The Report of China’s Automobile Industry Development 2016 points out that China’s automobile industry faces increasingly fierce competition from global peers, and domestic automobile manufacturers shall continuously improve independent innovation and industry innovative performance. Automobile manufacturers’ participation in distributed innovation and their research & development (R&D) capability play a key role in the growth of innovative performances of China’s automobile industry. With development of China’s automobile industry as the background, this study presents a theoretical framework of correlation of distributed innovation embedment, R&D capability and innovative performances of China’s automobile industry from the microscopic perspective of single industry. Several hypotheses were proposed and then verified by empirical method. Specifically, 117 effective questionnaires were collected through non-probability sampling and analyzed quantitatively, followed by hypothesis test. The results clarify the working principles of the three dimensions of distributed innovation embedment influencing innovative performances of China’s automobile industry and improvement of R&D capability of enterprises by distributed innovation. This study provides references to improvements of innovative performances of China’s automobile industry. Recently, distributed innovation embedment in enterprises have attracted great attention by scholars. The influences of distributed innovation on enterprises have been thoroughly investigated from the macroscopic perspective. It is believed that under the background of information explosion and intensified competition, distributed innovation embedment, which is characterized by cooperation, network, dynamics and sharing, can enhance innovation activities of enterprises and allow them to exploit comparative advantages, thus enhancing their performances. The rest of the paper is organized as follow. The following section shed light on the theoretical background and hypothesis development. Section 3 discusses the research design, methodology and data. Section 4 presents the results and findings of the study. Section 5 concludes the study.2.2.1.The innovative performances of China’s automobile industry shall be evaluated by quantitative indexes of enterprises and qualitative indexes of managers. 2.2.Previous studies of distributed innovation reveal that knowledge collaboration embedment has influences on innovative performances of enterprises. For instance, In summary, the following hypotheses are proposed:Hypothesis H1: Distributed innovation embedment has significant positive effects on innovative performances of China’s automobile industry.Hypothesis H1a: Distributed structure embedment has significant positive effects on innovative performances of China’s automobile industry.Hypothesis H1b: Distributed cognition embedment has significant positive effects on innovative performances of China’s automobile industry.Hypothesis H1c: Knowledge collaborative embedment has significant positive effects on innovative performances of China’s automobile industry.2.3.Owing to limited resources and capabilities, enterprises have to rely on distributed innovation systems to enhance R&D capability. A distributed innovation system is both a collaborative process of different organizations within the distributed innovation network and an interactive process of mutual adaptation and interactions between these organizations and the external environment. The distributed structure of distributed innovation system delivers a R&D network with peers in the automobile industry or upstream/downstream enterprises by modularization of complex system and strategy alliance. In this way, the R&D capability of an enterprise can be strengthened. The distributed cognition of distributed innovation system emphasizes common interest-based distributed cognition and trust, as well as dependence of upstream and downstream enterprises, resulting in a large-scale economy of China’s automobile industry. The knowledge collaboration is indeed the core part of distributed innovation system. R&D is originated from collaboration of vertical processing and horizontal modularization of knowledge. Creativity, knowledge and technical guidance can be obtained from collaborations, resulting in an effective R&D system.In summary, the following hypotheses are proposed:Hypothesis H2: Distributed innovation embedment has significant positive effects on R&D capability of China’s automobile industry.Hypothesis H2a: Distributed structure embedment has significant positive effects on R&D capability of China’s automobile industry.Hypothesis H1b: Distributed cognition embedment has significant positive effects on R&D capability of China’s automobile industry.Hypothesis H1c: Knowledge collaborative embedment has significant positive effects on R&D capability of China’s automobile industry.2.4.In summary, the following hypothesis is proposed:Hypothesis H3: R&D capability has significant positive effects on innovative performances of China’s automobile industry.3.3.1.The proposed model see  includes3.2.Innovative performances of China’s automobile industry are affected by distributed innovation embedment, including knowledge collaborative. This study further explores the significance of distributed innovation  to enterprise development. Hence, innovative performances of China’s automobile industry, distributed innovation and R&D capability were measured. Innovative performances of China’s automobile industry have been investigated based on sales volume, market shares, financial indexes, evaluation of innovation capability by managers and consumer satisfactory on new products. Considering differences in strategy and management of enterprise innovation, we measured innovative performances of enterprises from perspectives of product innovation, product update, technological innovation and managers, which are denoted by IP1, IP2, IP3 and IP4, respectively. Six items were designed for this measurement as R&D capability was measured from perspectives of ratio of investment in R&D, patent quantities, R&D employees, which are denoted by RDA1, RDA2 and RDA3, respectively. Six items were designed for this measurement as The variables of distributed innovation embedment were measured by methods reported elsewhere. Specifically, the variables are divided into categories of distributed structure embedment, distributed cognition embedment and knowledge collaborative embedment, which are denoted by DI1, DI2 and DI3, respectively. The distributed structure embedment focuses on geological distributions and project modularized allocation; the distributed cognition embedment focuses on trust, cognitive learning and cooperation among members; the knowledge collaborative embedment focuses on knowledge transfer, knowledge sharing and innovation management. Sixteen items were designed for this measurement as 3.3.via e-mail and 117 of them completed by respondents from automobile manufacturers participated in distributed innovation were regarded as effective ones. The study used non-probability sampling technique for the sample selection. All respondents are managers and engineers who have a thorough understanding of the enterprise and the industry. The manufacturers involved are mainly located in Hubei, Shanghai, Beijing, Chongqing, Guangzhou and Tianjin. Additionally, 90% of respondents have distributed innovation as a part of their enterprise strategy. In summary, samples are widely distributed and representative, thus meeting the requirements.Questionnaires were designed according to the measurement variables and sent to representative automobile manufacturers in China, including FAW-Volkswagen, Dongfeng Motor Corporation, Dongfeng Peugeot Citroen Automobile Company Ltd. (DPCA), Renault S.A., Volvo and Dongfeng Yueda Kia (DYK). One hundred eighty-seven questionnaires were allocated 4.4.1.In order to ensure the validity of this study, validity and reliability of samples shall be verified before data analysis. In this study, validity and reliability of samples were verified by factor analysis and internal consistency test, respectively. As shown in 4.2.Since reliability and validity of samples have been demonstrated to be reasonable, a theoretical model is proposed according to the hypotheses see  to invesp values in the theoretical model are summarized to obtain path coefficients and verification results.Model verifications of different variables demonstrate that fit indexes of the proposed theoretical model meet the requirements. Meanwhile, model verifications indicate that the goodness of fit index (GFI), the comparative fit index (CFI) and the root mean square error approximation (RMSEA) are 0.942, 0.973 and 0.035, all of which meet the requirements, demonstrating good fitting of the model and practical data. Hence, the results are statistically significant. via R&D capability.As shown in 4.3.Hypotheses H1a is valid. According to H1a, China’s automobile enterprises shall pay more attention to project modularized allocation and geographical distribution of 4S stores in order to increase their market shares.Hypotheses H1b is valid. According to H1b, learning cognition and identification of corporate culture by employees have positive effects on innovative performances of China’s automobile industry.Hypotheses H1c is valid. According to H1c, sharing, transfer and creation of key resources, namely knowledge, have significant effect on success rate and frequency of new product development and automobile industry shall pay more attention to knowledge management to develop intelligent enterprises and creative employees.Hypotheses H2 is valid. According to H2, distributed innovation has positive effects on R&D capability of China’s automobile industry.Hypotheses H3 is valid. According to H3, enhanced R&D capability has positive effects on technical content, success rate and frequency of new product development in China’s automobile industry.In summary, China’s automobile enterprises shall enhance their innovative performances and competitiveness by participating in distributed innovation and improving R&D capability.5.With the current situation of China’s automobile industry as the background, the correlation of distributed innovation embedment, R&D capability and innovative performance is investigated. Seven hypotheses are proposed and empirically verified based on practical data of China’s automobile industry. The results demonstrate that innovative performances of China’s automobile industry are highly dependent on distributed innovation embedment and R&D capability.In terms of theory, the correlation of innovative performances in automobile industry, R&D capability and distributed innovation embedment is investigated from a microscopic perspective  for the first time. Empirical and theoretical analyzes demonstrate positive correlations of these factors.This study has multiple practical implication for the Chinese automobile industry and for the policy making authorities. First the study motivates the companies to engage their stakeholders in the decision-making process and motive the knowledge sharing from bottom to top. Secondly the management of the companies must manage the knowledge coming from different ends such as within the organization and outside the organization’s boundaries. The management of this information and manage the flow of the information is critical and challenging for the firms. The managers must stress on the need on the R& D especially in the new product development which resulted in the better financial performance of the companies. The management must promote the learning culture within organizations and promote the knowledge sharing among the employees. This study also facilitates the regulatory bodies by introducing and promoting the open innovation laws and support the firms practicing this approach. These practices will not only enhance the Chinese industry performance overall but also set the benchmark for the rest of the industries in China specifically and for the rest of growing economies in the neighbor generally. Additionally, this study provides references to improvements of innovative performances of China’s automobile industry. Automobile enterprises shall strengthen project modularization, trust between employees and knowledge collaboration. Meanwhile, automobile enterprises shall attach great importance to knowledge sharing and innovation management.This study has several limitations. On the one hand, it is based on correlations of distributed innovation embedment, R&D capability and innovative performances of China’s automobile industry, while other factors affecting variable correlations have not been considered. On the other hand, the conclusions are only applicable to automobile industry in certain regions in China due to the insufficient sample size.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethical committee of the Hubei University ref. 20/NSF/684201. The patients/participants provided their written informed consent to participate in this study.JL initiated the idea and conceptualize the idea, and wrote the initial draft. GL manage the data and collected the data, and validate the analysis. HC performed the data analysis. YC supervise the project and managed the resources and final document, and took supporting role in the data analysis. All authors contributed to the article and approved the submitted version.This project is funded by The Impact of Technology Lock-in on the Dynamic Upgrading of China’s Manufacturing Industry in Global Value Chain and Its Solution 20AGL006.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.


