic acid to AsA. AsA prevents symptoms of AsA deficiency, such as reduced growth rate, impaired feed conversation, skeletal deformities , and reduced immunity [2− and •OH radicals by donating electrons, thereby maintaining redox homeostasis and protecting oxidative damage [immunity . An adeqimmunity  and is aimmunity . As an ae damage . In addie damage .Platichthys stellatus, is euryhaline fish that inhabits the marine environments of many countries, including Korea, Japan, and the US states, Alaska and California. In Korea, P. stellatus is one of the commercially-farmed fish due to its high salinity tolerance and disease resistance. P. stellatus production in Korea has steadily increased from 2377 tones in 2014 to 3373 tones in 2018 [P. stellatus. Therefore, this study aimed to evaluate the toxic effects of dietary Cd and mitigative effects of AsA supplementation by measuring the growth performance, bioaccumulation, hematological parameters, plasma components, and antioxidant responses of P. stellatus.The starry flounder, 2  and 340 g α-cellulose and used to design Cd concentrations  (L-ascorbyl-2-monophosphate (AMP), purchased from WOOSUNGFEED Co., Ltd., Daejeon, Korea, as the AsA source. The AMP premix was made with 50 g AMP and 450 g α-cellulose and used to design AsA concentrations . Concomitant with the increase in the amounts of Cd and AMP, the equivalent amount of α-cellulose was reduced to maintain the nutrient ratio. All ingredients were thoroughly mixed using a bakery mixer  and extruded as a 2 mm diameter module using a pellet machine . All pellets were left to dry for 48 h at 28 °C, sorted in a plastic bottle, and kept at −20 °C until further use. The actual concentration of Cd in the diet was measured using inductively coupled plasma mass spectrometry (ICP-MS)  and expressed as μg/g. A pure standard Cd solution (PerkinElmer) was used for the standard curve.All diets contained 62% white fish meal, 10% casein, 19.42% dextrin, 2% fish oil, 2% squid liver oil, 1% carboxymethylcellulose, 1% vitamin premix (AA-free), 1% mineral premix, and 0.5% choline salt. The Cd premix was prepared with 10 g CdCland C80) . We usedP. stellatus  were purchased from a commercial farm . Fish were acclimated to four 1000 L cylindrical water tanks (100–130 fish per tank) on the flow-through system for one week and were fed a commercial diet at a ration of 4% body weight per day. After acclimation, 180 fish were chosen at random from four 1000 L tanks and divided into nine groups (n = 20) for the experiment according to the Cd-AsA dietary concentrations  for two periods (two and four weeks), and each group was reared in a separate 500 L cylindrical tank under the experimental conditions . Fish were fed the Cd-AsA diet at a ration of 4% body weight per day during the exposure period, and the residuals were removed to avoid the effect of Cd or AsA derived from the dissolution in the rearing water. No mortality was observed throughout the study period. At the end of each exposure period, ten fish per tank were selected and anesthetized in a diluted tricaine methanesulfonate solution . After anesthesia, fish were weighed to evaluate their growth performance. Blood samples were collected from the caudal vein of the fish using a 1 mL syringe with heparin  and centrifuged at 3000× g for 10 min at 4 °C to obtain the plasma  which was kept at −20 °C until analysis of the plasma components. The intestine, kidney, liver, and gills were dissected and frozen at −80 °C until further use for the analysis of tissue accumulation and antioxidant response. This experiment was repeated three times. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Pukyong National University.Samples of 3, Guaranteed Reagent grade) and 2 mL of perchloric acid  at 28 °C for 4 h. The reagents were then evaporated on a hotplate at 105 °C overnight until a clear solution was obtained. At the end of the digestion, the solutions of the digested samples were filtered through a membrane filter  to remove impurities. The filtrate was analyzed using ICP-MS  equipped with argon gas. Cd bioaccumulation in tissues was calculated using a pure Cd standard solution (PerkinElmer) and expressed as μg/g dry weight.Tissue samples from the intestine, kidney, liver, and gills were rinsed with ultrapure water and freeze-dried for 48 h to obtain the dry weights of the samples. After freeze-drying, 0.1 g of the tissue samples were digested in 6 mL of nitric acid  counts, hemoglobin (Hb), and hematocrit (Ht) were diluted 400-times with Hayem’s diluting solution, and RBC counts were analyzed using a hemocytometer . Blood samples were placed into micro-hematocrit capillary tubes . The capillary tubes were sealed at one end with a tube sealing compound and subsequently centrifuged at 12,000 rpm for 5 min using a micro-hematocrit centrifuge . Ht values were expressed as percentages. Hb levels were analyzed using a clinical kit  according to the manufacturer’s instructions. Plasma components were used to evaluate the level of glucose, total protein, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT), using a clinical kit , according to the manufacturer’s instructions. Glucose was analyzed using the GOD/POD method, and the absorbance was measured at 500 nm and expressed as mg/dL. Total protein was quantified using the biuret method, and the absorbance was measured at 540 nm and expressed as g/dL. GOT and GPT were analyzed using the Reitman-Frankel method, and the absorbance was measured at 505 nm and expressed as karmen/mL. g for 30 min at 4 °C, and the supernatants were used for the analysis of antioxidant responses. The SOD and CAT activity was measured using the SOD Assay Kit  and CAT Assay Kit , respectively, according to the manufacturer’s instructions. GST activity and GSH levels in tissues were analyzed as described previously [The intestine, kidney, liver, and gills were placed in a micro-centrifuge tube  with cold 0.1 M phosphate buffered solution  and stainless bead and were homogenized using a GeneReady Standard homogenizer . The homogenates were centrifuged at 10,000× eviously . Quantitp values of <0.05. All data were expressed as mean ± standard error (S.E).Data and statistical analyses were performed using SigmaPlot 12.0  and the SPSS Statistics, version 27 . The between-groups analysis was performed at two and four weeks using one-way analysis of variance (ANOVA) and Tukey’s multiple-range test and classified as significant differences at P. stellatus exposed to dietary Cd-AsA after two and four weeks is shown in Cd accumulation in the liver, kidney, gills, and intestines of 40A1000 and Cd80A1000 was 33% and 42% at two weeks and 45% and 37% at four weeks, respectively. In the kidney, the reduction ratio of Cd accumulation in the groups exposed to Cd40A1000 and Cd80A1000 was 43% and 57% at two weeks and 57% and 47% at four weeks, respectively, which was the maximum level of reduction among the other tissues. In the gills, the reduction ratio of Cd accumulation in the groups exposed to Cd40A1000 and Cd80A1000 was 33% and 40% at two weeks and 27% and 53% at four weeks, respectively. In the intestine, the reduction ratio of Cd accumulation in the groups exposed to Cd40A1000 and Cd80A1000 was 24% and 29% at two weeks and 26% and 36% at four weeks, respectively.The reduction ratios of the Cd accumulation are presented in P. stellatus exposed to dietary Cd-AsA for two and four weeks is shown in The growth performance of P. stellatus exposed to dietary Cd-AsA after two and four weeks are shown in The hematological parameters of P. stellatus exposed to dietary Cd-AsA after two and four weeks are shown in The plasma components of P. stellatus exposed to dietary Cd-AsA after two and four weeks are shown in The antioxidant responses in the liver, kidney, gills, and intestines of Cyprinus carpio, Nile tilapia, and Oreochromis niloticus due to its chelating ability [The analysis of metal accumulation assists in evaluating the toxic effects on fish and determining the routes of metal uptake, biotransformation, and excretion . The gil ability ,30, whicThese results are consistent with our results, which indicated that Cd accumulation in tissues decreased as the level of AsA increased, suggesting that dietary AsA could assist in reducing the Cd accumulation.Pelteobagrus fulvidraco [Oncorhynchus mykiss [3+ to Fe2+ and the formation of stable chelates with iron [Growth performance is used as an indicator of chronic toxicological investigations as it is sensitive to environmental contaminants . Cd expolvidraco , and rais mykiss . In conts mykiss  and imprith iron . The effO. niloticus [Carassius auratus gibelio [P. stellatus exposed to dietary AsA showed a significant increase in RBC counts, Hb concentration, and Ht levels, thereby mitigating the toxic effects of Cd on the hematological parameters. AsA prolongs the life span of erythrocytes by protecting the lipid peroxidation of unsaturated fatty acids in the cell membrane from metal toxicity [The analysis of hematological parameters is an important indicator for evaluating the health status of fish, as it serves as a useful and reliable indicator of physiological and pathological changes . It can iloticus  and gibe gibelio . Similartoxicity . Moreovetoxicity . These etoxicity ,40. Our The analysis of plasma components was used to evaluate the potential adverse effects of metal exposure on the fish stress response, hepatic function, and hydromineral balance ,42. MetaAnalysis of antioxidant responses is an indirect method for assessing oxidative stress in tissues . The ant2− radical to H2O2, which is subsequently converted into H2O and O2 by CAT. Cd exposure induces the decrease of antioxidant enzyme activities by inducing ROS production, resulting in oxidative stress. Cd decreased SOD and CAT activities in the liver of C. carpio, which might be due to the imbalance between ROS and the antioxidant capacity of Cd [P. stellatus were significantly reduced in a dose-dependent manner after Cd exposure. These results suggest that excessive ROS production due to Cd toxicity causes an imbalance between antioxidants and ROS, thereby inhibiting SOD and CAT activities. SOD and CAT activities act as the first defense mechanism in the antioxidant system; SOD catalyzes the detoxification of the Oty of Cd . Zebrafity of Cd . Our resC. punctatus and GST activity in the liver and kidney of Labeo rohita, both of which were due to the conjugate reactions that occur in phase II of xenobiotic metabolism. Cellular GSH levels in fish exposed to Cd have been reported to decrease in many studies. The Japanese flounder, Paralichthys olivaceus, exposed to Cd showed a decrease in GSH levels in the gills and liver, which might be due to the high Cd accumulation in the cell and the formation of the GS-metal complex [O. niloticus exposed to Cd decreased during the 40 days exposure period, except at day 20, which indicated that changes in GSH metabolism were attributed to oxidative stress and associated with the exposure period [P. stellatus were significantly decreased in a dose-dependent manner following Cd exposure. The decreased GST and GSH levels in tissues might be due to the direct conjugation of GSH with Cd and the oxidative stress caused by continuous Cd exposure.GST comprises a group of phase II enzymes that facilitate the excretion of endogenous  and exogenous  substrates by binding to the conjugates of GSH with electrophilic metabolites . GSH is  complex . GSH leve period . Our resO. niloticus, and these results were due to the direct reaction of AsA with superoxide hydroxyl radicals and singlet oxygen. Kim and Kang [Sebastes schlegelii, exposed to dietary lead. Vijayavel et al. [Terapon jarbua, thus inhibiting the continuous ROS production and assisting in maintaining the normal status. Similar to previous studies, our results showed that AsA supplementation mitigated the antioxidant responses  in a dose-dependent manner and restored the antioxidant responses to control values. These results suggest that AsA supplementation mitigated the Cd-induced toxicity in terms of SOD, CAT, GST, and GSH levels in P. stellatus. The non-enzymatic antioxidant, AsA, deactivates the free radicals or scavenges the extracellular and intracellular ROS, which, consequently, protects the cells from oxidative stress . Severaland Kang  reportedl et al.  also demP. stellatus. Moreover, the AsA supplementation enhanced the antioxidant enzyme activities, and the high levels of AsA supplementation were effective in mitigating Cd-induced bioaccumulation and other toxic effects.Our results showed that dietary Cd and AsA had toxic and mitigative effects on the physiological and biochemical functions in a dose-dependent manner. Dietary Cd resulted in the highest accumulation of Cd in the intestine, followed by the liver, kidneys, and gills. Dietary Cd has adverse effects on growth performance , causing alterations in hematological parameters  and plasma components . In addition, dietary Cd decreased the antioxidant responses . In contrast, dietary AsA decreased Cd accumulation in proportion to an increase in AsA concentration. The altered growth performance, hematological parameters, plasma components, and antioxidant responses due to Cd toxicity were mitigated by dietary AsA. In conclusion, the results of this study indicate that dietary Cd exerts toxic effects on the growth performance, hematological parameters, plasma components, and antioxidant responses of


P < 0.001). In the subgroup analysis by ethnicity, STAT4 rs7574865 T allele was shown to be risk factor in SLE in Asian, European, and American origins. Our results do support STAT4 rs7574865 polymorphism as a susceptibility factor for SLE in populations of different ethnic and that its prevalence is ethnicity dependent.Previous studies have reported that STAT4 rs7574865 conferred the susceptibility to systemic lupus erythematosus (SLE). In this study, a meta-analysis (including 32 comparative studies of 11384 patients and 17609 controls) was conducted to investigate the role of STAT4 polymorphism in SLE in a comprehensive way. We found that the Asian population had the highest prevalence of the T allele than any other study population at 32.2% and that STAT4 rs7574865 polymorphism was associated with SLE in the overall population (OR = 1.579, 95%CI = 1.497-1.665,  Systemic lupus erythematosus (SLE) is classified as an inflammatory autoimmune disease characterized by production of autoantibodies to nuclear antigens and immune complex formation , leadingGenome-wide association studies have identified signal transducer and activator of transcription4 (STAT4) as a novel susceptibility gene associated with SLE . STAT4 cAs the investigation varied greatly among studies, the association between STAT4 rs7574865 polymorphism and SLE risk has reported mixed results , which mWe conducted a comprehensive literature search for available articles in which STAT4 polymorphism were analyzed in SLE patients, using the databases of PubMed, Web of Science, CNKI , and Wanfang. The relevant articles were retrieved by keyword combinations as follows: “STAT4,” “Signal transducer and activator of transcription 4,” “rs7574865,” “polymorphism,” and “systemic lupus erythematosus.” The references in the obtained studies were also examined to identify additional studies not included by the electronic databases, with no language or country restrictions.The inclusion criteria were listed as following: case-control studies, original data (independence among studies), and enough data to calculate an odds ratio (OR) with 95% confidence interval (CI). We excluded the following: studies in which the number of risk alleles could not be ascertained or contained overlapping data. These studies were assessed by two investigators independently. In case of any disagreement, it could be resolved by consultation with a third author.We extracted the following information from each article: first author, year of publication, demographics, ethnicity, the numbers of cases and controls, and allele frequency of the STAT4 rs7574865 polymorphism.Z-test, and P < 0.5 was considered statistically significant. In addition, Cochran, s Q statistics was used to assess within-study and between-study variation and heterogeneities. This heterogeneity test assesses the null hypothesis that all studies evaluated the same effect. The effect of heterogeneity was quantified using I2. In our meta-analysis, P < 0.10 for the Q-test and I2 more than 50% was considered that significant heterogenicity among studies could not be ignored. Data were combined using both the fixed effects model (the inverse variance-weighted method) and the random effects model (DerSimonian and Laird method) [We performed meta-analyses to estimate the association between STAT4 rs7574865 polymorphisms and SLE risk by odds ratios (ORs) and its 95% confidence interval (CI). The significance of the pooled ORs was determined by the  method) , 28. We Subgroup analyses were performed by ethnicity, to examine the potential sources of heterogeneity