Immune checkpoint inhibitors targeting the programmed cell death protein‐1 (PD‐1) and programmed death ligand‐1 (PD‐L1) axis (collectively referred to as PD[L]1i) have demonstrated clinical benefits in non‐small cell lung cancer (NSCLC) patients. The purpose of this United States‐based real‐world study is to examine changes in the landscape of first‐line therapies for NSCLC since the introduction of PD(L)1i.Patients with NSCLC initiating first‐line treatment between May 1, 2017, and October 31, 2020, were identified in the IBM MarketScan® database. Patients were assigned groups based on first‐line therapy: PD(L)1i monotherapy, chemotherapy alone, PD(L)1i with chemotherapy, or targeted therapy for patients with actionable driver mutations.A total of 5431 patients with NSCLC starting first‐line treatment were identified: chemotherapy alone 2568 (47%), PD(L)1i with chemotherapy 1364 (25%), PD(L)1i monotherapy 790 (15%), and targeted therapy 709 (13%). The use of PD(L)1i monotherapy and targeted therapy remained consistent, while the percentage of patients receiving PD(L)1i with chemotherapy more than doubled. Over a third of patients in 2019 and 2020 received chemotherapy alone. Patients aged ≥65 years , females , and those with respiratory  or kidney  disease were less likely to have received PD(L)1i with chemotherapy than patients that received chemotherapy alone.Since the approval of PD(L)1i for NSCLC, their use has significantly increased for first‐line treatment, especially when used in combination with chemotherapy. A significant proportion of patients received chemotherapy alone. Since the approval of PD(L)1i for NSCLC, their use has significantly increased for first‐line treatment, especially when used in combination with chemotherapy. A significant proportion of patients received chemotherapy alone. In 2015, pembrolizumab (PD‐1 inhibitor) was approved as monotherapy initially for high PD‐L1 expressing tumors (>50%), and then in 2019 for PD‐L1 expressing tumors (≥1%) based on the Keynote‐024 and Keynote‐042 trials, respectively.Despite the well‐established clinical efficacy and tolerability of PD(L)1i, little is known about the current patterns of use of PD(L)1i in clinical practice as a first‐line treatment for NSCLC, either as a monotherapy or in combination with platinum‐based chemotherapy. Previous studies, conducted shortly after the approval of the first PD(L)1i in the United States and Canada, suggested that their use was increasingly being adopted; however, these studies did not extend beyond 2018.22.1This observational study utilized administrative claims data from the IBM MarketScan® Research database which includes the Commercial Database and the Medicare Supplemental Database.Newly diagnosed patients with NSCLC were identified in the IBM MarketScan® Commercial and Medicare Supplemental databases during the study period of May 1, 2017 , through October 31, 2020. Eligible patients met the following criteria: (a) at least two claims within 1 year for NSCLC  during the study period; (b) no NSCLC claim in the database prior to the study period; (c) continuous enrollment at least 90 days prior to the first NSCLC claim; (d) continuous enrollment for at least 60 days after first NSCLC claim; (e) ≥18 years old, (f) began NSCLC therapy with first‐line,; and (g) did not receive therapy for small cell lung cancer (claim for etoposide or irinotecan hydrochloride). For those patients that met the eligibility criteria, the date of the first NSCLC diagnosis served as the index date.PD(L)1i monotherapy: patients whose first claim was a PD(L)1i  and had no claim for chemotherapy  within 45 days of diagnosis; (2) chemotherapy alone: first claim for chemotherapy and no PD(L)1i claim within 45 days as above; (3) PD(L)1i with chemotherapy: claim for a PD(L)1i and chemotherapy within 45 days, and (4) targeted therapy: patients that had a claim for an EGFR , ALK/ ROS1 , BRAF V600E plus MEK inhibitors , or NTRK  and no claim for chemotherapy or PD(L)1i within 45 days. Patients on dual check point blockade therapy with ipilimumab (anti‐CTLA4 inhibitor) and nivolumab were not included given that FDA approval occurred at the end of the study period, providing very limited data for this regimen.Patients were assigned to one of four groups: (1) We obtained pretreatment information including age, sex, insurance type, and treatment facility setting . We further collected clinical characteristics over the 180‐day period prior to first NSCLC diagnosis that may have influenced treatment selection, including cerebrovascular disease, congestive heart failure, diabetes, liver disease, renal disease, and COPD. The presence of the comorbidity was scored as a “1” and absence as “0.” The burden of comorbidities was assessed by calculating the Charlson comorbidity index (CCI) for each study patient over the 180 days prior to diagnosis.2.2We used descriptive statistics to examine the proportion of patients in each treatment group according to each year of the study period. Baseline differences in demographic and clinical characteristics between the study groups were tested using chi‐square, ANOVA, or Kruskal–Wallis test as indicated. We next sought to evaluate the predictors of PD(L)1i use. As decisions related to PD(L)1i monotherapy are often driven by high tumor PD‐L1 expression by immunohistochemistry (>50%), we performed a single logistic regression analysis to examine predictors of using PD(L)1i in combination with chemotherapy versus chemotherapy alone. Predictors included in the model were age, gender, urban/rural, CCI, insurance type, and year of treatment as well the presence or absence of a number of comorbidities including cerebrovascular disease, congestive heart failure, diabetes, liver disease, kidney disease, and chronic obstructive pulmonary disease or emphysema.3n = 2568; 47%) followed by PD(L)1i with chemotherapy , PD(L)1i monotherapy , and targeted therapy . The proportion of patients who received PD(L)1i monotherapy and targeted therapy remained relatively stable over these 4 years. The percentage of patients receiving PD(L)1i with chemotherapy more than doubled during this same period. Finally, while the proportion of patients that received chemotherapy alone decreased by almost half over 4 years, a significant percentage of patients did not receive PD(L)1i treatment.There were 5431 newly diagnosed patients with NSCLC starting first‐line therapy . There was no statistical difference in insurance type across treatment groups (p = 0.31); most of the patients were covered by a traditional health plan such as a health maintenance organization or preferred provider organization. The number of days from diagnosis to initiation of first‐line therapy was statistically shorter in the PD(L)1i monotherapy and targeted therapy groups than the other two chemotherapy groups (p < 0.001); however, the absolute difference was nominal (range: 29.69–32.58 days).The PD(L)1i monotherapy and chemotherapy alone groups were older compared to those receiving combination PD(L)1i with chemotherapy and targeted therapy (p < 0.001). The proportion of patients with congestive heart failure was significantly higher in the PD(L)1i monotherapy group compared to the PD(L)1i with the chemotherapy group. Diabetes and COPD were more prevalent among the chemotherapy alone group than the other therapy groups. The proportion of patients with kidney disease was higher in the PD(L)1i monotherapy and chemotherapy alone groups than the other two therapy groups.The distribution of comorbidities was different according to which first‐line treatment regimen patients received (see Table 3.2While PD(L)1i monotherapy is primarily driven by PD‐L1 expression, predictors of combining PD(L)1i with chemotherapy compared to chemotherapy alone are less clear. In adjusted analyses, older patients , females , and patients with renal disease  and COPD  were less likely to have received combination PD(L)1i with chemotherapy than patients who received chemotherapy without a PD(L)1i Table . A highe4The current study is among the first to examine the real‐world utilization of first‐line therapies in patients with NSCLC since the approval of PD(L)1i as monotherapy or in combination with chemotherapy. We found that usage of PD(L)1i monotherapy and targeted therapies have remained unchanged, while PD(L)1i combined with chemotherapy has more than doubled during the 4‐year study period. Notably, while the use of chemotherapy without PD(L)1i has declined significantly, over a third of potentially eligible patients still did not receive PD(L)1i in 2019 and 2020. In the adjusted analysis, we further found that patients who receive chemotherapy without PD(L)1i are more likely to be older, female, and have renal and/or pulmonary comorbidities. Our findings provide important information for researchers, clinicians, and policy makers regarding the adoption trends of immunotherapy and highlight a substantial subset of patients not receiving these novel agents that are deserving of increased focus to ensure optimal management.Despite the well‐established clinical efficacy and tolerability of PD(L)1i, there may be several concerns for the universal adoption of these novel agents. Rapid approvals of new drugs given “breakthrough” designation by the FDA have allowed for accelerated development and early patient access to cancer therapeutics. However, many oncologists may be hesitant to adopt new drugs based on the