Case Presentation. Seven-day-old female neonate was brought by her parents after they observe reddish discoloration of her genitalia. Upon examination, there was a vertical perineal wet groove-like unkeratinized erythematous lesion with no bleeding, sign of infection, or inflammation that extends from the posterior vaginal fourchette to the anal rim. The newborn was sent home after counseling with a reassessment plan in case of complications.  Perineal groove is a rare congenital anomaly characterized by nonepithelialized mucous membrane that appears as an erythematous sulcus in the perineal midline, extending from the posterior vaginal fourchette to the anterior anal orifice. It is one of the uncommon anomalies of anogenital region that is unknown to many clinicians, and it is usually misdiagnosed as an anal fissure, perineal trauma, diaper dermatitis, infection, or sexual abuse.  Recognition of the congenital perineal groove at birth is important for the health care providers to deliver an appropriate parental counseling and appropriate follow-up. Perineal groove is a rare congenital anomaly characterized by nonepithelialized mucous membrane that appears as an erythematous sulcus in the perineal midline, extending from the posterior vaginal fourchette to the anterior anal orifice , 2. It iA seven-day-old female newborn from Bahir Dar city was brought to Afilas General Hospital by her parents with the complaint of reddish discoloration of the skin around the genitalia (like fresh wound). The neonate was born to a 30-year-old Para II mother through spontaneous vaginal delivery with no instrumentation at gestational age of 39 + 6 weeks with birth weight of 3400 grams and Apgar score of 8 and 10 in the first and fifth minutes, respectively. The total duration of labor was 10 hours and rupture of membrane was intrapartum. The mother had regular antenatal care follow-up and it was uneventful. The mother has no history of cigarette smoking, alcohol consumption, or substance abuse. Both the father and mother have no history of congenital birth defects in the family. There was no history of local trauma, defecation issues, and urinary problem with the newborn.Physical examination of the neonate was normal in other body systems. On genital examination, there was around 1.5 cm wet groove-like erythematous vertical lesion which extends from the posterior vaginal fourchette at 6 o'clock to the anal rim . The lesPerineal groove is generally a wet sulcus lesion extending from the posterior vaginal fourchette to the anterior anus . It is mIt is a congenital malformation that consists of three main features: (1) a wet groove in the midperineum between the fourchette and the anus, (2) normal formation of the vestibule including the urethra and vagina, and (3) hypertrophy of the minoral tails that skirt the perineum and course posterior to join at the anus or surround it .There are several controversial embryological theories regarding the formation of perineal groove , 4. ThisPerineal groove could be complete or incomplete. The complete perineal groove extends from the vaginal fourchette to the anus, while the incomplete type either extends from the vaginal fourchette to the middle of the perineum or from the anus to the middle of the perineum .Few cases have been published in medical literatures , 10. DefPerineal groove usually resolves spontaneously, and complete epithelialization occurs by the age of 1 to 2 years . SurgicaCorrect recognition is important to avoid misdiagnosis and unnecessary investigations. I report this case to raise awareness and for doctors to provide appropriate advice and follow-up recommendations for parents of affected infants.


Dysfunction of glial cell communication is involved in Alzheimer’s disease (AD) pathogenesis, and the recent study reported that astrocytic secreted interleukin-3 (IL-3) participated in astrocyte–microglia crosstalk and restricted AD pathology in mice, but the effect of IL-3 on the pathological progression of AD in human is still unclear.A total of 311 participants with cerebrospinal fluid (CSF) IL-3, soluble triggering receptor expressed on myeloid cells 2 (sTREM2), and AD biomarkers were included from the Alzheimer’s disease Neuroimaging Initiative (ADNI). We assessed the associations of IL-3 with sTREM2 and AD biomarkers at baseline, and with cognitive change in longitudinal study. The mediation models were used to explore the potential mechanism of how IL-3 affects AD pathology.We found that CSF IL-3 was significantly associated with CSF sTREM2 and CSF AD core biomarkers  at baseline, and was also markedly related to cognitive decline in longitudinal analysis. Moreover, mediation analysis revealed that CSF IL-3 modulated the level of CSF sTREM2 and contributed to tau pathology (as measured by CSF p-tau/t-tau) and subsequent cognitive decline. In addition, Aβ pathology (as measured by CSF Aβ42) affected the development of tau pathology partly by modifying the levels of CSF IL-3 and CSF sTREM2. Furthermore, the effect of Aβ pathology on cognitive decline was partially mediated by the pathway from CSF IL-3 and CSF sTREM2 to tau pathology.Our findings provide evidence to suggest that IL-3 is linked to sTREM2 and mediates the correlation between Aβ pathology to tau pathology. It indicates that IL-3 may be a major factor in the spreading from Aβ pathology to tau pathology to cognitive impairment.The online version contains supplementary material available at 10.1186/s12974-022-02679-5. Alzheimer’s disease (AD) is the most common type of dementia, characterized by two specific neuropathological profiles: extracellular amyloid-β (Aβ) deposition and intraneuronal neurofibrillary tangles (NFTs) consisting of aggregated hyperphosphorylated tau protein . Glial cInterleukin-3 (IL-3), a multifunctional cytokine involved in astrocyte–microglia communication, is believed to involve in inflammatory and autoimmune diseases . CerebroTo systematically determine the complex roles of IL-3 on AD pathogenesis and its specific interactions with microglial activity in the participants who had preclinical or clinical AD, we aimed to ascertain the interrelationships between CSF IL-3, microglial activation markers (as reflected by soluble triggering receptor expressed on myeloid cells 2 [sTREM2]), AD pathology, and cognitive change, and to explore effects of CSF IL-3 and CSF sTREM2 on AD pathology and cognitive change using a mediation model. We hypothesized that CSF IL-3 was associated with CSF sTREM2 and AD pathology as well as cognitive change and that association of CSF IL-3 and CSF sTREM2, as reflected in astrocyte–microglia communication, may be a key part of the progression of AD pathology, and even with subsequent cognitive impairment.www.adni-info.org. ADNI was approved by the institutional review boards of all participating institutions and all participants provided written informed consent.This study included participants from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. The ADNI project, a multicenter longitudinal study, aims to combine clinical, imaging, genetic, and biochemical biomarkers to develop and validate the measures of early diagnosis of late-onset AD. The detailed inclusion criteria in ADNI can be found at APOE4 status were grouped as carrying at least one APOE4 allele or no one APOE4 allele.A total of 327 received measurements of CSF IL-3 in the ADNI database. Among them, participants who completed the detection of CSF sTREM2, AD biomarkers , and followed up cognitive measures at least 1 years (baseline plus two follow-up visits) were included in this study .The CSF level of AD core biomarkers  was measured using the electrochemiluminescence immunoassays Elecsys immunoassays on a cobas 601 instrument. CSF sTREM2 levels were measured by the MSD platform-based assay, and previous publication reported and validated this detailed method . CSF IL3ADNI composite memory score (ADNI-MEM) and ADNI composite executive function score (ADNI-EF) were applied to reflect memory function and executive function, respectively . AlzheimT) and neurodegeneration (N) groups and then, subjects were categorized into different A/T/N categories: Stage 0 (A−TN−), Stage 1 (A+TN−), Stage 2 (A+TN+), Suspected Non-AD Pathology (SNAP) (A−TN+) [χ2 tests for categorical variables. The associations between IL-3 with sTREM2 and AD biomarkers were explored utilizing Spearman partial correlation and multiple linear regression (MLR), with adjustment for age, sex, education, and APOE4 status. Next, we tested whether baseline IL-3 levels were associated with longitudinal cognitive change using a linear mixed model, controlling follow-up time, random slope, intercept, age, sex, education, and APOE4 status.The normality of distribution for each biomarker was assessed by Kolmogorov–Smirnov test, and those variables that did not follow a normal distribution were normalized by the Box–Cox transformations. Outliers (outside three SD) were excluded from this study. Based on pre-established CSF AD biomarker cutoffs , partici (A−TN+) . BaselinAPOE4 status.Finally, five mediation models using structural equation model (SEM) were conducted in this study. First, we tested whether the association between Aβ42 and sTREM2 was mediated by IL-3. The second mediation model was used to explore whether the association between IL-3 and p-tau/t-tau was mediated by sTREM2. The serial mediation model was to explore whether associations of Aβ42 with p-tau/t-tau were mediated by IL-3 and sTREM2. We calculated the subject-specific slopes as the annual change rates of cognitive change in a linear model cognitive outcome by time and the fourth mediation model was tested whether association of IL-3 and cognitive change was mediated by sTREM2 and p-tau/t-tau. The last mediation model was used to explore whether association of Aβ42 with cognitive change was mediated by IL-3, sTREM2, and p-tau/t-tau. These mediation models were both adjusting age, sex, education, and p value < 0.05 was considered statistically significant. The “lmer”, “lme4”, “corrplot”, “lavaan”, “ggplot2”, and “car” packages in R version 4.1.0 software were used to perform the all above analyses.A two-sided APOE4 allele, MMSE scores, and AD diagnosis as well as CSF IL-3, CSF sTREM2, CSF Aβ42, CSF t-tau, and CSF p-tau, but not age, sex, and education  and showed a strong correlative trend with CSF Aβ42 and CSF p-tau as well as CSF t-tau , and ADAS13 scores   and sTREM2 . The indirect effect of Aβ42 on sTREM2 via IL-3 was significant   and p-tau  as well as t-tau   as well as t-tau   CSF Aβ42 → CSF IL-3 → CSF sTREM2; (2) CSF IL-3 → CSF sTREM2 → CSF p-tau/t-tau. In the first mediation model, Aβ42 had a positive association with IL-3  