observed in the meta-analyses. Because of the limitations of funnel plotting, publication bias was evaluated using the Egger linear regression test , which mAs shown in P < 0.001) in all participants. The heterogeneity among studies was moderately obvious . Thus, we performed stratified analysis by ethnicity, indicating that the association between risk factor T allele carriers and SLE was significant in Asians, Europeans, and Americans, with no obvious difference. The pooled ORs for the T allele of STAT4 rs7574865 were 1.706  for populations of American, 1.518  for Asian, 1.674  for Europeans. The results of subgroup analysis revealed that between-study heterogeneity was moderately obvious in European  and American , but no such heterogeneity was found in Asian . . .P value > 0.05). . .Sensitivity analysis was conducted to evaluate the influence of a single study on pooled OR by the “trim and fill” method. Corresponding pooled OR was not substantially changed when any individual study was deleted, which confirmed the stability of our overall result. The estimated number of missing studies is 5, and the adjusted pooled OR estimated is 1.514 (95%CI = 1.462-1.567), which showed still significantly increased risk for SLE .Systemic lupus erythematosus is a highly heterogeneous autoimmune disease, with influence of complex genetic. Studies in multiple ethnic populations have reported up to 100 risk loci of importance in SLE, although the mechanisms underlying these associations are still elusive –33.Previous studies suggest that several single-nucleotide polymorphisms (SNPs) in LD in the third intron of STAT4, tagged by rs7574865, were initially demonstrated as SLE risk variants . The minIn our comprehensive meta-analysis, we identified 21 relevant studies including 11384 SLE cases and 17609 healthy controls. The results do support a trend of association between STAT4 rs7574865 polymorphism and SLE risk in overall. When stratified by ethnicity for STAT4 rs7574865 polymorphism, positive correlations were observed in Asian, European, or American origin. Regarding STAT4 rs7574865 polymorphism in overall samples, we observed that the average frequency of T allele was 39.4% in patients with SLE compared with 29.5% in controls. The prevalence of the rs7574865 T allele was found to vary among ethnic populations.Nevertheless, several limitations presented in this meta-analysis should be considered. First of all, the association between the STAT4rs7574865 polymorphism and disease severity and/or clinical features could not be performed limitation of amount of available data. Secondly, our ethnicity-associated results are applicable only to Asian, European, and American patients, as the data obtained from these ethnic groups. In addition, the heterogeneity has been presented obviously in European and American population, in subgroup analysis. Due to population demographics and lower SLE risk predisposition, or publication bias, fewer relevant studies were obtained in European and American, which may have distorted the heterogeneity, even have affected the final result, despite performing the “trim and fill” method and Egger regression test. Last but not least, STAT4 polymorphism may interact with other potential confounding risk factors.In summary, our results stressed the importance of STAT4rs7574865 polymorphisms in increasing the risk of SLE in multiple ethnic groups and that the prevalence of the STAT4rs7574865 T allele may depend on ethnicity, which may improve our understanding of SLE pathogenesis. However, additional research with well-designed and large sample sizes is needed to be carried out about European, American, and other ethnicities in the future.


Coupled with the convolutional neural network (CNN), an intelligent Raman spectroscopy methodology for rapid quantitative analysis of four pharmacodynamic substances and soluble solid in the manufacture process of Guanxinning tablets was established. Raman spectra of 330 real samples were collected by a portable Raman spectrometer. The contents of danshensu, ferulic acid, rosmarinic acid, and salvianolic acid B were determined with high-performance liquid chromatography-diode array detection (HPLC-DAD), while the content of soluble solid was determined by using an oven-drying method. In the establishing of the CNN calibration model, the spectral characteristic bands were screened out by a competitive adaptive reweighted sampling (CARS) algorithm. The performance of the CNN model is evaluated by root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV), root mean square error of prediction (RMSEP), coefficient of determination of calibration ( Quality control in the manufacturing process is an important issue to guarantee the quality of end-products of botanical drugs ,2. In 20Salvia miltiorrhiza and Ligusticum chuanxiong hort. The extract of Salvia miltiorrhiza has the pharmacological effects of anti-platelet aggregation, anti-thrombosis, promoting fibrin degradation, anti-myocardial ischemia, and antioxidant [Ligusticum chuanxiong hort can dilate blood vessels, increase coronary flow, and improve microcirculation [The Guanxinning tablet is a botanical drug which is clinically used for the treatment of coronary heart disease and angina pectoris. The raw materials of the Guanxinning tablet are ioxidant ,5, whereculation . The conCompared with conventional PAT techniques, Raman spectroscopy has the merits of being non-destructive, fast, and portable. Also, Raman spectroscopy has been widely used in molecular fingerprinting ,8, pathoSince Raman spectroscopy alone is still unable to achieve real-time detection, it needs to be combined with machine-learning algorithms . The conIn this paper, an intelligent deep-learning algorithm driven Raman spectroscopy analysis methodology was established for the rapid and non-destructive determination of the contents of four bioactive compounds and soluble solid in water extract samples of Guanxinning tablets, which were collected from the manufacturing process. The model performance of CNN was compared with that of PLSR and SVR.A reliable HPLC-DAD method was established to determine the four bioactive ingredients. The chemical structures of the four analytes are shown in 2) in the range of 0.9995–0.9997. The LODs and LOQs of the four major compounds were in the range from 0.2042 to 0.5313 μg/mL and from 0.6807 to 1.7709 μg/mL, respectively. The method was fully validated. The precision, repeatability, stability, and recovery of the method are shown in 335 real samples were analyzed by the HPLC-DAD method. All four bioactive compounds were baseline separated and could be accurately determined. Calibration curves, correlation coefficients, linearity ranges, and LOD and LOQ data are shown in The content of soluble solid is an important indicator of the water extracts. It is also necessary to establish a reliable reference method for determining soluble solid content. 335 samples were assayed by using the oven-drying method. The soluble solid of the water-extract samples ranged from 418.7 μg/mL to 4882.7 μg/mL.−1. The major Raman peaks were assigned by comparing with the literature [−1 can be ascribed to Ar ring stretching. The Raman peak at 1250 cm−1 can be ascribed to asymmetric stretching of the C-O-C bond. The Raman peak at 1500 cm−1 can be ascribed to C=C bond stretching.The raw Raman spectra of Guanxinning water extract are shown in terature ,15. The The number of abnormal Raman spectra in 335 water-extract samples of Guanxinning was removed by using the Mahalanobis distance method. The Mahalanobis distance distribution of the samples is shown in Using the Kennard–Stone (K–S) algorithm, 330 real samples were divided into calibration set and validation set by 4:1. For each model, the calibration set consists of 264 samples, and the remaining 66 samples belong to the validation set. Four variable selection methods were used to select the characteristic bands of Raman spectra. CNN, PLSR, and SVR models were established, respectively. RMSEC, RMSEP, In the CARS algorithm, the Monte Carlo sampling rate was set to 0.8, and the sampling number was 50. The performance parameters of the different calibration models established with the optimal band selecting method are listed in The established method is used for routine analysis in the production process, with which three batches of unknown samples of Guanxinning water extract were acquired by a portable Raman spectrometer. The Raman spectra of the unknown samples were corrected and