following reasons: (1) endpoints used for approval only provide preliminary evidence of efficacy ; (2) lack of generalizability to patients with cancer treated in real‐world practices; and (3) lack of long‐term data regarding side effects. As such, a few prior studies have demonstrated that the extent of uptake of PD(L)1i in clinical practice has been suboptimal for several cancer types.Since FDA approval in 2015, PD(L)1i monotherapy has been typically used for patients with NSCLC that have high tumor expression of PD‐L1 tumors (≥50%). Several prior studies have shown that approximately 28% of NSCLC fall under this subgroup.Following the approval of PD(L)1i monotherapy, PD(L)1i with chemotherapy was approved for first‐line treatment of patients with NSCLC regardless of PD‐L1 expression. As a result, we observed a significant shift of practice pattern to combine PD(L)1i with chemotherapy. Yet, a significant proportion of patients only received chemotherapy alone. Our analysis examining predictors of PD(L)1i use in combination with chemotherapy indicated that older patients were less likely to have a PD(L)1i added in combination with chemotherapy. While there may be concerns of tolerability for older patients, pooled analysis of clinical trials has demonstrated that adding PD(L)1i to chemotherapy did not increase toxicity rates compared to chemotherapy alone.Whereas the real‐world results of this retrospective analysis provide important insight into patterns of PD(L)1i use, they should be considered alongside some limitations of the study. First, this observational analysis utilized retrospective administrative claims data, which lack potentially important clinical details for the reasons of therapy selection . Despite this, our study was still able to evaluate the associations and impact of several demographics  and individual comorbidities on treatment patterns. Second, due to the lack of granularity in ICD coding, information pertaining to the staging is not available. However, the treatment groups were defined using regimens for advanced NSCLC according to both FDA approvals and NCCN guidelines (with the exception of pembrolizumab's indication for unresectable stage III disease in patients who cannot tolerate definitive chemoradiation). Third, this study was limited to only those individuals with commercial health coverage or private Medicare supplemental coverage. Consequently, results of this analysis may not be generalizable to patients with NSCLC with other insurance or without health insurance coverage, or patients outside the United States. Finally, the proportion of patients over 65 years old in the MarketScan® database is lower than what is typically seen in the general lung cancer population and therefore may not comprehensively reflect the treatment patterns of older NSCLC patients. Despite these limitations, this study still provides important and up‐to‐date real‐world data describing practice patterns of PD(L)1i use for first‐line treatment of stage IV NSCLC.5The current study highlights that PD(L)1i therapy utilization in patients with NSCLC has steadily grown in the United States since the approval of the first PD(L)1i in 2015. Moreover, this study identified important demographic  and clinical  factors associated with the use of PD(L)1i in NSCLC treatment. Taken together, our findings demonstrate that while the adoption of PD(L)1i is increasing, a significant proportion of patients still do not receive PD(L)1i. Further qualitative research should be undertaken to elucidate the underlying reasons of slow adoption. As more immunotherapy agents and combinations are approved and used in clinical practice, practice patterns should continue to be evaluated to ensure NSCLC patients are receiving optimal treatment.RS, DH, BT, and BB are employees of and own stock in Beigene LTD. FRH reports the following conflict of interest: Bristol‐Myers Squibb, Genentech, AstraZeneca/Daiichi, Regeneron, Novartis, Sanofi‐Genzyme, Merck, OncoCyte, and Amgen. RRV has served on advisory boards for Bristol‐Myers Squibb, AstraZeneca, Merck, Novocure, on unbranded speaker's bureau of AstraZeneca, received consulting honorarium from Beigene and Onconova Therapeutics, and research grants from BMS, Astrazeneca, and EMD Serono. JW received consulting honorarium from Sanofi, GSK, and Banook and a research grant from Sanofi. JW reports consulting honorarium from Sanofi, Atea Pharmaceutical, and Banook and grants from Sanofi, Regeneron, and Arnold Consultants. ET reports no conflicts. Editorial assistance was provided by Jason C. Allaire, PhD of Generativity ‐ Health Economics and Outcomes Research, Durham, NC.Veluswamy contributed conceptualization, methodology, writing of original draft, and reviewing and editing. Hirsch, Taioli and Wisnivesky contributed to the interpretation of results, writing of original draft, and reviewing and editing. Strauss, Harrough, and Tang contributed conceptualization, methodology, writing of original draft, and reviewing and editing. Barnes contributed to conceptualization, methodology, writing of original draft, supervision, and reviewing and editing. All authors reviewed and approved the final version for submission.Institutional review board approval was not required as MarketScan data are recorded in such a manner that subjects cannot be identified, directly or through identifiers linked to the subjects. Meeting these conditions makes this research exempt from the requirements of 45 CFR 46.101 under the Department of Health and Human Services.


Transplant‐free survival was measured from the date of right heart catheterization until patients reached the composite endpoint of lung transplant or death. Multivariable cox‐pulmonary hypertension regression was used to study the effect of the time of diagnosis. The final cox model was fitted in both younger and older patients to evaluate the difference between these groups. During a median follow‐up time of 4.1 (interquartile range: 2.2–7.3) years, 9 patients underwent lung transplantation and 151 patients died. The median overall transplant‐free survival was 6.2 (5.5–8.0) years. Patients older than 56 years at baseline who were diagnosed in 2005–2009 showed better survival compared to patients diagnosed in 2010–2014 and 2015–2019 with an adjusted hazard ratio of, respectively, 2.12 (1.11–4.03) and 2.83 (1.41–5.69). Patients younger than 56 years showed neither an improved nor deteriorated survival over time. In conclusion, survival in patients with PAH did not improve over time, despite more available therapeutic options. This might be partly due to the changed demographic characteristics of the PAH patients and a still important diagnostic delay.The prognosis of pulmonary arterial hypertension (PAH) remains dismal. Over the years, multiple therapeutic advances have been introduced. This study evaluates the evolution of PAH survival over the past 15 years. We included 293 consecutive adult patients diagnosed with PAH between 2005 and 2019 . Patients were divided into three cohorts based on the time of diagnosis: 2005–2009, 2010–2014, and 2015–2019 (2005–2009:  Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature. It affects approximately 7 per million people per year.Multiple registries have been established over the past decades. The first one to report on survival was published in 1991 and reported a 5‐year survival of 34% in incident patients with IPAH. More recent reports showed improved survival rates. Humbert et al.Not only did the therapeutic options change, the epidemiological and demographic characteristics of the PAH patient changed over time too. Registries observed that the age of the patients diagnosed increased over time and that survival of elderly patients is worse compared to younger patients.Limited data exist on how the prognosis of PAH changed by this evolving landscape. The primary objectives of this study were 1) to evaluate the evolution of transplant‐free survival in patients with PAH over the past 15 years; 2) to provide an up‐to‐date overview of mortality; and 3) to evaluate the (development of) treatment strategies in patients with PAH.In this study, all consecutive incident adult patients diagnosed with PAH between 2004 and 2019 from two pulmonary hypertension (PH) expertise centers in the Netherlands were screened for inclusion. All patients were treatment naïve at baseline and were treated in accordance with the ESC/ERS guidelines for pulmonary hypertension after the diagnosis PAH was established.All patients underwent an inpatient screening visit, during which the following tests were performed: 6‐min walking test (6MWD), 12‐lead electrocardiogram, transthoracic echocardiography, venous blood sampling, pulmonary function tests, lung perfusion scintigraphy, chest computed tomography scan, and a right heart catheterization with a fluid challenge when diastolic dysfunction was suspected. The date of right heart catheterization was considered the date of diagnosis. PAH was defined as a mPAP of ≥25 mmHg and PVR ≥ 3 WU with a PCWP of ≤15 mmHg, according to the contemporaneous guidelines.The primary endpoint was a composite endpoint of all‐cause mortality or lung transplantation. The number of patients on PH‐specific mono‐, dual‐, or triple therapy was used as a secondary endpoint. The endpoints were evaluated at 1, 3, 5, and 10 years after diagnosis. To compare the evolution of the primary and secondary endpoints over time, patients were