CSF Aβ42 → CSF IL-3 → CSF p-tau/t-tau; (2) CSF Aβ42 → CSF sTREM2 → CSF p-tau/t-tau; (3) CSF Aβ42 → CSF IL-3 → CSF sTREM2 → CSF p-tau/t-tau. In the first mediation model, Aβ42 was positively associated with IL-3 and was negatively associated with p-tau/t-tau, and IL-3 was positively associated with p-tau/t-tau. The indirect effect of Aβ42 on p-tau/t-tau via IL-3 was significant  CSF IL-3 → CSF sTREM2 → CSF p-tau/t-tau → ADAS11 change; (2) CSF IL-3 → CSF sTREM2 → ADAS11 change; (3) CSF IL-3 → CSF p-tau/t-tau → ADAS11 change. The significant associations were observed between p-tau/t-tau with ADAS11 change. However, sTREM2 did not reach a significant association with ADAS11 change Fig. C, D. The CSF IL-3βp-tau = 0.008, p = 0.03; βt-tau = 0.01, p = 0.02)  Fig. A.Fig. 5MTo exclude the influence of comorbidities affecting IL-3 on our results, we repeated our primary statistical analysis in the population by excluding those diagnosed with comorbidities affecting IL-3. Details of the participants are presented in Additional file In the present study, we showed that CSF IL-3 were associated with CSF sTREM2, CSF Aβ42, CSF p-tau, and CSF t-tau as well as subsequent cognitive changes. Our mediation results found that CSF sTREM2 mediates association of CSF IL-3 with p-tau/t-tau, and CSF IL-3 and CSF sTREM2 as key mediators moderated the effect of CSF Aβ42 on CSF p-tau/t-tau. Importantly, the mediation results further demonstrated that both CSF sTREM2 and CSF p-tau/t-tau contributed to the association between CSF IL-3 and cognitive changes. Finally, we further showed that the influence of CSF Aβ42 on cognitive change was affected by the concurrence of CSF IL-3, CSF sTREM2, and CSF p-tau/t-tau. These results provided important human evidence to understand the relationship of cytokine IL-3 with microglial activity and AD pathogenesis as well as cognitive function and supported our hypothesis that glial cell crosstalk, especially astrocyte–microglia communication, may be downstream of amyloid pathology and may play a critical role in the subsequent tau pathology and neurodegeneration as well as cognitive decline in AD Fig. B.Although astrocytes serve important roles in maintaining the integrity of blood brain barrier (BBB), CNS immune homeostasis, synaptic plasticity, and normal neuronal communication –36, the Consistent with previous studies showing correlations between astrocyte secreted IL-3 and TREM2-signaling , we founThe amyloid cascade hypothesis was believed to be primary evidence for AD progression, suggesting that Aβ accumulation in the brain is an initial step in AD pathogenesis, followed by tau pathology and synaptic loss as well as cognitive decline . This hyOur results may have implications for the ATX(N) system that is based on an extension of the AT(N) system called the Aβ/tau/neurodegeneration to adapt our further knowledge of the pathophysiological mechanisms underlying the AD continuum . Since tThe mechanisms linking IL-3 to AD pathology remain elusive. IL-3 has been implicated in multiple biological processes, including neural development, stimulation of cell growth, suppression of apoptosis, and enhancement of hematopoiesis . One posThis study has significant strengths. It is the first study using population-based data to systematically the relationship between IL-3 with sTREM2 and AD pathology as well as cognitive function in a well-characterized cohort. Moreover, the possible pathway underlying this process, ranging from the initiated Aβ deposition to cognitive decline, was specified in the current study. Nevertheless, several caveats should be considered when interpreting the current results. First, there are numerous raw values of CSF IL-3 below the least detectable dose (LDD) in the ADNI database, which may cause some results bias. Second, IL-3 data used in this study derived from the Biomarkers Consortium Project that was designed as exploratory and meant for hypothesis and model generation, but not for hypothesis confirmation and model validation. In addition, the use of samples in this study was relatively small. Thus, future work should validate our results using high-sensitivity measurements of CSF IL-3 and large-scale cohorts. Third, the mediation model used in the current observational study gives us a clear explanation of the associations between IL-3 with microglial activation and AD pathology as well as cognitive function, but it cannot allow us to infer causality. Finally, the association between IL-3 and the specific brain regions of AD pathology cannot be detectable in our study due to the limited number of participants with Aβ PET and tau PET in ADNI1 project.In summary, our results suggested that IL-3 is linked to sTREM2 and mediates the correlation between Aβ pathology to tau pathology. It indicates that IL-3 may be a major factor in the spreading from Aβ pathology to tau pathology to cognitive impairment. These findings have important implications for clinical trials and may encourage future studies on enhancing IL-3 levels as a therapeutic intervention to slow down the development of AD pathology and cognitive decline.Additional file 1: Table S1. Associations of baseline IL-3 and longitudinal cognitive change. Table S2. Participant characteristics at baseline by biomarker-defined groups in samples excluding comorbidities. Table S3. Associations of baseline IL-3 and CSF biomarkers in samples excluding comorbidities. Table S4. Correlations between CSF biomarkers in samples excluding comorbidities. Table S5. Associations of baseline IL-3 and longitudinal cognitive change in samples excluding comorbidities. Figure S1. Mediation analysis. Figure S2. Mediation analysis between IL-3 and cognitive change. Figure S3. Mediation analysis in samples excluding comorbidities. Figure S4. Mediation in samples excluding comorbidities.


The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione  was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of  Escherichia coli, which has 175 amino acid residues with a molecular weight of 18.8 kDa  is mainly produced from 18.8 kDa . Recentl18.8 kDa . Howevermpliance . To overmpliance , proteinmpliance , and newmpliance  have beempliance ) have bempliance , immunogmpliance  and metampliance . PEGylatvia Fc receptor (FcRn)-mediated recycling  Fattecycling . 3) Fattecycling ; 4) Fattecycling . Therefoecycling , Degludeecycling , Liragluecycling  and Semaecycling ) have beecycling . This moecycling . Thus, tecycling .Taken together, the aim of this study was to develop a novel fatty chain modification strategy for the site-specific conjugation of the thiol group of Cys18 in rhG-CSF. Firstly, 1-Pentadecyl-1H-pyrrole-2,5-dione   was synt10k-MAL) was purchased from Aladdin . Acetonitrile of HPLC grade and trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich Corporation . All other chemicals used were analytical grade.Recombinant human granulocyte colony stimulating factor (rhG-CSF) and the murine myeloblastic NFS-60 cell lines were obtained from Jiuyuan Gene Engineering Co., Ltd. . Palmitic acid was purchased from Sangon Biotech . Monomethoxyl polyethylene glycols maleimide with Mw of 10 kDa  were purchased from the Experimental Animal Center of Shandong . The license number is scxk 2014–0007. The animal husbandry system was maintained under standard laboratory conditions. All animal experimental protocols were approved by the Animal Ethics Committee of Shandong University of Technology and operated according to the guideline for the care and use of laboratory animals of Shandong University of Technology.2), and finally the solid powder of pure C15-NH2 was obtained by vacuum distillation to remove the solvent, and drying. In the second step, C15-NH2 (23.45 mmol) and maleic anhydride (23.45 mmol) were dissolved in 15 ml anhydrous dichloromethane. The solution was heated reflux at 40°C for 30 min. Subsequently, the reaction mixture was cooled to room temperature, and the solid was filtered off, washed with cooled dichloromethane and dried to obtain the solid powder of n-pentadecyl maleic acid. Then, n-pentadecyl maleic acid (12.8 mmol) and sodium acetate (8.8 mmol) were dissolved in 15 ml acetic anhydride and heated reflux at 100°C for 2 h. After the reaction mixture was cooled to room temperature and added with 50 ml water, the solution was transferred to a separating funnel, and then successively washed with ethyl ether , 2% KOH solution  and water . The organic phase containing C15-MAL was vacuum distilled to remove the solvent, and then the residue solid was re-dissolved in 20 ml dichloromethane, dried over MgSO4. Finally, the solid of crude C15-MAL was obtained by vacuum distillation to remove the solvent.The synthetic route of C15-MAL was shown in The crude C15-MAL was further purified using a YMC-Pack ODS-A C18 semi-preparative column  on a Shimadzu SPD-M20 A system . The mobile phase A and B was pure water and acetonitrile, respectively. The column was equilibrated with 90% (v/v) B, followed by isocratic elution with 90% B over 30 min at a flow rate of 15 ml/min. The effluent was detected at 215 nm and the loading volume was 2 ml for each injection.The purity of the purified C15-MAL was evaluated by RP-HPLC analysis. The sample was injected into a Phenomenex C18  and purified using an isometric elution 90% phase B (acetonitrile and 0.1% TFA) over 60 min, phase A  at a flow rate of 1.0 ml/min with UV detection at 215 nm.1H NMR, the sample to be tested was dissolved in methanol. The experimental data were recorded by 500 MHz nuclear magnetic resonance (NMR).The molecular weight of C15-MAL was determined by mass spectrometry using Thermo ScientificITQ 1100TM . The chemical shifts of hydrogen atoms in C15-MAL were detected by rhG-CSF (1.0 mg/ml) dissolved in 20 mM sodium phosphate buffer  and C15-MAL of the desired concentration  dissolved in DMSO solution were mixed at a volume ratio of DMSO/phosphate buffer of 12:1, 10:1, 8:1, 6:1 or 4:1, and then, the reaction was carried out at temperature of 4, 20, 30 or 40°C for 0–120 min with a stirring speed of 200 rad/min. The standard condition for the analysis of each factor was as follows: volume ratio of DMSO/phosphate buffer of 10:1, mole ratio of C15-MAL to rhG-CSF of 5:1, pH 7.0, temperature 20°C and reaction time 30 min. Each sample (1 ml), taken at different time interval, was added with 50 μL of 1 M DTT to stop the reaction, and then, immediately analyzed by RP-HPLC to calculate the concentration of the desired product (C15-rhG-CSF) based on the peak area. To evaluate the conjugation efficiency, the yield of C15-rhG-CSF was calculated by The crude C15-rhG-CSF was purified using a Sepax Bio-C8 RP-HPLC column  on a Shimadzu SPD-M20 A system . The mobile phase A was