inputted into the established CARS-CNN model. The contents of the four bioactive compounds and soluble solid were obtained at the same time. The three different batches of samples were analyzed with this model. Water-extract samples of Guanxinning tablets were collected from a Chinese medicine pharmaceutical factory  in Deqing. In the extraction process during the production of the Guanxinning tablets, reflux extraction was carried out three times. The extraction time of the first reflux process was 2 h. The extraction time of the second reflux process was 1.5 h. The extraction time of the third reflux process was 1.5 h. 10 mL samples were collected every 5 min for the first 1 h, and 10 mL samples were collected every 10 min for the next 1 h. When the crude drug enters the second and third reflux processes, 10 mL samples were collected every 5 min for the first 1 h, and 10 mL samples were collected every 10 min for the next 0.5 h. A total of seven batches of samples (335 samples) were collected from the Guanxinning water-extraction module. The port of extractor was spun off at certain time points and the water extract was poured into a small beaker. Then, the water extract was transferred to a centrifuge tube. The flow chart of this study is shown in 2O (v/v) and (B) acetonitrile. The gradient elution procedure was as follows: initial 95% (A); 0–12 min, 5–38% (B); 12–20 min, 38–48% (B); 20–35 min, 48–100% (B). The re-equilibration duration between single runs was 6 min. The column temperature was 36 °C and the flow rate was 0.8 mL/min. The detection wavelength of danshensu, ferulic acid, rosmarinic acid, and salvianolic acid B was 288 nm. LODs and LOQs were determined by using diluted standard solution when the signal-to-noise ratios (S/N) of the standard substances were about 3 and 10, respectively. Variations were expressed by relative standard deviations (RSD).In order to determine the concentration of four bioactive compounds in Guanxinning water extract, a high-performance liquid chromatography method was established. The water extract was centrifuged at 13,000 rpm for 10 min. Then, the supernatant was sent for analysis under the following chromatographic conditions. An Agilent 1260 high-performance liquid chromatography system  was used, including a quaternary pump, a sample vial injector, a column oven, and a diode array detector (DAD). The column was Hanbon Sci & Tech Hedera ODS-2 , and the mobile phases consisted of (A) 0.1% HCOOH-HSc is the soluble solid content of the extract, W is the quality of the extract, W2 is the total mass of the sample and weighing bottle after drying, and W1 is the mass of the weighing bottle.In order to determine the content of soluble solid in the Guanxinning water extract, an oven-drying method was adopted. Guanxinning water extract was centrifuged at 2500 rpm for 10 min. Then, about 3 mL of the supernatant was placed in a flat weighing bottle, evaporated to dryness in a water bath, and then placed in a 105 °C oven for 6 h. Finally, the bottle was taken out, placed in a desiccator to cool for 1 h, and weighed. The soluble solid content was calculated according to Formula (1), where −1, resolution 2.83 cm−1, acquisition time 500 ms, and samples were collected three times each. The Raman spectrometer was controlled by a compatible flat panel, and the “Pharmaceutical” software (Version 1.0) was used for data acquisition.The Raman spectrum was collected by a Rapid OLRaman-2 portable Raman spectrometer equipped with a Raman fiber probe, a CCD detector, and a laser emitter . It is a dispersive (with a grating) type of instrument. The acquisition parameters are as follows: wavenumber range 176–3500 cmIn addition to the sample information, the data collected by the Raman spectrometer also include abnormal spectra that may have been generated due to errors of instrument, method, environment, or manual operation during the collection process. In order to obtain a reliable, accurate, and stable quantitative model, it is necessary to identify and remove abnormal spectra before modeling. In order to eliminate the interference of abnormal spectra, Mahalanobis distance method was used.−1. In order to best utilize the effective spectrum, the optimal characteristic bands should be screened out during the calibration process. To screen out the optimal spectral bands, competitive adaptive reweighted sampling (CARS), Uninformative Variable Elimination (UVE), Successive Projections Algorithm (SPA), and Synergy Interval Partial Least Square (siPLS) toolbox were used [The Raman signal shift of the Raman spectrometer is 176–3500 cmere used . The perThe algorithms used for building the calibration models were PLSR, SVR, and CNN. The principle and application of these algorithms were well documented in the references ,18,19. TThe construction detail of the CNN model is as follows. First, a convolution layer was created. The parameters of the convolution layer were as follows: 32 filters, the filter window size was 3 × 3, the scanning window moved with a step size of 1 each time, and the rectified linear units (ReLU) activation function was applied. Second, a batch-normalization layer was created. Third, a maximum pooling layer was created. The number of filters in the pooling layer was the same as that of the convolution layer 1. The filter window size was 2 × 2, and the scanning window moved with a step size of 1 each time. There was no maxpooling layer for ferulic acid. Fourth, four convolutional layers with parameters setting to 16, 3, and 1 were created successively. The ReLU activation function was applied. After that, a convolution layer with parameters setting to 32, 3, and 1 was created and the ReLU activation function was applied. Then, a convolution layer with parameters setting to 64, 3, and 1 was created and the ReLU activation function was applied. Finally, a flattened layer was created. Then, two fully-connected layers were created for danshensu, salvianolic acid B, and soluble solid, whereas three fully-connected layers were created for ferulic acid and rosmarinic acid. The number of output neurons is 1, and the linear activation function was applied. The mean squared error loss function was chosen and Adam (lr = le × −4) was used as the optimizer. The specified batch size was 50. The number of iterations was 200. If the loss was not improved after 40 iterations, Keras would stop training.The root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV), root mean square error of prediction (RMSEP), correlation coefficient of calibration (terature .An intelligent Raman spectroscopy methodology for the simultaneous determination of danshensu, ferulic acid, rosmarinic acid, salvianolic acid B, and soluble solid in water extract of Guanxinning was established. The calibration model has been validated with satisfactory


A single generalizable metric that accurately predicts early dropout from digital health interventions has the potential to readily inform intervention targets and treatment augmentations that could boost retention and intervention outcomes. We recently identified a type of early dropout from digital health interventions for smoking cessation, specifically, users who logged in during the first week of the intervention and had little to no activity thereafter. These users also had a substantially lower smoking cessation rate with our iCanQuit smoking cessation app compared with users who used the app for longer periods.This study aimed to explore whether log-in count data, using standard statistical methods, can precisely predict whether an individual will become an iCanQuit early dropout while validating the approach using other statistical methods and randomized trial data from 3 other digital interventions for smoking cessation (combined randomized N=4529).Standard logistic regression models were used to predict early dropouts for individuals receiving the iCanQuit smoking cessation intervention app, the National Cancer Institute QuitGuide smoking cessation intervention app, the WebQuit.org smoking cessation intervention website, and the Smokefree.gov smoking cessation intervention website. The main predictors were the number of times a participant logged in per day during the first 7 days following randomization. The area under the curve (AUC) assessed the performance of the logistic regression models, which were compared with decision trees, support vector machine, and neural network models. We also examined whether 13 baseline variables that included a variety of