divided into three cohorts based on their date of diagnosis .Baseline demographic data, type of PAH, 6MWD, N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) level, comorbidities , and hemodynamic measurements were retrieved for this study. We calculated the French registry noninvasive risk score based on the number of low‐risk criteria .t‐test or Mann–Whitney U test depending on their distribution and one‐way analysis of variance when more than two subgroups were compared. Categorical data were compared using a χ2 test or Fisher's exact test.Continuous variables were presented as mean ± standard deviation or median (interquartile range) depending on their distribution. Categorical variables were presented as counts (percentage). Continuous variables were compared using an unpaired p < 0.2. Missing data were imputed using multiple imputations via chained equations and 20 imputed datasets were generated. Predictive mean matching was used for numeric variables and logistic regression for binary variables. Supporting Information: Table Transplant‐free survival was measured from the date of right heart catheterization until patients reach the composite endpoint. Survival curves were obtained using the Kaplan–Meier estimator stratified according to time cohort. Patients who did not complete the 3‐ or 5‐year follow‐up in the cohort 2015–2019 were right‐censored on the first of July 2021. Transplant‐free survival between time cohorts was compared using a logrank test. A multivariable cox proportional hazards model was used to analyze the association between the time of diagnosis and transplant‐free survival when adjusted for other clinical characteristics. Variables were selected using stepwise selection with a significance level of https://www.R-project.org/) using the packages “survival,” “ggplot2,” and “mice.”Statistical analysis was performed using SPSS  and R (R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing. URL n = 56; 2010–2014: n = 111; and 2015–2019: n = 126). There was no significant difference in the amount of included patients between centers (Supporting Information: Table p = 0.003). Significantly less patients with PAH due to connective tissue disease were present in the cohort diagnosed between 2005 and 2009 . Furthermore, 15.4% of the patients were diagnosed with PAH due to congenital heart disease, 6.5% with portopulmonary hypertension, 4.4% with heritable PAH, 3.8% with the pulmonary venous occlusive disease, 3.1% with drugs and toxins induced, and 0.7% secondary to HIV infection. No significant differences were found between subgroups regarding hemodynamic parameters. The median mPAP was 46 (37–56) mmHg, with a median PCWP of 9 (6–13) mmHg and PVR of 7.4 (4.9–10.8) WU. No significant differences were found between cohorts in comorbidities .A total of 293 patients with PAH were included in this study .During a median follow‐up time of 4.1 2.2–7.3) years, 9 patients underwent lung transplantation and 151 patients died. A detailed overview of the causes of death per time and age cohort can be found in Supporting Information: Table  years, 9The univariable cox‐PH regression models can be found in Supporting Information: Figure This study demonstrated a median overall transplant‐free survival of 6.2 years with a 5‐year transplant‐free survival rate of 57% in incident patients with PAH. We observed a 10‐year transplant‐free survival of 35%. Treatment strategies changed over time, with an increase in the number of patients on combination therapy with PAH‐specific medication. Despite major developments in the available PAH medication and treatment strategies, we did not observe an improvement in transplant‐free survival over time.The overall transplant‐free survival improved compared to the U.S. NIH registry.p = 0.019). Our study covers another and a broader range of therapeutic advances, evolution in treatment strategies, and new guideline implementations. When we compared the effect of the time cohort in patients older and younger than 56 years at the time of diagnosis, a significantly worse transplant‐free survival rate was observed in patients >56 years old. However, in our younger patients, no significant effect on transplant‐free survival over time was observed.Comparing our three‐time cohorts, we did not observe an improvement in transplant‐free survival over time. Transplant‐free survival rates were significantly worse in patients diagnosed in 2010–2019 when compared with patients diagnosed in 2005–2009 when adjusted for baseline differences in etiology, comorbidities, and clinical prognostic factors. Ling et al. investigated the evolution of survival of PAH patients between 2001 and 2009. They found no difference in overall survival between the investigated time cohorts. Only when adjusted for age, functional class, 6MWD, pulmonary diffusion capacity, mean right atrial pressure, and cardiac index, a better survival was found in patients diagnosed between 2007 and 2009 compared to 2001 and 2003 . Despite therapeutic developments, transplant‐free survival in the younger patients did not improve in our study. Two meta‐analyses analysed randomized controlled trials of PAH‐specific medication and described the benefit of intervention groups compared to controls .This study has several limitations. Our study endpoint was transplant‐free survival. We did not investigate the quality of life in this study. It would be interesting to know, that even though patients do not seem to live longer, whether their quality of life did improve. Given the observational design of this study, missing data are inevitable. Although with less precision, complete case analysis did not yield different outcomes in survival analysis. A clear survival benefit is only proven for epoprostenol. In our study, we did not distinguish between different prostacycline analogues, intravenous epoprostenol and intravenous or subcutaneous treprostinil. This distinction is also complicated by the fact that a noticeable amount of patients switched between these treatment modalities; switching from treprostinil subcutaneously to intravenous treatment with either treprostinil or epoprostenol, because of side effects, mainly local injection side reactions and switching from intravenous therapy to subcutaneous therapy, mainly for practical reasons. We did not have sufficient numbers of patients for each individual etiology to perform a subgroup analysis. The median follow‐up time differed between our time cohorts. This could introduce bias when patients are more at risk earlier after diagnosis. However, the proportional hazards assumption of our cox‐PH regression model holds for all included variables including the time cohort. Therefore, we consider this possible bias to be limited.Our study did not demonstrate improved transplant‐free survival over the past 15 years of PAH treatment despite an expanded therapeutic arsenal. This leads to one of our core questions: are we treating the right patients in the right way? More detailed information is needed about PAH in the elderly population. Does this type of PAH pathophysiologically differ from PAH in younger patients? Do both groups benefit from the same medication regime? Treatment strategies in younger patients should also be evaluated. Have we become too cautious with the use of intravenous and subcutaneous prostacyclin therapy? Furthermore, we also observed that patients are still diagnosed in advanced disease stages, this continuing diagnostic delay will also attribute to a poorer prognosis. Future research is crucial for the optimization of PAH‐care to identify the right patient for the right treatment strategies.The 1‐, 5‐, and 10‐year transplant‐free survival rate for incident patients with PAH in our cohort was, respectively, 88%, 71%, and 35%. Treatment strategies changed over time with a significantly increased use of combination therapy. Patients older than 56 years showed a significantly worse transplant‐free survival when diagnosed between 2010 and 2019 compared to patients diagnosed between 2005 and 2009, which could be partly due to changed demographic characteristics. Patients younger than 56 years old showed neither improved nor deteriorated transplant‐free survival over time.Conceptualization: Paul M. Hendriks, Liza D. van de Groep, Annemien E. van den Bosch, Marco C. Post, and Karin A. Boomars. Patient inclusion: Robert M. Kauling, Leon M. van den Toorn, Prewesh P. Chandoesing, Annemien E. van den Bosch, Hans‐Jurgen Mager, Marco C. Post, and Karin A. Boomars. Data curation: Paul M. Hendriks, Diederik P. Staal, and Liza D. van de Groep. Data analysis: Paul M. Hendriks, Liza D. van de Groep, and Diederik P. Staal. Supervision: Robert M. Kauling, Leon M. van den Toorn, Prewesh P. Chandoesing, Hans‐Jurgen Mager, Annemien E. van den Bosch, Marco C. Post, and Karin A. Boomars. Writing, review, and editing: Paul M. Hendriks, Diederik P. Staal, Liza D. van de Groep, Robert M. Kauling, Leon M. van den Toorn, Prewesh P. Chandoesing, Annemien E. van den Bosch, Marco C. Post, and Karin A. Boomars. All authors read and approved the manuscript.The authors declare no conflict of interest.This study was performed according to the principles outlined in the Declaration of Helsinki. Informed consent was waived in accordance with Dutch national law. The study protocol was approved by the medical ethics committee of both participating centers .Supporting information.Click here for additional data file.