pure water with TFA adjusting pH 4.2, and mobile phase B was pure acetonitrile with TFA adjusting pH 4.2. The column was equilibrated with 40% (v/v) B, followed by elution with a linear gradient of 40%–55% B over 5 min and 55%–85% B over 30 min. The column temperature was maintained at 30°C and the flow rate was 10 ml/min, and the effluent was monitored at 280 nm. The collected fraction of the purified C15-rhG-CSF was diluted and dialyzed with 10 mM acetate buffers (pH 4.2), and then concentrated using Amicon Ultra-15 centrifugal filter devices with a 3 kDa MW cut-off , and stored at −20°C until use.10k-rhG-CSF was dialyzed with buffer A, and then concentrated using Amicon Ultra-15 centrifugal filter devices with a 3 kDa MW cut-off , and stored at −20°C until use.PEGylated rhG-CSF was prepared as control, referring to a previously reported method . BrieflyThe molecular weights of rhG-CSF and the purified C15-rhG-CSF were detected by MALDI-TOF-MS on a Brukerul Traflex III TOF-MS spectrometer  with a nitrogen laser. Each sample was spotted onto a 100-well sample plate and the R-cyano-4-hydroxycinnamic acid was the matrix. All spectra were acquired in a linear mode, and positive ions were monitored.10k-rhG-CSF and C15-rhG-CSF were determined on the J-815 circular dichroism spectropolarimeter . Each sample was adjusted to a rhG-CSF equivalent concentration of 0.2 mg/ml with 10 mM acetate buffers, pH 4.2. Spectra were recorded at wavelengths between 190 and 250 nm using a 0.1 mm path length cell at 25°C. Each spectrum was obtained from an average of three scans, and CD spectra were corrected for buffer contributions.The secondary structures of rhG-CSF, PEGThe reaction mixtures derived from the modification of rhG-CSF with C15-MAL and the purified C15-rhG-CSF were analyzed using a TSKgel Protein C4-300 RP-HPLC column  on a Shimadzu LC-20A System  at room temperature. The mobile phase A was pure water with 0.1% TFA, and mobile phase B was pure acetonitrile with 0.1% TFA. Each sample of 20 μl was analyzed at a linear gradient of 40–55% B (v/v) over 5 min, and then 55%–75% B over 20 min at a flow rate of 1 ml/min with UV detection at 280 and 215 nm.in vitro bioactivity of C15-rhG-CSF was determined by NFS-60 cell proliferation using the 3--2,5-diphenyl tetrazolium bromide (MTT) assay  was added to each well, and the plate was incubated overnight at room temperature to dissolve the formed formazan crystals. The absorbance at 570 nm was measured by a microplate reader .The T) assay . The unm10k-rhG-CSF and C15-rhG-CSF groups), and the method of subcutaneous administration and sampling time was shown in 10k-rhG-CSF and C15-rhG-CSF at a single dose of 1.0 mg rhG-CSF/kg body weight. After injection, blood samples were collected from orbit at selected times (n = 6 mice at each time) and mice were sacrificed by neck break. The blood samples were immediately centrifuged at 4,000 × g for 30 min at 4°C, and then the plasma samples were stored at −20°C until use. The rhG-CSF concentrations of plasma samples were measured using Human G-CSF DuoSet ELISA Kit , according to the manuals from the manufacturer. Pharmacokinetic parameters were calculated using DAS 2.0 software .Male KM mice (16–20 g) were randomly allocated into three groups  of 0.1 and 0.2 mg rhG-CSF/kg body weight, respectively, for five consecutive days. The mice in low- and high-dose PEG10k-rhG CSF and C15-rhG-CSF group were subcutaneously injected with single dose of 0.5 and 1.0 mg rhG-CSF/kg body weight, respectively. The mice in normal control group and model control group were subcutaneously injected with multiple doses (five times) of saline for five consecutive days. On day 6 after the D1 administration, the mice were sacrificed by neck break, and then the blood samples were collected and the WBC counts were measured using an automatic blood cell analyzer .Male KM mice (16–20 g) except normal control group were intraperitoneally injected with 0.2 ml cyclophosphamide (CTX) at a dose of 1.0 mg rhG-CSF/10 g body weight for three consecutive days, and mice (16–20 g) in normal control group were intraperitoneally injected with equal volume of saline. To determine whether the modeling was successful, five mice from normal control group and model group were randomly selected to measure the white blood cell (WBC) counts. The successfully modeled mice were randomly allocated into seven groups with 12 mice in each group. The method of subcutaneous administration was shown in p < 0.05, **p < 0.01 and ***p < 0.001 were considered significant for intergroup comparisons.Data were expressed as mean ± SD. Statistical inferences were conducted using Student’s t-test by GraphPad Prism six software . Two-sided values of *1H NMR were used to further characterize the C15-MAL. As shown in 1H-NMR spectrum showed that the proton resonance signals  were corresponding to main chemical groups in C15-MAL. Therefore, C15-MAL was successfully prepared.Since maleimide can highly specific react with the thiol group , C15-MALTo achieve an efficient process for the modification of rhG-CSF with C15-MAL, two issues should be addressed: 1) rhG-CSF is highly hydrophilic, while C15-MAL is highly hydrophobic. 2) the free thiol group of Cys 18 in rhG-CSF is buried in a hydrophobic cavity of the protein in aqueous solution. Therefore, suitable solvent systems should be selected to achieve high solubility of the two substrates as well as the adequate exposure of the buried thiol group of Cys 18 of rhG-CSF to react with C15-MAL. One previous study  reportedIn summary, optimized conditions for the modification of rhG-CSF with C15-MAL were the volume ratio of DMSO/phosphate buffer of 10:1, mole ratio of C15-MAL to rhG-CSF of 5:1, pH 7.0, temperature 20°C and reaction time 10 min, consequently, a C15-rhG-CSF yield of 89.2% was achieved.10k-rhG-CSF was also prepared as control, which was purified using cation exchange chromatography  had no significant effect on its secondary structure. Moreover, C15-rhG-CSF could restore the secondary structure of rhG-CSF after being purified into the aqueous solution. Similar result was observed during the PEGylation of rhG-CSF with PEG-MAL in DMSO solution  of C15-rhG-CSF was also 1.8-fold higher than that of rhG-CSF. These results demonstrated that fatty chain modification could prolong serum half-life and increase bioavailability of rhG-CSF. Moreover, serum concentration of C15-rhG-CSF reached peak was 2.15 ± 0.21 h, which was faster than that of rhG-CSF (2.15 ± 0.21 h). In contrast, serum concentration of C15-rhG-CSF reached peak was 11.87 ± 0.32 h, which was slower than that of rhG-CSF. This result indicated that fatty chain modification could promote the trans-membrane absorption/permeability of rhG-CSF after subcutaneous injection. On the other hand, the serum half-life of C15-rhG-CSF was shorter than that of PEG10k-rhG-CSF. To develop more long-acting fatty chain-rhG-CSF conjugates, the systematic investigation of the effects of the fatty chain properties    and the lengths)  should bin vivo efficacies of rhG-CSF, PEG10k-rhG-CSF and C15-rhG-CSF were evaluated based on their effects on the WBC counts in CTX treated mice on day 6 after the D1 administration (p < 0.001) than that of normal control group mice, indicating that the modeling of mice treated with CTX was successful. The mean WBC counts of the mice in all six experience groups were significantly increased, compared with model control group mice, indicating that all the rhG-CSF, PEG10k-rhG-CSF and C15-rhG-CSF had in vivo efficacies. The mean WBC counts of the mice in all three experimental high-dose groups were significantly higher than those of all three experimental low-dose groups, respectively, which showed dose-dependent manners. Compared with normal control group mice, the mice in both the single-dose PEG10k-rhG-CSF groups and multiple doses rhG-CSF groups at equal total dose of 1.0 mg rhG-CSF/kg body weight showed no significant differences in the mean WBC counts, indicating an equivalent in vivo efficacy between PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration at the same total dose. Interestingly, the mean WBC counts of the mice in both low- and high-dose C15-rhG-CSF groups were significantly increased, compared with PEG10k-rhG-CSF groups, which indicated that C15-rhG-CSF had a better in vivo efficacy than that of PEG10k-rhG-CSF, although the serum half-life of C15-rhG-CSF was lower than that of PEG10k-rhG-CSF  should be investigated in future studies.The stration . The mea-rhG-CSF . This waity, etc , and thein vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the half-life of C15-rhG-CSF in mice was 5.72 ± 0.43 h, which was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study showed the potential of C15-rhG-CSF to be developed as a candidate drug as well as an efficient process for site-specific fatty chain conjugation of protein and peptide drugs.In this study, we successfully developed a strategy for the site-specific modification of rhG-CSF with C15-MAL in mixed DMSO-aqueous solutions, in which both of the two substrates had high solubility and the thiol group of Cys18 located in a hydrophobic cavity of rhG-CSF could be adequately exposed to react with C15-MAL. Effects of volume ratio of DMSO/phosphate buffer, pH, temperature, molar ratio of C15-MAL to rhG-CSF and reaction time on the conjugation reaction were investigated and optimized. Consequently, the desired product (C15-rhG-CSF) achieved a high yield of 89.2% under the optimized conditions of volume ratio of DMSO/phosphate buffer 10:1, mole ratio of C15-MAL to rhG-CSF 5:1, pH 7.0, temperature 20°C and reaction time 10 min. The C15-rhG-CSF was efficiently separated from the reaction mixture by preparative liquid chromatography, resulting in a high HPLC purity of 99.0% and a yield of 84.6%. Circular dichroism spectroscopy analysis showed that there was no significant change in the secondary structure of C15-rhG-CSF compared with unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of