demographics  and smoking characteristics  might improve this prediction.The AUC for each logistic regression model using only the first 7 days of log-in count variables was 0.94 (95% CI 0.90-0.97) for iCanQuit, 0.88 (95% CI 0.83-0.93) for QuitGuide, 0.85 (95% CI 0.80-0.88) for WebQuit.org, and 0.60 (95% CI 0.54-0.66) for Smokefree.gov. Replacing logistic regression models with more complex decision trees, support vector machines, or neural network models did not significantly increase the AUC, nor did including additional baseline variables as predictors. The sensitivity and specificity were generally good, and they were excellent for iCanQuit .Logistic regression models using only the first 7 days of log-in count data were generally good at predicting early dropouts. These models performed well when using simple, automated, and readily available log-in count data, whereas including self-reported baseline variables did not improve the prediction. The results will inform the early identification of people at risk of early dropout from digital health interventions with the goal of intervening further by providing them with augmented treatments to increase their retention and, ultimately, their intervention outcomes. An imw weeks) ,11, the w weeks) -14. The A growing body of empirical literature aims to address this problem by examining variables that might predict early dropout and then comparing statistical models that improve the precision of these predictions -20. The P=.03). Such differences in cessation rates are substantial, especially when considering that, across all 4 interventions, 49%-65% of all users were 1-week users and indeed were the largest log-in trajectory group [Recently, we published 2 studies identifying a type of early dropout from digital interventions that is highly predictive of treatment outcomes ,25. Specry group ,25. The The goal of this study was to explore whether log-in count data using standard statistical methods can predict whether an individual will become an iCanQuit early dropout  while validating the approach using the randomized trial data from the 3 other digital interventions for smoking cessation (combined N=4529), namely, QuitGuide, WebQuit.org, and Smokefree.gov. We chose log-in count data from each of the first 7 days following random assignment to the treatment arm as log-ins are a simple metric that is objectively collected without requiring user self-report and is readily available to both researchers and developers deploying interventions in real-world contexts . We also examined whether 13 baseline variables that included a variety of demographics  and smoking characteristics  might improve this prediction. The potential value of this study is to focus limited resources on the development and testing of augmented intervention components that could be offered only to those at high risk of early dropout, thereby improving both their use of the intervention and, in turn, their treatment outcomes.For the main aim of training a model to predict early dropouts, data were drawn from the iCanQuit app arm of a 2-arm randomized controlled trial (RCT) comparing iCanQuit with the NCI QuitGuide app for smoking cessation, with full protocol details previously described . In brieFor validating the statistical approach predicting early dropouts across interventions, data were drawn from the QuitGuide app arm of the aforementioned RCT  as well across the 2 RCTs and, thus, were fully comparable across each of the 4 interventions [The eligibility criteria for each trial, fully described in each trial’s main outcome paper ,28, wereventions ,28—partiAll study activities for both trials were approved by the Fred Hutchinson Cancer Center Institutional Review Board . Participants provided consent on the web by clicking an “I accept” button option on the web-based consent form. All data were deidentified and collected on a secure web-based database. Participants received US $25 for completing each follow-up survey and an additional US $10 bonus if the web-based survey was completed within 24 hours of the initial email invitation to take the survey.Full descriptions of each digital intervention, which were double-blinded, are provided in their respective RCT main outcome papers ,28 alongThe overall differences between the 4 interventions were that (1) iCanQuit and WebQuit taught skills based on the principles of ACT  for smokThe iCanQuit smartphone app intervenes in the ACT-focused processes of acceptance of internal cues to smoke and enacting one’s values that guide smoking cessation . The accThe US NCI QuitGuide smartphone app content is delivered in four main sections: (1) “Thinking about quitting,” which focuses on motivations to quit by using reason and logic, such as identifying reasons to quit and providing information on the health consequences of smoking and quitting; (2) “Preparing to Quit,” which helps users develop a quit plan, identify smoking behaviors and triggers as well as reasons for being smoke free, and social support for quitting; (3) “Quitting,” which teaches skills for avoiding cravings to smoke; and (4) “Staying Quit,” which presents tips, motivations, actions to stay smoke free, and skills for coping with slips.The WebQuit website is based on ACT , an apprThe US NCI Smokefree.gov website follows the USCPG and provides a standard treatment that teaches skills to smokers to avoid urges. Users can navigate through all pages of the website at any time, and there are no restrictions on the order in which the content can be viewed. Smokefree has 3 main sections: “Quit today,” “Preparing to quit,” and “Smoking issues.” The “Quit today” section has 7 pages of content that provide tips for the quit day, staying smoke free, and dealing with cravings. This section also provides information on withdrawal and the benefits of quitting. The “Prepare to quit” section has 7 content pages providing information on various reasons to quit, what makes quitting difficult, how to make a quit plan, and using social support during a quit attempt. The “Smoking issues” section provides 5 pages on the health effects of smoking and quitting, depression, stress, and secondhand smoke, and coping with the challenges of quitting smoking for the lesbian, gay, bisexual, and transgender community.Engagement with the assigned digital intervention was objectively measured using either Google Analytics  or an internally hosted secure server . For each participant, time- and date-stamped log file records of opening each digital program were recorded. For the main analysis, we used the number of log-ins on each of the first 7 days following randomization as the predictor variables .The 13 self-reported baseline characteristics were (1) age, (2) gender, (3) race, (4) Hispanic ethnicity, (5) highest education level, (6) working status (full- or part-time employment vs any other), (7) sexual orientation, (8) whether they smoked half a pack of cigarettes per day (≤10 vs ≥11), (9) smoking within 5 minutes of waking (yes or no), (10) use of e-cigarettes in the past 30 days (yes or no), (11) confidence in being smoke free, (12) heavy drinking , and . The variables race, confidence in being smoke free, and heavy drinking status had missing values for some participants in the WebQuit and Smokefree data . Given these small proportions, participants with missing values were excluded from the analysis. Thus, the final sample sizes for this study were 1038, 1033, 1192, and 1266 for the iCanQuit, QuitGuide, WebQuit, and Smokefree arms, respectively. Analyses were conducted using R  [For the first step in the analysis, we compared 4 of the most common statistical approaches to classification problems: standard multivariate logistic regression, decision tree, support vector machine (SVM), and neural network models -43. Logisklearn (Python Software Foundation) [sklearn. The basis expansion used for SVM was the radial basis. The models for each arm were fitted independently.Each classification method was implemented to predict early dropout based only on log-in counts from days 1 to 7 after randomization. For each intervention, a training data set was used to fit the classification model, whereas the test data set was used to assess the performance of the model. Across all arms of the trials, 80% (3623/4529) of the participants were randomly selected into the training data set, and the remaining 20% (906/4529) were used as the test data set. The classification models were implemented using the Python package ndation) . Tuning