In contrast to the intra‐arterial nitroglycerin that showed nonstatistically significant results in preventing RAS and RAO ‐ ), respectively. Also, topical nitroglycerin did not prevent RAS . Compared with placebo, subcutaneous nitroglycerin during transradial catheterization reduced the incidence of RAS and RAO. Meanwhile, Intra‐arterial and topical nitroglycerin did not show statistically significant outcomes. Subcutaneous nitroglycerin may be a practical and cost‐effective technique to facilitate transradial catheterization; however, more RCTs are needed to evaluate the subcutaneous versus intra‐arterial nitroglycerin administration.Radial artery spasm (RAS) is the most common cause of transradial access site crossover and is a common intra‐procedural complication. RAS incidence can lead to radial artery occlusion (RAO) postprocedure, preventing the radial artery as a future access site. We evaluated the efficacy of nitroglycerin preventing RAS and RAO during transradial catheterization discussing the different routes of administration, including topical, subcutaneous, and intra‐arterial. A systematic review and meta‐analysis included all relevant articles until April 23, 2022. We searched six databases Google Scholar, Web of Science, SCOPUS, EMBASE, PubMed (MEDLINE), and CENTRAL. We registered our review protocol in PROSPERO with ID: CRD42022330356. We included 11 trials with 5814 patients. Compared to placebo, the pooled analysis favored subcutaneous nitroglycerin in preventing RAS (risk ratio [RR]: 0.57 with 95% confidence interval [CI] [0.43–0.77],  We used the following search terms: “nitroglycerin,” “vasodilators,” “radial artery spasm,” “radial artery occlusion,” and “transradial.” We did not use any search filters. The detailed search strategy and results are demonstrated in Table 2.3We included RCTs with the following PICO criteria: population (P): adult patients undergoing cardiac diagnostic or intervention transradial catheterization; intervention (I): nitroglycerin regardless of dosage and route of administration; control (C): placebo; outcomes (O): primary outcomes: incidence of RAS detected by various clinical criteria  during or pre‐catheterization, and incidence of RAO detected by the absence of anterograde flow detected by vascular duplex Doppler ultrasound following catheterization for up to 24 h. Our secondary outcomes are radial artery diameter, procedure time, radial artery puncture attempts, and incidence of adverse events .We excluded animal studies, pilot studies, observational studies , single‐arm clinical trials, in vitro studies (tissue and culture studies), book chapters, editorials, press articles, and conference abstracts.2.4Using Covidence,2.5Using a pre‐tested extraction sheet, four reviewers  independently extracted the following data from the included articles: study characteristics ; baseline information ; efficacy outcomes data ; safety outcomes data . Conflicts were resolved through discussion.2.6Four reviewers  independently assessed the included studies for the risk of bias (ROB) using The Cochrane Collaboration's tool for assessing the risk of bias in randomized trials,2.7I2 and Chi‐square tests; the Chi‐square test assesses whether there is significant heterogeneity, while the I‐square evaluates the magnitude of heterogeneity. According to the Cochrane Handbook (Chapter 9),I2 test is interpreted as follows: . With significant heterogeneity, we used the random‐effects model; otherwise, we used the fixed‐effects model.We conducted the statistical analysis using RevMan v5.3 software.In the case of significant heterogeneity, we performed a sensitivity analysis by removing one study at a time and repeating the analysis to analyze the influence of each study on the overall effect size of the outcomes. Moreover, we performed a subgroup analysis based on the route of nitroglycerin administration to test the stability of our results. We did not present funnel plots to identify publication bias because we only included less than 10 studies in each outcome, as recommended by Egger et al.33.1Two thousand six hundred ninety‐two records were retrieved after the searching process. Eight hundred fourteen duplicates were removed using Covidence. We screened 1878 titles and abstracts, excluding 1841 irrelevant records, then we screened 37 full‐text articles, and finally included 11 articles in our systematic review and meta‐analysis. The selection process is demonstrated in a PRISMA flow chart Figure .3.2We included 11 trials3.3According to Cochrane's risk of bias tool, the quality of the included trials ranged from moderate to high. We demonstrated the summary of the risk of bias in Figure 3.43.4.1p = .0001) . Pooled studies were homogenous  ; however, pooled risk ratio showed no difference between topical nitroglycerin (91 patients) and placebo (92 patients)  and placebo (1020 patients) ), respectively  (1829 patients) over placebo (1765 patients) in reducing the incidence of RAS  Figure , Table 3y Figure .3.4.2p = .02) . Pooled studies were homogenous  ; however, pooled risk ratio showed no difference between intra‐arterial nitroglycerin (1873 patients) and placebo (1873 patients)   (718 patients) over placebo (718 patients) in reducing the incidence of RAO  Figure , Table 3) Figure .3.53.5.1p = .01) . Pooled studies were not homogenous  ; however, pooled mean difference showed no difference between intra‐arterial nitroglycerin and placebo   over placebo in dilating the radial artery  Figure , Table 3) Figure . We cond) Figure .3.5.2p = .78) . Pooled studies were homogenous  . We conducted a subgroup analysis based on the route of administration to evaluate the effect of subcutaneous and intra‐arterial routes on the procedure duration; pooled mean difference showed no difference between subcutaneous or intra‐arterial nitroglycerin and placebo  – ), respectively  and placebo  . Pooled studies were not homogenous  ; however, pooled mean difference showed favored placebo over intra‐arterial nitroglycerin   and placebo  Figure , Table 3) Figure . We cond) Figure .3.5.4p = .12)  . We conducted a subgroup analysis based on the route of administration. The pooled mean difference showed no difference between subcutaneous nor topical nitroglycerin and placebo ((RR: 0.64 with 95% CI [0.39–1.04]. p = .07) – (RR: 1.43 with 95% CI [0.30–6.92]. p = .65)), respectively  Figure , Table 3–1.10. ) y Figure .p = .009)  . We conducted a subgroup analysis based on the route of administration. The pooled mean difference showed statistically significant result with intra‐arterial nitroglycerin administration . In contrast to subcutaneous nitroglycerin that showed nonsignificant result   Figure , Table 3.70, ) Fi) Figure .p = .01)   We conducted a subgroup analysis based on the route of administration. The pooled mean difference showed statistically significant result with subcutaneous nitroglycerin administration . In contrast to topical nitroglycerin that showed nonsignificant result   Figure , Table 3) Figure .4Our meta‐analysis involving 11 RCTs demonstrated that nitroglycerin is effective in reducing the incidence of RAS and RAO by using subcutaneous nitroglycerin. Moreover, nitroglycerin was effective in increasing the radial artery diameter. However, we found no difference between nitroglycerin and placebo in reducing radial artery puncture attempts and procedure duration.Regarding RAS incidence Table , subcutaSimilarly, subcutaneous nitroglycerin was effective in reducing RAO, while intra‐arterial nitroglycerin was not. Three studies , it possesses a thicker tunica media than many other arteries, hence classified as a type III artery graft.Furthermore, the variability in heparin doses may affect the pooled analysis of RAO. The heparin dose was 3000u in Chen et al.Nitroglycerin's vasodilatory effect is hypothesized to be the mechanism through which it may reduce the rate of RAO.Regarding the safety of nitroglycerin, on one hand, our analysis showed more incidence of hypotension and headache in the nitroglycerin group compared with the placebo. We can attribute this to the vasodilatory effect of nitroglycerin. Hence, headache and hypotension are more likely to happen after intra‐arterial nitroglycerin because of the high dose and the greater effect on general circulation.5To the best of our knowledge, this is the first systematic review and meta‐analysis to discuss the effect of nitroglycerin with the various routes of administration to prevent RAS and RAO during transradial catheterization, hence providing the most subtle evidence in this regard. Moreover, we strictly followed the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) statement6Our study has a few limitations. First, we included two non‐peer‐reviewed trials.7Future trials should assess the following: first, the effect of different hemostasis protocols, especially hemostasis duration on nitroglycerin effect in preventing RAO to reach the most proper time for hemostasis compression after transradial