The features of preoperative systemic anticancer therapy associated with best outcomes after resection of initially-irresectable liver metastases from colorectal cancer are yet to be identified. We harnessed data from a prospective international surgical database (LiverMetSurvey) to explore the duration and modalities of preoperative systemic anticancer therapy associated with longer overall survival in this clinical setting. Our study included 2793 patients having undergone liver surgery after preoperative systemic anticancer treatment for initially irresectable disease. We found that short  duration was associated with longer survival outcomes, independently from other prognostic factors. Conversely, all the comparisons between different conventional active regimens displayed similar results. Our findings support the recommended onco-surgical approach of aiming at performing liver surgery as soon as technically feasible after response to preoperative systemic anticancer therapy in hepatic metastases from colorectal cancer, initially not amenable to surgery. The results of this study also suggest that, provided the systemic anticancer therapy regimen is active, the choice of the drugs used bears overall little if any impact on the outcomes. Background: Prognostic factors have been extensively reported after resection of colorectal liver metastases (CLM); however, specific analyses of the impact of preoperative systemic anticancer therapy (PO-SACT) features on outcomes is lacking. Methods: For this real-world evidence study, we used prospectively collected data within the international surgical LiverMetSurvey database from all patients with initially-irresectable CLM. The main outcome was Overall Survival (OS) after surgery. Disease-free (DFS) and hepatic-specific relapse-free survival (HS-RFS) were secondary outcomes. PO-SACT features included duration (cumulative number of cycles), choice of the cytotoxic backbone , fluoropyrimidine  and addition or not of targeted monoclonal antibodies (anti-EGFR or anti-VEGF). Results: A total of 2793 patients in the database had received PO-SACT for initially irresectable diseases. Short (<7 or <13 cycles in 1st or 2nd line) PO-SACT duration was independently associated with longer OS (HR: 0.85 p = 0.046), DFS  and HS-RFS . All other PO-SACT features yielded basically comparable results. Conclusions: In this international cohort, provided that PO-SACT allowed conversion to resectability in initially irresectable CLM, surgery performed as soon as technically feasible resulted in the best outcomes. When resection was achieved, our findings indicate that the choice of PO-SACT regimen had a marginal if any, impact on outcomes. Colorectal cancer remains the third cancer in terms of incidence and mortality in both sexes . Liver iIn this context of uncertainty regarding the impact of the type and modalities of pre-operative systemic anticancer therapy (PO-SACT) on patients’ outcomes and aware of the clinical relevance of real-world data , we harnLiverMetSurvey is an international, internet-based registry designed to assess the efficacy of multimodality treatment options for colorectal liver metastases (CLM) ,17. FromIrresectability was defined as the technical inability to completely remove all metastases while leaving at least 30% of normal liver parenchyma. Thus, in these patients, PO-SACT was administered with downsizing conversion intent.The oncologist in charge selected the most appropriate chemotherapy regimen after discussion in a multidisciplinary staff meeting according to local experience, tumor genotype, medical and biological condition and individual clinical treatment history, abiding by the most up-to-date international recommendations. Basically, all patients received fluoropyrimidines (leucovorin-modulated 5-fluorouracil or capecitabine), in association with irinotecan, oxaliplatin or both (triplet combination), and with or without targeted therapies .The response to chemotherapy was evaluated every four to six cycles of treatment with computed tomography of the thorax, abdomen and pelvis according to the Response Evaluation Criteria in Solid Tumours Criteria . PreoperDuring surgery, the consensual policy was to resect all the lesions detected at diagnosis, including remnant calcifications or scar lesions, with the aim to completely clear the liver of palpable and visible tumoral tissue, sparing the highest amount of liver parenchyma possible, as recommended ,19.The indication of the regimen choice and the duration of postoperative therapy were decided by the lead oncologist following a multidisciplinary team discussion after surgery. In the absence of standardized guidelines , the mosPatients were followed at 1 month postoperatively and then every 3 to 4 months with serum tumor markers (carcinoembryonic antigen [CEA] and CA 19.9), clinical examination and diagnostic imaging, as per local practice. Repeat resection of intra- and/or extrahepatic disease recurrence was performed when curative resection could be achieved. Further chemotherapy was administered if deemed indicated.The primary endpoint was overall survival (OS), calculated from the time of surgical resection till death or last follow-up visit. Secondary endpoints included: disease-free survival (DFS), calculated from the time of surgical resection till recurrence in any site or death, and hepatic-specific relapse-free survival (HS-RFS), calculated till the occurrence of liver recurrence only or death.The main aim of the study was to identify the features of preoperative therapy associated with the longest OS after liver resection. In particular, we focused on the duration and type of PO-SACT. For the first aim, we categorized chemotherapy duration into short (6 cycles or less) or long (7 cycles or more) if the first-line regimen . In the We first determined OS probabilities as a function of different features of PO-SACT with the Kaplan-Meier method and compared them using the log-rank test. Then we identified the features of PO-SACT independently predicting for longer OS using multivariable Cox proportional hazard models, accounting for other relevant known potential clinical prognostic factors, including period of surgery (cutoff year: 2005) and Institution (continent-wise).For the secondary endpoints (DFS and HS-RFS), we proceeded with the same approach.2 test for categorical data and the independent-samples t-test or analyses of variance for continuous data.Comparisons of baseline clinical-demographic characteristics between subgroups of patients defined by PO-SACT features were performed using the Chip value ≤ 0.05. All statistical analyses were performed using SAS software version 9.1.3 .The threshold for statistical significance was set for a At the time of LiverMetSurvey database analysis (December 2020), our study population included 2793 patients having received conversion PO-SACT for initially non-resectable disease . The maiAltogether, the most frequent PO-SACT regimen used was FOLFOX: in 28.3% of patients, it was used alone, and with bevacizumab or cetuximab/panitumumab in further 14.2% and 10.4% of patients, respectively. In comparison, FOLFIRI was used alone, in combination with anti-VEGF or with anti-EGFR monoclonal antibodies (mAb) in 10.8%, 10.7% and 8.6%, respectively. Over a third of the patients received less than 7 pre-operative cycles, and resection after 3rd or more line therapy was rare.Postoperative chemotherapy was administered to slightly less than half of the patients in this setting.p = 0.021; p < 0.05).Shorter (as defined above) PO-SACT duration was associated with significantly better OS  and the addition or not of targeted agents , whereas all the other comparisons yielded similar outcomes (not shown).DFS, however, was marginally longer with doublet (median: 15.2) than with triplet regimen . Additionally, HS-RFS was significantly longer without mAbs (median unattained) than with anti-EGFR (33.0) or anti-VEGF ; this secondary outcome, too, did not seem to be influenced by choice of the backbone agent or of the fluoropyrimidine (not shown).For HS-RFS, too, there was a trend towards better outcomes on doublet therapy .p = 0.046); moreover, at multivariable analysis, the use of no targeted agent or of anti-VEGF mAb was retained as a predictor of shorter OS . The other independent negative prognostic factors are shown in Longer  PO-SACT duration was independently confirmed as a prognostic factor of poorer OS  and HS-RFS .Longer PO-SACT was also an independent predictor of shorter DFS  pre-operative systemic anticancer therapy duration as associated with longer OS, DFS and HS-RFS after surgery of initially-irresectable hepatic metastases ; Table 2Thus, in initially unresectable disease, a longer duration of PO-SACT could be assumed as a surrogate of insufficient down-sizing capability due to an extensive tumor load or to a chemoresistant biological tumoral phenotype. Additionally, prolonged pre-operative treatment could increase the risk of disappearing metastases, which might not have been completely sterilized by the PO-SACT ,29. ThusIn accordance with observed outcomes in metastatic disease independently of any surgical strategy ,33,34, oIn accordance with expected antitumor activity being highly comparable between oxaliplatin- and irinotecan-based chemotherapy . We did Somewhat unexpectedly, in our surgical database we only observed independent survival prolongation with intensified conversion PO-SACT, from the adjunction of either cetuximab or panitumumab c, whereaAltogether, since intensive conversion PO-SACT, including triplet with biologicals or hepatic artery infusion, is associated with high response and secondary resection rates ,47, evenThis introduces what we believe to be the main limitation of the current study: the intrinsic nature of our heterogeneous surgical database does not consider the patients referred to the oncologist for optimal chemotherapy for metastatic colorectal with hepatic involvement and never resected. Hence, we are unable to gauge with this “surgical” registry to which extent each type of conversion PO-SACT regimen was able to downsize the hepatic disease enough to allow secondary liver resection. Additionally, we did not routinely collect information about molecular subtypes classification, individual mutational genotyping, or immune-microenvironment, all of which play a role in prognosis and response to medical therapy ,53,54,55Nevertheless, all the patients in this cohort were discussed in multidisciplinary team meetings involving both oncological and surgical expertise, as recommended ; moreoveIn summary, our real-world data in a large international prospective registry revealed that best OS, DFS and HS-RFS are achieved when resection is performed after 6 or fewer cycles of pre-operative first-line treatment (12 for second-line) in patients with the non-resectable disease. Furthermore, our study did not identify the best pre-operative regimens, but suggested that, whenever indicated, the addition of anti-EGFR mAbs to the cytotoxic backbone ought to be preferred. Therefore, optimally active chemotherapy for the individual patient and a limited number of cycles should be aimed towards in as much as the main objective of the strategy. We recommend that reviewing each patient every 2 months at a multidisciplinary meeting as recently proposed by an expert panel is the best means to achieve this objective.