pROC [The receiver operating characteristic curve and corresponding area under the curve (AUC) were used to evaluate the performance of each predictive classification model . AUC valpROC .Next, we assessed whether the inclusion of baseline variables in the logistic regression could improve the prediction of early dropout. All 13 baseline variables (see the Baseline Characteristic Predictors section for a complete list) were added to the log-in counts from days 1 to 7 in the full model. Finally, we assessed whether a more parsimonious logistic regression model could improve the prediction of early dropout. In this model, the 13 baseline variables were added to the log-in counts of days 1 to 7 as inputs to stepwise logistic regression using the Bayesian information criterion for variable selection ,49. As wThe baseline characteristics of each digital intervention treatment group are reported in For the iCanQuit test data, the AUC values were 0.94 (95% CI 0.90-0.97) for the logistic regression model, 0.92 (95% CI 0.88-0.96) for the decision tree, 0.93 (95% CI 0.89-0.97) for SVM, and 0.94 (95% CI 0.90-0.97) for the neural network model. The results of these analyses, along with those from the QuitGuide, WebQuit, and Smokefree data sets, are shown in P=.62). The results were similar for the other 3 digital therapeutic data sets. No baseline variables predicted early dropout across any of the 4 interventions, as shown in Table S3 in As shown in Table S2 in At the standard classification threshold of 0.5, the sensitivity and specificity of the test data were 0.91 and 0.85, respectively, for iCanQuit; 0.90 and 0.64, respectively, for QuitGuide; 0.95 and 0.55, respectively, for WebQuit; and 0.55 and 0.58, respectively, for Smokefree. By decreasing the classification threshold to 0.3, the sensitivity increased to ≥0.95 for all interventions, whereas the specificity decreased. These results are presented in The overall aim of this study was to identify a small number of highly generalizable and easily collected variables that can predict early dropout with high precision using statistical models that can be readily implemented by other researchers and intervention developers. To address this aim, this study explored whether log-in count data using standard statistical methods can precisely predict whether an individual will become an iCanQuit early dropout  while validating the approach using the randomized trial data from 3 other digital interventions for smoking cessation, namely, QuitGuide, WebQuit.org, and Smokefree.gov. We also examined whether a limited number of commonly used self-reported baseline demographic variables  might improve this prediction. Overall, the results showed that the AUC for each logistic regression model using only the first 7 days of log-in count variables was 0.94 (95% CI 0.90-0.97) for iCanQuit, 0.88 (95% CI 0.83-0.93) for QuitGuide, 0.85 (95% CI 0.80-0.88) for WebQuit.org, and 0.60 (95% CI 0.54-0.66) for Smokefree.gov. Replacing logistic regression models with the more complex methods of decision trees, SVM, or neural network models did not significantly increase the AUC, nor did including additional baseline variables as predictors. The sensitivity and specificity were generally good, and they were excellent for iCanQuit.The results generally supported the overall study aim, showing that the first 7 days of log-in count data were sufficient to predict early dropout from a digital intervention. This prediction was achieved using a standard logistic regression model and validated with large randomized trial data sets testing 3 other digital interventions. Indeed, the pattern of results was generally robust across different clinical content , structure of intervention delivery , and delivery platforms . The final models showed that a higher number of log-in counts in the first 7 days after randomization predicted a lower likelihood of being an early dropout, as demonstrated by the significant negative coefficients. The pattern of results was generally more pronounced for the log-ins occurring on later days of the week, which showed higher-magnitude negative coefficients. For example, in the iCanQuit data, the number of log-ins on days 5, 6, and 7 was especially predictive of being an early dropout. Across the 4 interventions, the number of log-ins on day 7 was generally the strongest significant predictor of early dropout.This study provides 2 major advances on previous research on predicting early dropout in digital interventions. The first advance is that 1 type of variable that is straightforward, objective, and commonly measured provided reliable prediction of early dropout as compared with previous studies that have used a complex set of variables that are difficult to generalize and replicate  or have relied on self-reporting, which requires extra resources to collect, has higher missing data rates, and has subjective measurement biases -20. In tTo build on this study’s findings, future research could focus on replicating this approach in other digital intervention data sets as well as testing adaptive treatment augmentations for early dropouts. Specifically, replications of the model could focus on an array of possible digital intervention platforms , types of behavior change interventions , and types of study design . In an SMS text messaging context, an analogous engagement metric to “daily login” would be a time-stamped SMS text message engagement with the user . Regarding adaptive interventions, this study has implications for developing a randomized trial that offers an adapted, augmented intervention for individuals who are likely to drop out of their assigned digital intervention. The Sequential Multiple Assignment Randomized Trial is an increasingly common type of factorial design that could be applied to the problem of early dropout from a digital intervention by experimentally manipulating the offer (vs not) of a specific treatment augmentation -55. The This study has important limitations. First, as fitting the study aims, the study was an exploratory analysis as participants were not randomized to log-in a different number of times each day. Thus, caution should be taken regarding making causal inferences from the analysis. Second, because of a Google Analytics error, there was approximately 11% of missing use data for the iCanQuit trial. Finally, the model performance predicting early dropout was generally weakest for the NCI Smokefree digital intervention. Although there is nothing unusual about this intervention in terms of its content or structure, it would be worthwhile for future research to conduct diagnostics into the features of this intervention to learn why model performance was weaker for the prediction of early dropouts. For example, our previous research identified 3 log-in trajectory groups in Smokefree: 1-week , 4-week, and 5-week users . HoweverThis study has important strengths. First, the sample size for each digital intervention was large (range 1038-1266), with a total combined sample size of 4529. Second, the sample was geographically diverse, recruited from all 50 US states, and had a high percentage of participants with minority race and ethnic backgrounds. Third, the type of variable that predicted early dropout was objectively collected, thereby reducing measurement bias. Related, the predictor variable is commonly collected by other researchers, thus making the analyses more comparable and readily replicable. Fourth, the pattern of results found in the primary sample of the iCanQuit data was validated in 3 other digital interventions with different clinical content , structure of intervention delivery , and delivery platforms . Finally, a highly generalizable quality of these trials is that they were conducted in the real world (as opposed to a laboratory), where users were completely free to log-in at will and in the context of their daily lives.In conclusion, logistic regression models using only the first 7 days of log-in count data are generally good at predicting early dropouts. These models performed well when using simple, automated, and readily available log-in count data, whereas including self-reported sociodemographic baseline variables did not improve the prediction. The results will inform the early identification of people at risk of early dropout from digital health interventions with the goal of intervening further by providing them with augmented treatments to increase their retention and, ultimately, their intervention outcomes.