catheterization. Second, future trials should depend on the objective assessment of radial artery lumen using duplex ultrasound, which is the gold standard in detecting thrombosis and flow disturbance, rather than the subjective clinical assessment to accurately determine the extent of post‐procedural RAO.8Our current review indicates that nitroglycerin is a feasible economic strategy to facilitate transradial catheterization, decreasing the incidence of RAS and subsequent RAO, especially subcutaneous nitroglycerin, while the evidence of nitroglycerin's efficacy in reducing the incidence of RAO, radial artery puncture attempts, and procedure duration is uncertain. More trials are still required to assess the intra‐arterial route's potential efficacy through different puncture techniques, post‐puncture cocktails, equipment, and hemostasis protocols.Basel Abdelazeem and Mohamed T. Abuelazm conceived the idea. Basel Abdelazeem designed the research work and plan. Basel Abdelazeem and Mohamed Gamal searched the databases. Sarya Swed and Mohamed Gamal screened the retrieved records, and Mostafa Atef resolved the conflicts. Sarya Swed, Mohamed Gamal, and Mostafa Atef extracted relevant data, assessed the quality of evidence, and resolved the conflicts. Mohamed T. Abuelazm and Basel Abdelazeem performed the analysis. Mohamed T. Abuelazm and Muhammad A. Noori wrote the final manuscript. Annabelle S. Volgman and Anthony Lutz reviewed and edited the manuscript. All authors have read and agreed to the final version of the manuscript.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.


Through development, multisensory systems reach a balance between stability and flexibility: the systems integrate optimally cross-modal signals from the same events, while remaining adaptive to environmental changes. Is continuous intersensory recalibration required to shape optimal integration mechanisms, or does multisensory integration develop prior to recalibration? Here, we examined the development of multisensory integration and rapid recalibration in the temporal domain by re-analyzing published datasets for audio–visual, audio–tactile, and visual–tactile combinations. Results showed that children reach an adult level of precision in audio–visual simultaneity perception and show the first sign of rapid recalibration at 9 years of age. In contrast, there was very weak rapid recalibration for other cross-modal combinations at all ages, even when adult levels of temporal precision had developed. Thus, the development of audio–visual rapid recalibration appears to require the maturation of temporal precision. It may serve to accommodate distance-dependent travel time differences between light and sound. Because sensory systems develop at different rates, some have suggested that multisensory coordination and integration build on intersensory calibration and continuous recalibration during development3. Presumably, the intersensory recalibrations should be extensive at younger ages and then decrease as maturation progresses, because with age, the systems become more specialized to native stimuli and neural plasticity decreases. Recently, however, Rohlf, Li, Bruns, and Röder4 demonstrated a reversed trend by showing that audio–visual integration in the spatial domain develops prior to the emergence of spatial recalibration to newly experienced spatial disparity. Here we examine this surprising developmental trend in the temporal domain among three sensory combinations—audio–visual, audio–tactile, and visual–tactile—by re-analyzing previously published data7.Neural plasticity enables the human brain to adapt to the environment throughout the lifespan. From birth, we learn to associate sensory signals from different modalities when they arise from the same event in the external world13. Temporal simultaneity provides one crucial cue in determining which cross-modal signals belong together15, while those integration benefits tend to decline when temporal asynchronies are introduced between the component signals17. In real world contexts, the relative timing of signals generated by an external event can vary significantly between sensory modalities. Specifically, the timing of audio–visual signals is dependent on source distance in audition but not in vision because sound travels much slower than light; on the other hand, a tactile stimulus is always applied to the body surface . Once at the sensory periphery, there are internal factors such as differences in neural transduction times18: visual processing is the slowest19, and tactile processing time is positively correlated with the neural transduction distance20. Temporal asynchrony is therefore inevitable between stimulus signals arising from a common event.The ability to integrate cross-modal signals supports our survival in a multisensory world: multisensory integration improves precision, accuracy, and processing speed of perception15, for a review). Temporal recalibration is manifested by shifts in the observer’s point of subjective simultaneity (PSS)—the time lag at which sensory signals are perceived as most likely to be simultaneous. For example, adapting to a visual-leading pair biases the PSS toward visual-leading presentations29. There are two forms of temporal recalibration at different time scales. Slow recalibration involves an exposure phase during which observers are presented repeatedly with a fixed time lag between signals  for a period of time  before demonstrating a change in tested simultaneity perception22. Following several minutes of adaptation, the recalibration effect lasts for about a minute24. Rapid recalibration, on the other hand, involves a change after a time lag on the single previous trial, but is influenced far less by the time lag of one further trial back25. Hence, slow and rapid recalibrations demonstrate quick adaptations and recoveries from stimulus variations of multisensory systems—such dynamic malleability is separate from plasticity which often involves experience-dependent changing of neural substrates. In adults, both rapid and slow recalibrations are more reliable for audio–visual than in audio–tactile or visual–tactile pairings31.To deal with the temporal variations of multisensory signals that originate from the same event, the multisensory system realigns cross-modal signals according to previous experience, a phenomenon known as temporal recalibration . Precision improves with age and reaches adult levels by 9 years for audio–visual pairings and by 11 years for visual–tactile and audio–tactile pairings. The PSS, however, is adult-like by the youngest ages tested .From a developmental perspective, the capability to detect temporal proximity between multisensory stimuli is observed at birth or even earlier in the fetal stage. However, in practice, testing temporal recalibration would be an enormous procedural challenge with children, especially the long adaptation periods for the slow recalibration. On the other hand, the development of temporal processing throughout childhood suggests that rapid temporal recalibration may be present and linked to the maturation of simultaneity perception35 . Rapid recalibration is easily uncovered by a sequential analysis of the data of simultaneity judgments35.Given the early maturation of the PSS, it remains an open question whether young children exhibit temporal recalibration, and if so, how it might vary through development and for different sensory combinations. The sensitivity to temporal correspondence at very early ages and the early maturation of the PSS suggest that temporal recalibration may be present early in life7 that measured simultaneity perception in children and compared those data to adult groups. If cross-sensory calibration serves as the basis for the development of multisensory integration2, then we would expect rapid recalibration to emerge early and then decrease as multisensory simultaneity perception matures35. Alternatively, rapid recalibration might develop after the system matures, providing a form of malleability to accommodate the temporal variations among multisensory events4.The current study examined the developmental trajectories of rapid temporal recalibration for three cross-modal pairs  = 3.15, p = 0.02, d = 0.72, bootstrapped p < 0.001; 11-year-olds: t(19) = 4.44, p = 0.001, d = 0.99, bootstrapped p < 0.001; adults: (t(19) = 3.23, p = 0.02, d = 0.72, bootstrapped p = 0.004), but not at 7 years of age (t(18) = 1.83, p = 0.08, d = 0.42, bootstrapped p = 0.053). For the audio–tactile or visual–tactile pairings, none of the age groups differed significantly from 0 ms , suggesting that rapid temporal recalibration processes remain weak for audio–tactile and visual–tactile pairings throughout development.When each age group was compared to a hypothesized PSS shift of 0 ms see Fig. a, audio–t(30) = 0.38, p = 0.71, d = 0.14, bootstrapped p = 0.65) and visual–tactile pairings (t(33) = 1.89, p = 0.07, d = 0.64, bootstrapped p = 0.05). Similarly, in the 