Anti GM-CSF autoantibodies (aAb) have been related to acquired pulmonary alveolar proteinosis (PAP) and described in cases of severe infections such as cryptococcosis and nocardiosis in previously healthy subjects. Whether there are different anti-GM-CSF autoantibodies corresponding to these phenotypes is unclear. Therefore, we examined anti-GM-CSF autoantibodies to determine whether amount or neutralizing activity could distinguish between groups.Plasma samples gathered in the National Institute of Health from patients with anti GM-CSF aAb and either PAP (n = 15), cryptococcal meningitis (n = 15), severe nocardiosis (n = 5) or overlapping phenotypes (n = 6) were compared. The relative amount of aAb was assessed using a particle-based approach, reported as a mouse monoclonal anti-human GM-CSF as standard curve and expressed in an arbitrary Mouse Monoclonal Antibody Unit (MMAU). The neutralizing activity of the plasma was assessed by inhibition of GM-CSF-induced intracellular phospho-STAT5 (pSTAT5) in monocytes.vs 197 ± 159 MMAU, p = 0.02); there was no difference with patients with nocardiosis (430 ± 493 MMAU) nor between the two types of infections.Anti-GM-CSF aAb relative amounts were higher in PAP patients compared to those with cryptococcosis (mean 495 ± 464 MMAU 50) did not vary appreciably across groups of patients . Nor was the concentration of GM-CSF necessary to induce 50% of maximal GM-CSF-induced pSTAT5 in the presence of 10 MMAU of anti-GM-CSF aAb (EC50). When studying longitudinal samples from patients with PAP or disseminated nocardiosis, the neutralizing effect of anti-GM-CSF aAb was relatively constant over time despite targeted treatments and variations in aAb levels.The dilution of plasma resulting in 50% inhibition of GM-CSF-induced pSTAT5 (approximate ICDespite different clinical manifestations, anti-GM-CSF antibodies were similar across PAP, cryptococcosis and nocardiosis. Underlying host genetics and functional analyses may help further differentiate the biology of these conditions. Anti-cytokine autoantibodies are emerging entities that may explain some severe infections in previously healthy adults . The mosHowever, the case of anti-granulocyte–macrophage-colony-stimulating-factor (anti-GM-CSF) autoantibodies appears to be more complex. Anti-GM-CSF antibodies were first described in 1999 and are now recognized as the main cause of acquired pulmonary alveolar proteinosis . In pulmC. gattii is vastly overrepresented compared to its more common relative, C. neoformans [Neutralizing anti-GM-CSF autoantibodies have also been described as risk factors for disseminated infections such as cryptococcal meningitis and disseminated nocardiosis in previously healthy subjects , 12. In oformans , suggestIn most cases of anti-GM-CSF autoantibody-mediated infection, pulmonary function is normal at the time of infection diagnosis, excluding significant concomitant PAP at that time. In some cases, however, PAP has occurred years after the first infectious manifestation . FurtherBlood samples from 41 patients with proven anti-GM-CSF autoantibodies were collected between March 2010 and April 2019 at the Laboratory of Clinical Immunology and Microbiology at the National Institutes of Health (NIH). Twenty-three of these patients were seen at the NIH; blood samples of the others had been referred for screening from collaborating sites in the US or abroad. Normal PBMCs were obtained though the NIH Blood Bank. Written informed consent was provided by all participants or their guardians under appropriate IRB-approved protocols .2 of interpolation slopes > 0.98), secondarily revealed by a PE-labeled goat anti-mouse IgG (ThermoScientific). The relative amount of antibodies in patient samples was determined by the mean fluorescence intensity (MFI) of samples compared to those of the standard curve. Because of the use of a mouse monoclonal antibody as standard instead of purified human polyclonal antibodies, we chose to express the concentration results in an arbitrary Mouse Monoclonal Antibody Unit (MMAU). Each patient sample was tested on three distinct occasions with at least three different dilutions between 1/5,000 and 1/500,000 and results from the most reproducible dilution fitting the most points of the standard curve were averaged.Plasma samples were screened for anti-GM-CSF autoantibodies using a particle-based approach as previously described . BrieflyPlasma from each subject was separated from whole blood and stored at − 80 °C until testing. Effort was made to choose samples from dates distant from interventions that might influence antibody levels  and before lung transplantation, where relevant.6 cells/ml in complete RPMI 1640  and 10% fetal calf serum (FCS). For each set of experiments, PBMCs from the same healthy donor were used in order to maintain a constant cellular background.PBMCs from healthy donors were obtained by density-gradient centrifugation, frozen in media at − 80 °C. Thawed cells were cultured at 106 cells/ml) were cultured in complete RPMI 1640 with 10% FCS with plasma from normal subjects or patients and left unstimulated or stimulated with recombinant human GM-CSF (Peprotech) for 30 min at 37 °C. Monocytes were identified by CD14 (BD Pharmingen) surface staining before being fixed and permeabilized for intracellular staining with p-STAT5 (Y694) Ab (BD Bioscience), as described previously [Neutralizing activity of plasma against GM-CSF was performed by assessing STAT5 phosphorylation in cells stimulated with GM-CSF in the presence of plasma from patients or controls. Briefly, PBMCs (1 × 1050 (concentration of antibodies responsible for 50% of inhibition of GM-CSF-induced STAT5 phosphorylation), the patient plasmas were used at different dilutions (diluted in AB serum from 0.01 to 30%) with a constant concentration of GM-CSF for stimulation (10 ng/ml).In order to approximate the IC50 (concentration of GM-CSF responsible for 50% of STAT5 phosphorylation in the presence of antibodies), the volume of plasma was chosen to get a constant final concentration of antibodies in each tube at 10 MMAU (completed with AB serum); stimulation with GM-CSF varied from 10–1 to 104 ng/ml.In order to assess the ECData were collected using a BD LSR Fortessa (BD Biosciences), analyzed using FlowJo software (TreeStar), and graphed with Prism8 (GraphPad).t test. Correlation analyses were done calculating nonparametric Spearman’s correlation coefficient. The threshold for statistical significance was set to p < 0.05.Differences between groups were evaluated using unpaired Student’s gattii species) and 5 patients with severe nocardiosis. Six patients had overlapping phenotypes at the time of sampling: 3 with both PAP and cryptococcal meningitis, 2 with PAP and disseminated nocardiosis and 1 patient had sequential nocardiosis then cryptococcosis. In patients with PAP and infections, the infection had preceded the PAP for several years except for one patient who presented PAP and Cryptococcus meningitis concomitantly. The clinical cases of some of these patients have already been reported [We identified 15 patients with circulating anti-GM-CSF autoantibodies and PAP only, 15 patients with cryptococcal meningitis . No statistical difference was observed between PAP patients and subjects with Nocardia infection alone  or between subjects with cryptococcosis and Nocardia infections .The relative amounts of autoantibody were determined in patient plasmas using a particle-based approach with GM-CSF-coupled magnetic beads and compared between groups Table . We restp = 0.05. The plasma dilution sufficient for 50% inhibition of GM-CSF-induced pSTAT5 was negatively correlated with the antibody relative amounts (r = -0.88), meaning that the higher the dilution the more neutralizing the plasma. Anchoring the plasma dilution to the antibody level determined in each plasma, we calculated the IC50 as the concentration of anti-GM-CSF autoantibody resulting in 50% inhibition of GM-CSF-induced pSTAT5. This latter was not significantly different between the groups of patients: mean ± SD of 2.46 ± 2.3 MMAU for PAP patients, 3.7 ± 3.3 MMAU for cryptococcosis patients and 1.94 ± 1.1 MMAU for patients with Nocardia infections  in circulating monocytes from healthy donors after a fixed dose of GM-CSF (10 ng/ml) in the presence of varying dilutions of patient plasmas (ranging 0.01% to 30%). The global neutralizing effect of patient plasma (dilution of plasma sufficient for 50% inhibition of GM-CSF-induced pSTAT5) tended to be higher in patients with PAP compared to patients with cryptococcal infections: mean ± SD dilution of 1.6 ± 3.1% vs 3.9 ± 6%, –1 to 104 ng/ml) to stimulate normal circulating monocytes. The effect of GM-CSF on STAT5 phosphorylation in monocytes in the presence of a constant antibody concentration led to determination of an EC50 for each sample. The mean EC50 was not significantly different between groups of patients with different phenotypes: 50.08 ± 69.5 ng/ml in PAP patients, 35.5 ± 22.4 ng/ml in patients with cryptococcal infections and 25.6 ± 18 ng/ml in patients with Nocardia infections  and varied the GM-CSF amount   Fig. A.Fig. 3ENocardia infection in 2011 involving the lungs and the brain (previously reported in ref 11)  Fig. B. BankedAnti-GM-CSF antibodies have been described in various and not necessarily overlapping clinical situations. We queried whether the characteristics of the anti-GM-CSF antibodies  varied between patients with autoimmune PAP and those with autoantibody-related severe infections. We found that (i) patients with autoimmune PAP tended to have higher levels of anti GM-CSF autoantibodies, (ii) levels and global neutralizing activity of plasma correlated, and (iii) there was no significant difference between groups in terms of neutralizing activity of anti-GM-CSF autoantibodies.–1) exists above which anti-GM-CSF antibodies are likely to disrupt GM-CSF-stimulated functions in alveolar macrophages and blood leukocytes [The disease induced by anti-cytokine antibodies is the result of a complex combination of several factors including the characteristics of the antibody , level of patient exposure to certain irritants and pathogens and potentially individual genetic susceptibility. Characteristics of the antibody have been shown to contribute to disease activity and phenotype in other autoantibody-mediated diseases , 19. Antukocytes , 25 and ukocytes . Monocloukocytes , 28. Furukocytes –31. The ukocytes . The antukocytes , 33.Cryptococcus or Nocardia infections, as is true for autoimmune PAP [Cryptococcus or Nocardia infection from each other and from patients with PAP alone remains obscure. Although our study was not designed to determine absolute levels of antibodies, it allowed us to compare the relative amounts and activities of autoantibodies in the plasmas of these three groups of patients. No dramatic differences were found between groups, except for a slightly higher level in PAP patients. The in vitro neutralizing effects of anti-GM-CSF autoantibodies were not statistically different by phenotype. Plasma from PAP patients tended to be more effective at blocking GM-CSF signaling (effective at 1.6% dilution) than plasma from patients with cryptococcosis . The use of whole plasmas cannot exclude other possible non-antibody inhibitory effects. However, anchoring the plasma dilution to the antibody level, we did not find any difference in IC50 determined as the concentration of anti-GM-CSF autoantibody responsible for 50% inhibition of GM-induced pSTAT5. We also did not find it regarding the EC50 for pSTAT5 experiments. For each set of experiments, PBMCs from the same healthy donor were used in order keep a constant genetic background underlying the cellular response.Our study is the first to address and compare anti-GM-CSF antibody characteristics in diseases other than autoimmune PAP. Previous reports have noticed a predominance of IgG1 subclass antibodies in patients with mune PAP , 12. GM-mune PAP . HoweverThese in vitro effects may not perfectly reflect the neutralizing activities of the antibodies in vivo . FurtherCryptococcus or Nocardia [Almost every case of anti-GM-CSF–related disseminated infection in our cohort involved the CNS. The search for anti-GM-CSF antibodies is now recognized as part of the workup in “non-immunocompromised” patients with brain infection with Nocardia , 40.Recent studies suggest that GM-CSF, elicited by IL-23-driven T cells, favors CNS invasion by phagocytes and secondarily myeloid-mediated tissue immunopathology –43. AntiLongitudinal samples may be particularly informative. In our patients who developed different manifestations of anti-GM-CSF autoantibodies, the infections usually preceded PAP. In only one patient was PAP diagnosed concomitant with cryptococcal meningitis. Globally, PAP seems to occur later in clinical evolution. As it does not appear to be a matter of circulating antibody levels or global neutralizing effect, this temporality could be due to cumulative effects over time or changes in exposure.Our study was retrospective, gathering samples from different centers; some clinical data may have been overlooked. The precise date of onset of the initial manifestation may have been missed. Additionally, the delay between diagnosis and date of sampling was variable between patients, with some sampling made at intervals from onset and after several lines of treatment. These realities make the value of our rare longitudinal samples greater. Regardless of these shortcomings, which might serve to artifactually amplify differences, we found very few.Nocardia infection. We found no explanatory differences between the groups in terms of relative amounts of circulating antibody or in vitro neutralizing effect on GM-CSF-induced STAT5 phosphorylation in monocytes. Genetic analysis, affinity assays on various cellular targets and longitudinal samples will be needed to help delineate the biology of these anti-GM-CSF autoantibodies.This study is the first to gather and compare cohorts of patients with the various anti-GM-CSF antibody-related clinical manifestations of PAP, cryptococcal meningitis, and disseminated