Advanced genomic techniques have now been incorporated into diagnostic practice in neuro-oncology in the literature. However, these assays are expensive and time-consuming and demand bioinformatics expertise for data interpretation. In contrast, single-gene tests can be run much more cheaply, with a short turnaround time, and are available in general pathology laboratories. The objective of this study was to establish a molecular grading scheme for adult gliomas using combinations of commonly available single-gene tests. We retrospectively evaluated molecular diagnostic data of 1,275 cases of adult diffuse gliomas from three institutions where we were testing for IDH1/2 mutation, TERTp mutation, 1p19q codeletion, EGFR amplification, 10q deletion, BRAF V600E, and H3 mutations liberally in our regular diagnostic workup. We found that a molecular grading scheme of Group 1 , Group 2 , Group 3 , Group 4 , Group 5 , and Group 6  could significantly stratify this large cohort of gliomas for risk. A total of 1,028 (80.6%) cases were thus classifiable with sufficient molecular data. There were 270 cases of molecular Group 1, 59 cases of molecular Group 2, 248 cases of molecular Group 3, 27 cases of molecular Group 4, 117 cases of molecular Group 5, and 307 cases of molecular Group 6. The molecular groups were independent prognosticators by multivariate analyses and in specific instances, superseded conventional histological grades. We were also able to validate the usefulness of the Groups with a cohort retrieved from The Cancer Genome Atlas (TCGA) where similar molecular tests were liberally available. We conclude that a single-gene molecular stratification system, useful for fine prognostication, is feasible and can be adopted by a general pathology laboratory. There has been a vast increase in our knowledge of the molecular pathology of central nervous system (CNS) tumors, and a new WHO classification will soon be published . The WHONew England Journal of Medicine, in addition to using IDH and 1p19q, introduced also TERT mutation to stratify low-grade gliomas (Isocitrate dehydrogenase (IDH) genotype has been shown to be a major classifier and prognosticator for gliomas , 11 and  gliomas . BRAF is gliomas –18. Phil gliomas –24.We hypothesized that combinations of genes IDH1/2, 1p19q, TERTp, EGFR, BRAF, TERTp, 10q, and H3 status can stratify gliomas for risk and may even be superior to conventional histological grading for prognostication. We recognize that this gene list is not exhaustive as regards the vast amount of literature concerning the molecular pathology of gliomas. However, since there is a limit as to how many single-gene tests an in-house facility can possibly carry out with a reasonable turnover time, we hypothesize that such a list would adequately molecularly grade a glioma and that it could be superior to conventional histological grading in some instances.A total of 1,275 adult diffuse gliomas diagnosed as astrocytic or oligodendroglial from patients aged 18 years or above were unselectively and retrospectively retrieved from the pathology archives of three institutions: Prince of Wales Hospital, Chinese University of Hong Kong, Huashan Hospital, Fudan University, Shanghai and First Affiliated Hospital of Zhengzhou University, Zhengzhou. Pediatric gliomas were excluded due to the vastly different molecular pathology between adult and pediatric gliomas , 26. SomDemographics and survival data were obtained from the hospital information systems of the three hospitals. The protocol for treatment for adult gliomas of Grades II–IV in all three hospitals was maximal safe resection in the first instance. Some but not all patients received adjuvant radio-chemotherapy due to individual clinical situations Table 1,in situ hybridization (FISH) for CDKN2A homozygous deletion was also evaluated in some cases as previously described  containing the genomic sequences of the 7p11.2 and a centromere probe  was used for EGFR amplification detection. Vysis LSI PTEN/CEP 10 FISH Probe Kit was applied for detection of 10q loss. Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Vysis) was used to examine 1p19q codeletion. Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes (Vysis) was employed to study CDKN2A. At least 100 non-overlapping signals were counted and analyzed in each case. EGFR amplifications were considered when >5% recorded cells displayed many tight clusters or a ratio of the target (red) to reference (green) signal >2 , 36. DelA cohort with a similar gene set from TCGA was also retrieved , 14. GraStatistical analysis was conducted on IBM SPSS software v22. A chi-square test was used to identify the correlation between molecular grade and clinical parameters. Survival curves were evaluated by the Kaplan–Meier (KM) method, and the log-rank test was done to compare survival distribution between groups. All statistical tests performed were two-sided, with p-values less than 0.05 as the threshold for significance.https://www.surgery.cuhk.edu.hk/btc/hsbc/.A summary of the clinical characteristics of this study cohort is shown in We stratified the adult gliomas into 6 molecular grades based on IDH mutations, 1p19q codeletion, TERTp mutation, BRAF mutation, EGFR amplification, 10q loss, and H3 mutations. The molecular grading scheme was as follows:Molecular Group 1: IDH mutant, 1p19q codeletedMolecular Group 2: IDH mutant, 1p19q non-codeleted, TERT mutantMolecular Group 3: IDH mutant, 1p19q non-codeleted, TERT wild typeMolecular Group 4: IDH wild type, BRAF mutantMolecular Group 5: IDH wild type, BRAF wild type, with no evidence showing TERTp mutation, EGFR amplification, 10q loss, and H3 mutationMolecular Group 6: IDH wild type, positive for TERTp mutation, EGFR amplification, 10q loss, or H3 mutationsThe numbers of different tests carried out on this cohort are shown in Overall, there were 270 cases of molecular Group 1, 59 cases of molecular grade 2, 248 molecular grade 3, 27 cases of molecular grade 4, 117 cases of molecular Group 5, and 307 cases of molecular grade 6 Table 1.As shown in We also tried to compare the power of prognostication of the molecular groups against histological grades in different scenarios. In three scenarios, the molecular groups were better than histological grades in prognostication. We next retrieved an unselected adult glioma cohort with relevant clinical and molecular data from TCGA . The proThe diagnostic protocols for brain tumors have been vastly changed in the advanced centers due to the advent of next-generation sequencing, for both DNA and RNA and also genome-wide methylation profiling. The former can evaluate gene mutations or gene fusion on a panel of a large number of genes, while the latter can map all brain tumors according to their methylation profiling into 91 methylation classes . Copy nuMost general pathology laboratories in the world are not in a position to carry out next-generation sequencing or genome-wide methylation profiling routinely on all their neurosurgical specimens, as these tests remain expensive and demanding on expertise for interpretation, e.g., t-distributed stochastic neighbor embedding (t-SNE) plots. Most national types of health finance systems are unable to support such extensive genomic tests regularly. However, most pathology departments in the more advanced societies will have the capacity for single-gene studies and molecular tests like Sanger sequencing, for example, for BRAF V600E, IDH1 R132, and IDH2 R172H. Similarly, FISH is also widely available in pathology departments, especially in the context of Her-2 testing for breast cancers. We therefore believe that there is a need to develop a relatively simple molecular grading algorithm for the gliomas based on single-gene testing based on current literature on the molecular pathology of gliomas , 40–42. Mutations of the promoter region of TERT have been suggested to be a key terminal event in the evolution of glioblastomas, but it has an intriguing property of being mutated also in oligodendrogliomas, which are mostly low-grade gliomas . The reaOther than lower-grade gliomas, there have also been a few studies in the literature devoted to molecular-grade special groups of gliomas by single-gene studies , 33, butThe biomarkers used in this study were known in the literature on their own. Group 1, IDH mutant, 1p19q codeleted is the regular oligodendroglioma as currently defined by WHO Classification, and Group 6 constitutes the bulk of the regular IDH wild-type glioblastomas as defined by cIMPACT-NOW update 3 . Group 5For glioblastomas, cIMPACT and the new WHO 2021) Classification recommended the use of EGFR amplification, TERT, and chromosome 7+/10− as criteria for diagnosis of molecular glioblastoma in IDH wild-type adult gliomas, which do not show diagnostic histological features like necrosis and microvascular proliferation  Classifi. For IDHThe molecular grading scheme we propose can easily be made available in general pathology departments. Adoption of this system provides a fine prognostication for gliomas and in, some instances, is superior to the histological grading, as shown in We also recognize the limitations of our study, as we used retrospective analyses of existing data, and it would be impossible for us to re-do a new panel of single-gene markers for 1,275 cases according to the recommendation of the new WHO Classification. Ours is not a proposal to replace the new WHO Classification (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by The Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee; the Ethics Committees of Huashan Hospital, Shanghai; and The First Affiliated Hospital of Zhengzhou University, Zhengzhou. The patients/participants provided their written informed consent to participate in this study.The authors confirm contribution to the paper, as follows: study conception and design: AC, W-SP, HL, and H-KN. Data acquisition: Z-FS, KL, W-WW, HC, NC, DC, X-ZL, Z-YZ, and YM. Analysis and interpretation of data: AC, Z-FS, KL, NC, and H-KN. Drafting of the manuscript: AC and H-KN. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by the Health and Medical Research Fund (HMRF), the Food and Health Bureau of Hong Kong (07180736); the National Natural Science Foundation of China ; and Shanghai Municipal Science and Technology Major Project (2018SHZDZX01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is a growing need for advanced treatment in children with persistent and severe asthma symptoms. As a matter of fact, between 2 and 5% of asthmatic children experience repeated hospitalizations and poor quality of life despite optimized treatment with inhaled glucocorticoid plus a second controller. In this scenario, mepolizumab, a humanized monoclonal antibody, has proven to be effective in controlling eosinophil proliferation by targeting interleukin-5 (IL-5), a key mediator of eosinophil activation pathways. Mepolizumab is approved since 2015 for adults at a monthly dose of 100 mg subcutaneously and it has been approved for patients ≥ 6 years of age in 2019. Especially in children aged 6 to 11 years, mepolizumab showed a greater bioavailability, with comparable pharmacodynamics parameters as in the adult population. The recommended dose of 40 mg every 4 weeks for children aged 6 through 11 years, and 100 mg for patients ≥ 12 years provides appropriate concentration and proved similar therapeutic effects as in the adult study group. A marked reduction in eosinophil counts clinically reflects a significant improvement in asthma control as demonstrated by validated questionnaires, reduction of exacerbation rates, and the number of hospitalizations. Finally, mepolizumab provides a safety and tolerability profile similar to that observed in adults with adverse events mostly of mild or moderate severity. The most common adverse events were headache and injection-site reaction. In conclusion, mepolizumab can be considered a safe and targeted step-up therapy for severe asthma with an eosinophilic phenotype in children and adolescents. Severe asthma is a highly heterogeneous disease due to the interaction between genetic predisposition, immune response, and environmental risk factors. This complex interaction influences the onset of symptoms, the clinical evolution, and the severity of illness , 2. SeveA multidisciplinary assessment is required to exclude comorbidities and modifiable factors, confirm the diagnosis, and initiate the most appropriate treatment for these patients . The ideRecently, several innovative monoclonal antibody agents have been approved to target specific inflammatory type 2 mediators and improve uncontrolled severe asthma when added to basal treatment . HoweverIn this review, especially focused on mepolizumab, we present all current evidence on the indications, use, safety, and efficacy of this fully-humanized anti-interleukin-5 (IL-5) antibody for children with severe asthma.This review is based on the most significant results from an extensive PubMed search, conducted independently by three different researchers, using the search terms: “treatment in severe pediatric asthma,” “severe asthma,” “mepolizumab,” “anti-IL-5 therapy,” “anti–IL-5 antibody,” “children,” “adolescents,” and all different synonyms or word combinations. More relevance was given to clinical trials especially focused on mepolizumab treatment.There is no uniformly accepted definition of severe asthma . DiffereAccording to the Global Initiative for Asthma (GINA) guidelines 2022, severe asthma isindicated by needing a high dose of ICS-LABA to maintain symptom control or uncontrolled asthma despite steps 4 and 5 of the care and management of contributory factors .The European Respiratory Society and the American Thoracic Society (ERS/ATS) definitions of severe asthma require that patients have needed therapy with a high dose of ICS and a second controller, such as LABA, leukotriene modifier, or theophylline, for the previous year or have needed OCS for 50% or more of the year to prevent asthma from becoming uncontrolled or that cannot be controlled despite this therapy .Patients that need to be moved to GINA steps 4 and 5 care have to be referred to a tertiary center for a specialist evaluation first to confirm the diagnosis and subsequently to identify the best personalized treatment . In the Moreover, the presence of comorbidities, including allergic conditions , sinus disease, gastroesophageal reflux, obesity, obstructive sleep apnea, anxiety, and depression, may reduce the response to therapy –23.Finally, the exclusion of asthma-mimicking conditions , such asOnce all the following steps are carried out, that is, revising treatment issues, excluduing or treating comorbidities or other differential diagnoses, and confirming that it is severe asthma, it is possible to consider add-on medications, including biologics.The introduction of biologics has revolutionized the care of severe asthma in adult and pediatric populations –29. ReceThe phenotype is the summation of clinical features while the endotype is determined by biomolecular mechanisms leading to the pathogenesis of the disease . To dateThe T2-low endotype is more frequent in adults and is characterized by the following: neutrophilic or paucigranulocytic inflammation, T-helper lymphocytes type 17, innate lymphoid type 3 cells, and IL-1, IL-8, IL-17, and IL-23 are the respective implied molecules , 35.The T2-high endotype typically affects children and it is characterized by two specific characteristics: allergic sensitization and eosinophilia . AllergiIn the T2-high endotype, T-helper lymphocytes type 2 signal the production of IL-4, IL-5, and IL-13, while innate lymphoid cells type 3 are activated by the epithelial alarmins TSLP, IL-33, and IL-25. Specifically, IL-4 and IL-13 promote B lymphocyte activation, inducing plasma cell formation, isotype switching to IgE, and their production. On the other hand, IL-5 induces chemoattraction, proliferation, and activation of eosinophils and also decreases their apoptosis .Current research has suggested that these molecular pathways of severe asthma should guide the choice of the most adequate biologic treatment . The ide50 of <1 nM, a dissociation constant of 4.2 pM, and stoichiometry of 2.2, so that two IL-5 dimers are cross-linked by two molecules of mepolizumab. Therefore, through this mechanism of action, mepolizumab effectively inhibits IL-5 ligation to IL-5Rα  has been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in patients aged 12 years and older since November 2015 and was licensed in September 2019 in patients aged 6 years and older as an add-on maintenance therapy for severe eosinophilic asthma , 41. Mepo IL-5Rα . As ment9/l in the absence of OCS treatment and > 0.3 × 109/l in the previous year plus two or more asthma exacerbations requiring hospitalization or OCS treatment. In contrast to adults, among children with severe asthma, a smaller proportion of patients appear to have clear features of type 2 mucosal inflammation, including greater eosinophilia  ; MepolizThe impact on health-related quality of life has been objectified through several validated questionnaires. The Asthma Control Questionnaires (ACQ-5 and ACQ-7) have been used in most studies , 49, 50wAnother tool, the St. George's Respiratory Questionnaire (SGRQ), offers a wider perspective on treatment effectiveness and shows confirmed clinical improvement in all the domains  after 6 months of therapy compared to the placebo group . It is nAlthough the clinical experience with mepolizumab in pediatric asthma is often heterogeneous and with high rates of treatment discontinuation , this dr1) compared to the placebo group  higher blood eosinophils, (2) higher number of severe exacerbations in the previous year, (3) adult-onset asthma, (4) adult-onset asthma, (5) nasal polyposis, (6) maintenance oral corticosteroids, and (7) low lung function.To date, mepolizumab is also approved for the treatment of pediatric patients aged ≥12 years with hypereosinophilic syndrome (HES) at a dose of 300 mg every 4 weeks. The recommended patient population includes those with ≥ 6 month duration of HES without an identifiable non-hematologic secondary cause , 41.1 is not the most appropriate marker to detect mepolizumab's beneficial effects. Due to its capacity to target specific inflammatory type 2 mediators, mepolizumab represents a milestone in the application of personalized medicine. The prescription of mepolizumab for the treatment of severe asthma has expanded rapidly and this drug, currently used in adults, has also been registered for children, despite most of the scientific evidence in literature coming from adults. More data on efficacy in pediatric patients would be necessary to confirm its promising effects. Further clinical trials in the pediatric population are also important to prove long-term safety and the impact of this medication on the natural history of the disease. Finally, the identification of new biomarkers could be useful to predict real benefits from therapy with mepolizumab and to establish its optimal duration.From a clinical point of view, the main purpose of asthma treatment is to reduce symptoms and the recurrence of exacerbations. Mepolizumab proves its efficacy in the specific phenotype of asthmatic patients with intense eosinophilic airway inflammation and recurrent exacerbations. As often stated in children, we believe that FEVRC and NU discussed the writing project. RC, NU, FPe, and OF wrote the manuscript with significant support from FPo, EP, and AO. NU and RC supervised the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.