9-year-olds, audio–visual PSS shifts did not differ significantly from audio-tactile (t(36) = 1.32, p = 0.19, d = 0.42, bootstrapped p = 0.18) and visual–tactile (t(36) = 0.79, p = 0.43, d = 0.25, bootstrapped p = 0.44) pairings. However, by 11 years of age, PSS shifts were significantly larger in audio–visual conditions than in audio–tactile (t(38) = 3.25, p = 0.002, d = 1.06, bootstrapped p < 0.001) and visual–tactile (t(38) = 3.12, p = 0.003, d = 0.99, bootstrapped p = 0.001) conditions. The larger PSS shifts in audio–visual than audio–tactile (t(38) = 2.00, p = 0.05, d = 0.63, bootstrapped p = 0.03) and visual–tactile (t(38) = 1.88, p = 0.07, d = 0.59, bootstrapped p = 0.04) pairings continued in adulthood.Figure F = 5.63, p = 0.002, ηp2 = 0.19, bootstrapped p < 0.001), audio–tactile pairings  = 5.54, p = 0.002, ηp2 = 0.20, bootstrapped p = 0.001), and visual–tactile conditions  = 8.83, p < 0.001, ηp2 = 0.28, bootstrapped p < 0.001). Post-hoc pairwise comparisons showed that for audio–visual and audio–tactile presentations, main effects were driven by the 7-year-old groups, which had wider windows than the older age groups (ps < 0.05). Similarly, for visual–tactile pairings, 7- and 9-year-olds had larger window sizes than the other ages (ps < 0.05). In contrast, age did not have a significant effect on PSS shifts for the audio–visual pairing  = 1.44, p = 0.24, ηp2 = 0.06, bootstrapped p = 0.06), the audio–tactile pairing  = 0.17, p = 0.92, ηp2 = 0.01, bootstrapped p = 0.49), and the visual–tactile pairing  = 1.71, p = 0.15, ηp2 = 0.07, bootstrapped p = 0.20).The effect of age on the width of the simultaneity window is contrasted with its effect on the PSS shift in Fig. ß = 0.36, t(18) = 3.47, p = 0.003, bootstrapped p = 0.009; adult: ß = 0.34, t(18) = 5.29, p < 0.001, bootstrapped p < 0.001) but not reliable in the younger age groups  = 2.07, p = 0.05, bootstrapped p = 0.02; 9-year-olds: ß = −0.06, t(17) = -0.55, p = 0.59, bootstrapped p = 0.71). The other cross-modal pairings did not demonstrate a convincing relation between the PSS shift and the width of the simultaneity window  = 3.33, p = 0.005, bootstrapped p = 0.04).Further within-age-group analyses showed that the correlation between the PSS shifts and the widths of the simultaneity window was dependent on age and the modality combination. Specifically, positive correlations between larger PSS shifts and wider audio–visual simultaneity windows were demonstrated reliably by 11 years of age , suggesting a developmental trend of the audio–visual system from malleable to stable. However, Noel et al. had only nine participants in the age range of 7 to 10 years—the critical age range according to our data—and used a broad sliding window that averaged across 7–11 years of age. Doing so would miss the critical changes we observed with 20 children per age group for ages 7-, 9-, and 11-year-olds (see Footnote 1 in7).For the audio–visual pairing, rapid recalibration first appeared at age 9 and continued with age; in contrast, children’s precision of simultaneity perception  improved with age up to age 9, at which point it was adultlike4) suggests that plasticity/malleability of the audio–visual system does not decrease monotonically with age. Children’s wider simultaneity window5 and larger spatial ventriloquism effect4 indicate that they tend to integrate visual and auditory information originating from disparate timings or locations more often, and less selectively, than do adults. During development, the audio–visual system is plastic in order to accommodate physical growth of the body and the maturation of each sensory system. In addition, intersensory calibration based on daily audio–visual experience will lead to changes based on the more accurate  modality, the precision of each sensory system, and expectations about signals that ought to be integrated . In turn, the audio–visual system develops a statistically optimal algorithm for signal integration in both spatial and temporal domains that is useful for most daily events41. During this developmental period, accommodating to particular audio–visual events which may occur occasionally or exceptionally  might lead to a misrepresentation of the optimal window or even a prolonged developmental trajectory if the window keeps changing.The earlier development of the precision of audio–visual perception than rapid recalibration in both the temporal and spatial domains . The first is a long-term scale beginning at birth and lasting until late childhood during which the multisensory system is tuned by general events from an overly broad system into optimal precision. This development of optimal integration is based on continuously improving sensory reliability and establishing the prior of common cause for multisensory signals48 . Afterward, when the system is relatively stable, the second time scale takes effect with mild short-term influences from recent experience. The rapid recalibration underpinned by short-term malleability may rely mainly on accommodating the prior of common cause and not depend as much on signal reliability53. For example, rapid recalibration occurs when the asynchronous audio–visual stimuli are supposed to originate from the same event , and thence they are realigned54.After the audio–visual system achieves optimal precision for integration, it remains malleable in both the temporal and spatial domains, even in adulthood. This is demonstrated in the phenomena of rapid recalibration55, suggesting that short-term malleability is more pronounced for audio–visual than for audio–tactile or visual–tactile pairings. Note that in experimental settings, like those we used, the locations of the audio and visual stimuli were very close5, whereas the locations of the audio–tactile and visual–tactile stimuli differed7. This difference in locations may have reduced the likelihood of multisensory integration9 and therefore the necessity of rapid recalibration. However, the potential influence of the locations of multisensory stimuli on rapid recalibration has been ruled out in prior studies55.We found that the development of rapid recalibration emerged specifically for the audio–visual pairing, and not for the audio–tactile and visual–tactile pairings at any age. These results are consistent with previous studies in adults56. For the audio–visual pairing, because both stimuli are distal and the speed of light is much faster than sound, the signal arrival time difference is distance-dependent. To accommodate rapidly such variations of arrival time differences, the audio–visual system can modify the PSS according to the estimated distance of the source59 or, even more straightforwardly, according to the asynchrony of the most recent experience . For audio–tactile and visual–tactile pairings, the tactile stimulus is proximal on the body’s surface, and therefore the variability of the arrival time differences is small, caused mainly by the different neural transduction times when stimulating different locations on the body31. Thus, the fact that rapid recalibration develops mainly in audio–visual simultaneity perception but only mildly, or not at all, in audio–tactile or visual–tactile simultaneity perception appears to indicate that greater malleability is maintained for the audio–visual pairing because of the greater variation in daily experience.The need for rapid recalibration may stem from the nature of processing of each cross-modal combination. In cross-modal simultaneity perception, the arrival time difference between each signal is determined by their physical transmission time to receptors and neural transduction time to the associated brain areas35, who demonstrated a decrease of rapid recalibration and the width of audio–visual simultaneity window  from childhood to adulthood, we did not observe a similar effect in any cross-modal pairings . This correlation suggests that the wider window  of audio–visual simultaneity perception in older children and adults may be attributable partly to higher susceptibility to the recent audio–visual asynchrony , indicating a need to rethink the ecological meaning of the precision of audio–visual simultaneity perception. The correlation in the audio–visual pairing contrasted with the other cross-modal presentations, which demonstrated a general reduction in variance and a null correlation in later stages of development. Interestingly, we found a significant inverse relation in the youngest visual–tactile condition, which indicates a repulsive rapid recalibration when the window of simultaneity perception is wide. This trend may be spurious because of the higher variance in the young children’s behavioural measures, the unique performance of three children, or an overall preference for touch in visual-tactile judgments by 8 years of age49.Our findings also reveal that the relation between rapid recalibration and precision in multisensory simultaneity perception is not straightforward through development. Contrary to Noel et al.see Fig. c. Furthe36, whereas