Exercise fatigue is a common physiological phenomenon in human activities. The occurrence of exercise fatigue can reduce human power output and exercise performance, and increased the risk of sports injuries. As physiological signals that are closely related to human activities, surface electromyography (sEMG) signals have been widely used in exercise fatigue assessment. Great advances have been made in the measurement and interpretation of electromyographic signals recorded on surfaces. It is a practical way to assess exercise fatigue with the use of electromyographic features. With the development of machine learning, the application of sEMG signals in human evaluation has been developed. In this article, we focused on sEMG signal processing, feature extraction, and classification in exercise fatigue. sEMG based multisource information fusion for exercise fatigue was also introduced. Finally, the development trend of exercise fatigue detection is prospected. Exercise fatigue is a physiological phenomenon that originates from human activities and results in decreased physical performance . The maiMuscle fatigue is divided into central and peripheral components. Peripheral fatigue is caused by changes in the neuromuscular junction. Central fatigue originates in the central nervous system (CNS). The production of skeletal muscle force depends on contractile mechanisms, and failure of nervous, ion, vascular, and energy systems can contribute to the development of muscle fatigue . During Exercise fatigue can be detected through surface electromyography (sEMG) . ConstanThis review offers an overall summary of the assessment of human exercise fatigue based on sEMG signals and highlights signal processing, feature extraction, and classification. The possibilities for future work on exercise fatigue with sEMG will also be discussed in this study.The sEMG is a biological electric signal generated by muscle contraction that can be harvested by electrodes. The sEMG signals are the most intuitive physiological signals of muscle activity and the best means to detect muscle fatigue. The sEMG signal is a kind of pseudorandom physiological signal that is very weak. The voltage of the sEMG signal range from 50 μV to 100 mV and the frequency is varied from 10 to 500 Hz . The eleExact electrode positioning is vital for obtaining reliable EMG signals. A study showed high correlations between all electrode sites and clavicular movements. The traditional electrode site record more informative signals in subjects . All truThe raw sEMG data collected will inevitably be mixed with power line interference and motion artifacts . TherefoThe increase from baseline represents the onset of muscle activity. Muscle activity onset can be estimated from EMG and ultrasound . Gupta eFeature extraction from the sEMG signal plays an important role in the accuracy of fatigue detection. Time domain, frequency domain, time-frequency domain, and nonlinear parameters are four major types in sEMG-based signal processing .The calculation of time-domain features is the most popular approach for sEMG feature extraction that is directly calculated with time as the independent variable. In the time domain, the sEMG signal is usually regarded as a random signal whose mean value is zero and variance varies with signal intensity. As the calculation of time-domain features is simple and intuitive, it is a widely used feature extraction method of sEMG signal in human movement . The typWith the occurrence of muscle fatigue, the time domain features of sEMG generally show an upward trend over time . RMS andThe frequency-domain features are spectrum or power spectrum (PS) features that are obtained by fast Fourier transform (FFT) to the original sEMG signals, thus the frequency band distribution of the signals can be observed directly . ResearcTo make up for the deficiency of Fourier transform analysis of non-stationary signals, time-frequency features of sEMG signals are introduced. The analysis of time-frequency features is important to the estimation of muscle fatigue state, and it is usually necessary to analyze both the time-domain and frequency-domain features of sEMG signals to obtain comprehensive information of muscle physiological changes. Traditional sEMG time domain and frequency domain analysis methods only describe the time domain or frequency domain features, but the time-frequency domain feature analysis method can overcome this limitation. At present, the available time-frequency analysis methods for sEMG signal mainly include Short-time Fourier Transform (STFT), Wavelet Transform (WT), Choi William distribution (CWD), and Wigner-Ville distribution (WVD), used for visual observation of signal frequency content evolution over time. WVD is a common bilinear time-frequency distribution, which is the most common type of Cohen’s time-frequency distribution. Instantaneous frequency parameters commonly used are instantaneous mean frequency (IMNF)  and instThe sEMG complexity and entropy decrease linearly with the increase of fatigue degree. The complexity of Lempel-Ziv [C (n)]  is the sWith the development of machine learning, machine learning algorithms were widely applied to exercise fatigue classification. Classification in this article normally refers to supervised learning where individuals are classified based on their features. Some classification algorithms based on sEMG are listed in Support vector machine is a popular machine learning classification method because it is simple, fast, and stable, and shows better accuracy than other methods . Since tMulti-sensor fusion based on sEMG can collect more dimensions of human activities from multiple dimensions. Thus, it is more comprehensive than fusion methods that use data from a single sensor. Fatigue detection based on surface EMG has important application value in sports training, rehabilitation treatment, and movement recognition. This article aimed to provide an overview of sEMG signal processing, feature extraction, and classification in exercise fatigue. In real-time detection, portability of the device, removal of artifacts, feature extraction, and classification techniques should be properly investigated. Using appropriate methods can remove noise to improve EMG signal quality. With the increase in the number of EMG channels and features, it is necessary to choose a reasonable dimensionality reduction method. The methods should greatly reduce the computational complexity of the classifier and preserve maximum information of the signal. Classification techniques have been extensively studied, but their timeliness and generalization still have research significance. A combination of processing methods and pattern recognition techniques may be helpful to increase the classification speed and accuracy. And the adaptability of good algorithms to fresh samples needs further study. Finally, we propose that the current review can be used as a guide for further improving exercise fatigue assessment based on sEMG for various applications of the human body.JS mainly wrote the manuscript under the guidance of GL and JC. YS, KL, and ZZ participated in the analysis. 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.


Do preoperative sedatives such as midazolam and dexmedetomidine have a protective effect against perioperative respiratory adverse events (PRAEs) while achieving adequate sedative effect?In this randomized clinical trial that included 384 children undergoing tonsillectomy and adenoidectomy, the incidence of PRAEs was 24.2% in the dexmedetomidine (2.0 μg/kg) group, 56.5% in the midazolam (0.1 mg/kg) group, and 40.8% in the normal saline group.Intranasal midazolam used for premedication was associated with increased incidence of PRAEs, whereas premedication with dexmedetomidine was associated with reduced incidence of PRAEs. This randomized clinical trial investigates the effect of intranasal dexmedetomidine or midazolam used for preoperative sedation on the occurrence of perioperative respiratory adverse events (PRAEs) among children undergoing tonsillectomy and adenoidectomy. Perioperative respiratory adverse events (PRAEs) are the most common complication during pediatric anesthesia, and they may be affected by the administration of preoperative sedatives.To investigate the effect of intranasal dexmedetomidine or midazolam used for premedication on the occurrence of PRAEs.This single-center, double-blind, randomized clinical trial was conducted among children aged 0 to 12 years undergoing elective tonsillectomy and adenoidectomy from October 2020 to June 2021 at Children’s Hospital of Xuzhou Medical University, Xuzhou, China. Data analysis was performed from June to October 2021.Children were randomly assigned to 3 groups: the midazolam group received intranasal midazolam (0.1 mg/kg), and the dexmedetomidine group received intranasal dexmedetomidine (2.0 μg/kg) for premedication. The normal saline group received intranasal 0.9% saline for control.The primary outcome was the difference in the incidence of PRAEs among the 3 groups. The secondary outcomes were the frequency of the individual PRAEs, including the incidence of such events during the induction and recovery periods, postoperative emergence delirium, postoperative pain score, sedation success rate, and heart rate values.A total of 384 children  were enrolled and randomized; 373 data sets were available for intention-to-treat analysis . After the data were adjusted for age, sex, American Society of Anesthesiologists physical status, body mass index, obstructive sleep apnea, upper respiratory tract infection, and passive smoking, children in the midazolam group were more likely to experience PRAEs than those in the normal saline group , whereas the dexmedetomidine group had a significantly lower PRAEs incidence than the normal saline group . Compared with the dexmedetomidine group, the midazolam group had a higher risk of PRAEs , but no other serious clinical adverse events were observed.In this randomized clinical trial, intranasal midazolam used for premedication was associated with increased incidence of PRAEs, whereas premedication with intranasal dexmedetomidine was associated with reduced incidence of PRAEs. Where clinically appropriate, anesthesiologists should consider using intranasal dexmedetomidine for sedation in children undergoing tonsillectomy and adenoidectomy.ChiCTR2000038359Chinese Clinical Trial Register Identifier:  The occurrence of these complications can prolong hospitalization time, increase hospitalization costs, and bring varying degrees of physical and psychological trauma to children and parents.4 A substantial proportion of children undergoing tonsillectomies experience PRAEs, with a prevalence of up to 50%.6 Independent risk factors include age 6 years and younger, upper respiratory tract infection (URTI), lung disease, obesity, obstructive sleep apnea (OSA), and passive smoking.7 These factors are very common in children undergoing tonsillectomy and adenoidectomy.Perioperative respiratory adverse events (PRAEs) are the most common complication during pediatric anesthesia,10 However, preoperative strategies are needed to provide anesthesiologists a comprehensive approach for high-risk children.Clinicians have explored various strategies to minimize PRAEs, including but not limited to the use of laryngeal mask airways, intravenous induction of anesthesia (vs mask induction), and lidocaine topicalization of the airway.13 but their effect on PRAEs is still unclear.Pediatric patients can experience substantial anxiety and distress during the perioperative period. The use of sedative premedication may help to reduce