the rapid and slow recalibrations tend to be dissociable in terms of their underlying mechanisms63 and developmental trajectories4. Inspired by the results in the spatial domain, it would be interesting to measure the development of slow recalibration together with rapid recalibration and the simultaneity window in order to gain a full picture in the temporal domain. Van der Burg et al.24 have developed a novel method to measure the rapid and slow recalibrations together, which is promising for future developmental studies.A limitation in the present study was that we examined only the relation between the development of simultaneity perception and rapid recalibration in the temporal domain, while the development of slow recalibration was not studied. Recent studies in the spatial domain suggest that audio–visual integration and rapid recalibration share a common neural substrate31. The uniqueness of rapid recalibration in the audio–visual system highlights its malleability to accommodate the stimulus arrival time difference depending on the distance of the source. Future investigations should examine whether maturation of audio–visual simultaneity perception is a prerequisite for the development of rapid recalibration; for example, it would be interesting to examine rapid recalibration in patients treated for congenital cataract because they show abnormal audio–visual simultaneity perception later in life64. The result will provide a contrast to the spared audio–visual integration and rapid recalibration in the spatial domain after early visual deprivation65.In conclusion, we demonstrate that cross-modal rapid recalibration in the temporal domain emerged after multisensory simultaneity perception achieved adult precision for audio–visual presentations. In contrast, rapid recalibration in the audio–tactile and visual–tactile pairings did not develop at any age7. All experimental protocols of these studies were approved by the McMaster Research Ethics Board. Methods in each study conformed to the Canadian Tri-Council Statement on Ethical Conduct of Research Involving Humans and the Declaration of Helsinki. All three studies recruited twenty participants for each age group that were balanced approximately by sex at birth. All three studies recruited independent groups of participants. The audio–visual experiment tested 5-, 7-, 9-, 11-year-olds, and adults. The age groups recruited in the audio–tactile and visual–tactile studies were similar, in that 7-, 9-, 11-year-olds, and adults were tested, with the exception that 13-year-olds were also tested in the visual–tactile study. Written consent was obtained from adult participants. For child participants, verbal assent was obtained from the child in conjunction with informed, written consent from their parents.Cross-modal simultaneity judgment data were re-analyzed from three previous developmental studies5, audio–tactile7, and visual–tactile6. The following, however, provides a brief description. In the audio–visual experiments, participants fixated on the middle of a visual grey ring (~ 2° inner diameter), where a visual white disk (~ 2° diameter) was presented for about 17 ms on each presentation. The auditory stimulus was a 17 ms white noise burst presented from speakers on either side of the monitor. Audio–tactile stimuli were a 10 ms white noise burst presented through closed-ear headphones and a 10 ms dull tap delivered to the right index finger using a solenoid-based mechanical device aligned with the body midline. The same visual stimulus from the audio–visual experiment was used to record the visual–tactile simultaneity judgments, and the tactile stimulus was a 17-ms tap delivered to the right index finger, situated 20° below the visual stimulus and aligned with the participant’s body midline. In all three studies, participants reported orally if they perceived the individual onsets of the cross-modal stimuli to be synchronous or asynchronous. An experimenter seated beside participants recorded their responses and ensured that each participant adhered to the task requirements. A total of 13 stimulus onset asynchronies (SOAs) were tested for each cross-modal pairing: ± 1200, ± 800, ± 400, ± 300, ± 200, ± 100 or 0 ms. Negative SOAs indicate an auditory leading stimulus in audio–visual and audio–tactile experiments and a tactile leading stimulus in visual–tactile experiments. A total of 130 trials  were collected for each 5-year-old participant, whereas a total of 260 trials  were collected for older participants.The procedures used to acquire each dataset are described in detail in the respective studies, that is, audio–visualA–PSSB). Following earlier studies, we label A-leading SOAs as negative and B-leading as positive. Using this convention, rapid temporal recalibration exhibits a positive relationship with SOA25. That is, if modality A led on the previous trial, then the current trial PSS is shifted toward negative SOAs, and if modality B led on the previous trial, then the current trial PSS is shifted toward positive SOAs.In our reanalysis of the data, we estimated the magnitude of rapid temporal recalibration for each participant’s dataset  by first dividing the data into two bins based on the leading modality in the preceding trial. This resulted in two subsets for a specific age group and cross-modal condition, where modality A  or modality B was the leading stimulus in the previous trial see Fig. . A GaussR2 < 0.60) on either subset. This eliminated data from the 5-year-olds because of fewer trials. Because of poor Gaussian fits, data were excluded for one 7-year-old and one 9-year-old were excluded from the audio–visual condition. For the audio–tactile condition, data collected from seven 7-year-olds, and one 9-year-old were excluded from the analysis. Finally, for the visual–tactile condition, data from four 7-year-olds, one 9-year-old, and one 13-year-old were excluded.To ensure that the estimated PSS shifts were representative of the participants’ responses, we selected age groups with the same number of trials and excluded individuals with poor Gaussian fits  and a negative bootstrap test may indicate a biased sample.We conducted three analyses. The purpose of the first analysis was to determine the age at which rapid temporal recalibration becomes significant. PSS shifts of each condition were compared against a hypothesized PSS shift of 0 ms, which represented no rapid temporal recalibration. These comparisons were conducted using two-tailed, one-sample t-tests and bootstrap hypothesis tests. To conduct the bootstrap tests, 1500 bootstrap samples were generated for each pairwise comparison by resampling individual PSS shifts with replacement. The mean PSS shift of each iteration was then compared against 0 ms. Statistical significance was determined using the formula:23, but it is unclear if a similar advantage can be observed across the different age groups. To investigate, PSS shifts of audio–tactile and visual–tactile pairings were evaluated against the PSS shifts of audio–visual pairings at the age groups common to all three datasets . As before, these comparisons were performed using two-tailed, independent-sample t-tests and bootstrap hypothesis tests. Similar bootstrap procedures were performed, with the exceptions that the test statistic was the difference in mean PSS shift and the comparison value was a mean difference of 0 ms.The second analysis aimed to evaluate the differences in PSS shifts among three cross-modal pairings at a given age. Adult observers experience larger audio–visual rapid temporal recalibration effects than audio–tactile or visual–tactile pairings25; however, it is unclear if this correlation applies to younger age groups and other cross-modal presentations. Studies have shown that younger children have a wider window in which they make cross-modal simultaneity judgments35, but the wider windows might result from factors independent of the larger PSS shifts, such as needing more practice, inattention, or fatigue. One-way ANOVAs were conducted on each type of cross-modal presentation to examine the effect of age on the width of the simultaneity window or PSS shift. Separate linear regression analyses were also performed within each age group for each cross-modal pairing, and this allowed us to test how well the PSS shifts were correlated with the width of the simultaneity window. Similar bootstrap procedures were performed, and the same sample selections were used for PSS shifts and the width of the simultaneity window. In the within-age-group analysis, regression slopes were estimated for each of the bootstrapped samples, after which the probability of samples with slopes greater or less than 0 was computed. Main effects were assessed using bootstrapped F ratios, and we estimated the p value by computing the probability of obtaining ratios smaller or equal to 1 .In the final analysis, we investigated the correlation between the widths of the simultaneity window and PSS shifts across and within age groups. Broader audio–visual temporal simultaneity windows have been linked to larger PSS shifts in adults