their anxiety and minimize the emotional trauma, but there are no preferred recommendations or well-documented clinical studies to guide our choice of a certain sedative to decrease the incidence of PRAEs. Midazolam and dexmedetomidine, which are the most common preoperative sedatives used for children, have been commonly used in recent years,15 showed that preoperative midazolam seemed to increase the incidence of PRAEs, but a multicenter trial16 showed that midazolam had a preventive effect on PRAEs. Dexmedetomidine has been proven to be effective in reducing the occurrence of PRAEs in children with congenital heart disease,17 but there is no evidence to support this protective effect in the general population undergoing tonsillectomy and adenoidectomy. However, those previous studies are mostly observational, and, to our knowledge, there are still no randomized clinical trials with high-quality evidence to study the effect of the preoperative sedatives midazolam and dexmedetomidine on PRAEs.There are contradictory studies of midazolam and PRAEs: previous studiesTo address this inconsistency and the small samples included in previous studies, we conducted a prospective, single-center, double-blind, randomized clinical trial to investigate the effect of intranasal dexmedetomidine or midazolam on the occurrence of PRAEs in children undergoing tonsillectomy and adenoidectomy. We hypothesized that preoperative sedatives, intranasal dexmedetomidine or midazolam, would reduce the occurrence of PRAEs.CONSORT) reporting guideline for randomized studies.18 The full trial protocol is available in This trial was performed at the Children’s Hospital of Xuzhou Medical University, Xuzhou, China, from October 1, 2020, to June 30, 2021. The study protocol was approved by the Medical Ethics Committee of the Children’s Hospital of Xuzhou Medical University and was registered in the Chinese Clinical Trial Registration Center on September 21, 2020. This report follows the Consolidated Standards of Reporting Trials  was determined by the responsible anesthetist independently. Preoxygenation was routinely used. Tracheal tubes for airway management were used in all children. After the end of surgery, secretions and intraoperative irrigation fluid in the mouth were sucked out to avoid aspiration, and then children with a tracheal tube were transferred to the postanesthesia care unit (PACU). Tracheal extubation was undertaken when the child was awake by a specialized pediatric anesthesiologist in the PACU. Neostigmine was used if the child presented with residual muscle relaxation. After extubation, postoperative pain was assessed using the Wong-Baker Pain Scale (eAppendix 1 in 19), postoperative emergence delirium, and heart rate values , postoperative pain score, sedation success rate . The Kolmogorov-Smirnov test was used to determine whether the continuous data conformed to the normal distribution. The quantitative variables that obey normal distribution are presented as mean (SD), and nonnormally distributed data are represented by median and IQR. Binomial variables are expressed as rate. The continuous data of normal distribution were analyzed by 1-way analysis of variance, and the continuous data of nonnormal distribution among the 3 groups were analyzed by the Kruskal-Wallis rank-sum test. Categorical data were analyzed using the χ2 test or Fisher exact test, and the crude odds ratio (OR) and 95% CI were calculated. The adjusted OR (aOR) and 95% CI were calculated for both primary and secondary outcomes. Age, sex, American Society of Anesthesiologists physical status, body mass index, URTI, passive smoking, and OSA were adjusted for using binary logistic regression.Outcome analyses were performed in the intention-to-treat population, and a per-protocol analysis was also performed for the primary end point. The primary outcome was analyzed using χA total of 384 children  were recruited . Of thesThere was no significant difference in extubation time, time spent in the PACU after the extubation, or postoperative hospital stay among the 3 groups. Wong-Baker Pain Scale scores and Pediatric Anesthesia Emergency Delirium Scale scores were similar among groups, but fewer children in the dexmedetomidine group required postoperative analgesics than in the midazolam group. The rate of emergence delirium in the dexmedetomidine group was lower than those in the midazolam group and normal saline group . Compari20 Direct trauma to the airway during the operation causes swelling of the upper respiratory tract and surrounding tissues in children; as a result, secretions are retained in the airway, thus greatly increasing the risk of PRAEs.Tonsillectomy and adenoidectomy are among the most frequently performed surgical procedures in children.16 have shown that midazolam has a protective effect on airway contraction. A number of vitro studies have shown that midazolam has a spasmolytic effect on histamine-induced bronchoconstriction, and it has been confirmed that midazolam has an in vitro bronchiectasis effect in animal experiments.23 Therefore, midazolam may have a protective effect against bronchospasm. However, midazolam was associated with increased incidence of desaturation and also was associated with a higher risk of laryngospasm and airway obstruction. The current research on the mechanism of midazolam mainly is focused on bronchospasm, and the association of midazolam with other adverse events is still unclear. This performance may be related to the paradoxical reaction of midazolam24; that is, the children show irritability and other behaviors that are completely opposite to sedation and hypnosis. Children with sympathetic nerve excitement and increased stress reaction may be responsible for the high incidence of PRAEs associated with midazolam; thus, more experiments are needed to further explore its mechanism.In this randomized clinical trial, children who were sedated with midazolam preoperatively had a higher risk of PRAEs compared with those sedated with dexmedetomidine and those in the saline control group. Previous studies were mostly observational, and the results were contradictory. The different routes of administration, sedation time, dose and depth of sedation, and intraoperative management strategies may all affect the outcome. In our study, the influence of confounding factors was effectively controlled. Compared with the normal saline control group, the midazolam group had a significantly increased risk of PRAEs, although previous studies26 Second, the direct airway smooth muscle effect of dexmedetomidine may have also contributed. Accordingly, it has been demonstrated that dexmedetomidine attenuates both exogenous acetylcholine-induced and C-fiber–mediated isolated tracheal ring contraction, suggesting that it has the effect of relaxing airway smooth muscle and suppressing cough.27 Finally, dexmedetomidine may have modulated the inflammatory process, which is associated with increased airway sensitivity; both interleukin-6 and tumor necrosis factor–α levels have been shown to be substantially reduced after the use of this drug.28 In our study, the dexmedetomidine group had less demand for fentanyl because of the mild analgesic effect.29 It is evident that intravenous fentanyl is associated with coughing and respiratory depression.30 Therefore, dexmedetomidine may also be associated with reduced incidence of coughing and desaturation by reducing the use of fentanyl.Premedication with intranasal dexmedetomidine was associated with a significant decrease in the incidence of PRAEs, especially the incidence of oxygen desaturation and coughing. Several mechanisms may underlie this beneficial effect. First, dexmedetomidine may have increased the depth of anesthesia and, thus, reduced airway reflexes.There was no significant difference in the overall incidence of PRAEs in the 3 groups during the induction period. This was because preoxygenation was routinely used in our institution, which greatly decreased the incidence of oxygen desaturation, the most common PRAE, during the induction period. However, the midazolam group was more likely to experience laryngospasm and coughing. Owing to the use of tracheal tubes for airway management, almost no PRAEs were observed during the maintenance period.We conducted a post hoc analysis in children with URTIs in the previous 4 weeks. The occurrence of URTI in the entire cohort was 33.0%, and the incidence of PRAEs in the midazolam group with URTI was 64.4% , whereas the incidence of PRAEs in the dexmedetomidine group with URTI was 20.0% . These findings may be associated with increased airway sensitivity and may be caused by potential chronic airway inflammation.31 dexmedetomidine can reduce the airway reflex and suppress a sharp increase in heart rate during extubation, which could be explained by the markedly decreased sympathetic activity.Our results show that dexmedetomidine facilitated tolerance of the endotracheal tube and significantly reduced coughing during extubation without affecting the extubation time, an effect possibly mediated via its sedative and analgesic properties. Consistent with research conducted in Turkey,32 it was confirmed that 2.0 μg/kg dexmedetomidine may be a better choice than 1.0 μg/kg. The dose of intranasal midazolam is typically 0.2 mg/kg, but it has a burning sensation and will irritate the nasal cavity. In our pre-experiment, children refused nasal drops, and a large number of midazolam preparations leaked. Thus, we decided to try low-dose midazolam preparations, and the results showed that the onset time of a 0.1 mg/kg nasal drip was longer than that of a dose of 0.2 mg/kg; it took effect after approximately 10 to 15 minutes but could also achieve a satisfactory sedative effect after 30 minutes. It turns out that a reduction in the dose can relieve nasal irritation. In previous studies,29 it was reported that approximately 36.1% of patients receiving midazolam would have nasal discomfort and tearing, but in our study, only 15 patients (12.1%) who received midazolam exhibited the aforementioned symptoms.Finally, we chose intranasal midazolam 0.1 mg/kg and dexmedetomidine 2.0 μg/kg for preoperative sedation. In previous studies,34 Some studies have considered midazolam as a risk factor for emergence delirium,35 and others have considered midazolam to be useful as a pharmacological prevention strategy.36 Dexmedetomidine has been confirmed to have a preventive effect against emergence delirium,37 and the incidence in the dexmedetomidine group in our study was significantly lower than that in the midazolam and normal saline group. Therefore, dexmedetomidine may be a better choice than midazolam.In our study, the incidence of emergence delirium in the midazolam group was higher than that in the normal saline group, but the difference was not significant. The use of midazolam as pharmacological prevention for emergence delirium is controversial.This study has limitations that should be considered. In this trial, the researchers were blinded to the treatments, but experienced anesthesiologists would easily be able to differentiate between the different sedatives by simply observing patient behavior, especially during the induction period. This might have led to investigator bias in which those diagnosing the outcome were aware of the group allocation and/or the study hypothesis. However, it is important to note that none of the anesthesiologists who participated in this study were aware of the study hypothesis; therefore, this risk of bias was reduced.We did not routinely use antagonists because neostigmine causes gastrointestinal spasm, and the children often show persistent discomfort and crying. We did not monitor the train of 4 simulation, although the clinical manifestations provide a possible basis, we still cannot ensure that all children are completely unaffected by residual muscle relaxation.In the process of the experiment, we found that the individual differences of the children themselves were also obvious, which may be related to the education level of the parents. In this experiment, the education level of the parents was not evaluated, so this factor may be ignored. Moreover, we acknowledge that the OSA status was assessed by the otolaryngologist from the clinical history rather than by polysomnography, which is the standard for diagnosis and quantitative description of OSA, and we did not grade the severity of OSA.The findings of this study suggest that both midazolam and dexmedetomidine can achieve satisfactory sedative effects intranasally before surgery, and dexmedetomidine may have a protective effect against the occurrence of PRAEs, whereas midazolam increases the risk of PRAEs during the perioperative period. Therefore, if there are no special contraindications, we recommend dexmedetomidine sedation before surgery for children undergoing tonsillectomy and adenoidectomy.