Primary outcomes included changes in body mass index standard deviation score (BMI SDS) and percentage above the International Obesity Task Force cut‐off for overweight (%IOTF‐25). Secondary outcomes included changes in sleep, physical activity, and eating behaviour. From pre‐ to posttreatment there was a statistically significant difference in change in both BMI SDS  and %IOTF‐25  between FBSFT and TAU groups. FBSFT participants achieved significant reductions in mean BMI SDS  and %IOTF‐25 , whereas in TAU nonsignificant changes were observed in BMI SDS  and %IOTF‐25 . More FBSFT participants (31.5%) had clinically meaningful BMI SDS reductions of ≥0.25 from pre‐ to posttreatment than in TAU . Regarding secondary outcomes, only changes in sleep timing differed significantly between groups. FBSFT improved weight‐related outcomes compared to TAU.To compare the effectiveness of family‐based behavioural social facilitation treatment (FBSFT) versus treatment as usual (TAU) in children with severe obesity. Parallel‐design, nonblinded, randomized controlled trial conducted at a Norwegian obesity outpatient clinic. Children aged 6–18 years referred to the clinic between 2014 and 2018 were invited to participate. Participants were randomly allocated using sequentially numbered, opaqued, sealed envelopes. FBSFT ( Family‐based behavioural treatment is recommended as an evidence‐based treatment for childhood obesity.Family‐based behavioural treatment delivered in research clinics, has been shown to yield clinically significant weight loss in children with obesity.Delivered at an obesity outpatient clinic, family‐based behavioural social facilitation treatment (FBSFT) improved weight‐related outcomes significantly more than treatment as usual (TAU) among children (ages 6–18 years) with severe obesity.Investigation of individual treatment response showed that significantly more children receiving FBSFT achieved a clinically meaningful body mass index standard deviation score reduction of ≥0.25 compared to children receiving TAU.The beneficial changes in weight outcomes exhibited in FBSFT compared to TAU were not explained by differences in sleep, physical activity, or eating behaviour.1Paediatric obesity is one of the major global health challenges of the 21st century. Effective treatment options are urgently needed, given the increasing prevalence of paediatric obesity, the risk of adverse health consequences, and the fact that obesity‐related risk factors track into adulthood.To date, various treatment options have been tested, including interventions focusing on lifestyle modification, as well as pharmacological therapy and bariatric surgery,Treatment programmes targeting multiple lifestyle behaviours whilst applying behavioural techniques in a family‐based approach have shown the most promise and are considered best practise in obesity treatment for children aged 6–17 years.Moreover, emerging data suggest that obesity risk is influenced by sleep patterns. Several aspects of sleep, including duration and timing, have been identified as contributors to the development and maintenance of childhood obesity.The aim of the present study was to compare the effectiveness of FBSFT to TAU in severe childhood obesity treatment delivered in a public healthcare setting. Outcome measures included BMI‐related metrics, sleep measures, physical activity, and eating behaviour. We hypothesized that FBSFT would yield greater improvements in BMI‐related metrics as well as sleep and eating behaviour, compared to TAU, with similar improvements in physical activity due to a comparable focus on this component in both treatment programmes.22.1The FABO study is a parallel‐design, nonblinded, randomized controlled trial (RCT), conducted at the Obesity Outpatient Clinic at Haukeland University Hospital, Bergen, Norway. All children aged 6–18 years) referred by their general practitioner to the clinic between February 2014 and October 2018 were invited to participate. Written informed consent was obtained after an initial clinic assessment. Participating families were randomized to either FBSFT (Arm A) or TAU (Arm B). Randomization was in 1:1 ratio, and sequentially numbered, opaque sealed envelopes were used to conceal the randomization sequence. Figure 8 years rClinicalTrials.gov (NCT02687516).The study was approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (number 2013/1300) and was registered on 2.22) or for obesity (≥30 kg/m2)One hundred fourteen children and adolescents  participated, with 59 participants in Arm A and 55 in Arm B. Inclusion criteria were BMI above the IOTF cut‐off for severe obesity  for paediatric obesity, which is the most documented approach in childhood obesity treatmentTraditionally, FBT is implemented in a mixed  format,Families assigned to TAU (Arm B), a lifestyle intervention targeting the child, were provided with a personalized plan for changing specific lifestyle behaviours and were advised to participate in monthly counselling sessions with their local healthcare nurse. TAU was delivered over the course of 12 months2.42): BMI standard deviation score (SDS) and percentage above the IOTF cut‐off for overweightHeight and weight were measured by trained assessors in clinic. The assessors were informed about study participation, but not treatment assignment. Height was measured to the nearest 0.1 cm with an electronic wall‐mounted seca 264 stadiometer (Seca), and weight was measured to the nearest 0.1 kg using a digital InBody720 scale (Biospace). Measurements were taken with participants wearing light indoor clothing only (without socks and shoes).2.5Sleep was objectively measured using an Actiwatch 2 (Philips Respironics). Actiwatch 2 devices are wrist‐worn accelerometers with a light sensor and an event marker, which record all uniaxial movement over 0.05G.Sleep statistics were calculated using Respironics Actiware software, version 6.0.9. The rest interval (time in bed) associated with the main sleep period in the 24‐h day was manually set, according to a standardized scoring protocol.2.5.1The mean sleep duration over 7 consecutive days was used in the analyses. For inclusion, participants completed recordings of at least 5 (out of 7) days, including at least three school nights and two weekend nights. At baseline 105 of 114 participant presented with valid sleep recordings, 96 with 7 nights, 7 with 6 nights and 2 with 5 nights. Posttreatment the numbers where 79 of 114 participants in total, 66 with 7 nights, 11 with 6 nights and 2 with 5 nights.2.5.2Sleep timing  was calculated as a 7‐day mean of the mid‐sleep time, i.e., the midpoint between sleep onset time and wake‐up time: (sleep onset time + sleep offset time)/2. For participants with only five or six nights of recordings, the mean of these nights was used. Furthermore, sleep onset and final wake‐up times were reported for additional information about sleep timing. Seen together, these parameters give valuable information about sleep hygiene. Sleep during daytime was not assessed in the study.2.6am and 9:00 pm) was objectively assessed using data from the same device (Actiwatch 2). Wrist‐worn accelerometers have been validated for measuring physical activity in children,Daytime physical activity , moderate (524–811 counts/30‐s epochs), and vigorous intensity (>812/30‐s epochs).2.72.7.1α coefficient for the 33‐item version was .76 for restrained, .87 for external and .96 for emotional eating at baseline. For the 20‐item version, the Cronbach's α coefficient was .79 for restrained, .73 for external and .85 for emotional eating at baseline. These indicate acceptable (>0.7) to excellent (>0.9) internal consistency for the three subscales in the current sample.The Dutch Eating Behaviour Questionnaire (DEBQ) is a measure of disturbed eating patterns in children and adolescents.2.8SD) for continuous variables, and as frequency and percentage for categorical variables. Baseline differences between FBSFT and TAU participants, and between children who completed the intervention and those who did not, were tested using t‐tests and χ2 tests of independence, with the significance level set as 0.05.Data were analysed with IBM SPSS version 26 (IBM Corp.) and Stata version 17 . Descriptive statistics are expressed as the mean and standard deviation (Linear mixed models were used to estimate and compare changes from pre‐ to posttreatment under the two treatment conditions. The mixed models included the treatment condition (FBSFT vs. TAU), time (baseline and posttreatment), and a treatment‐by‐time interaction, and were fitted with an unstructured residual covariance structure for all primary and secondary outcomes. Models were checked for heteroscedasticity and normality of residuals. Differences in means and 95% confidence intervals were computed from the fixed effect model parameter estimates. This analytical approach deviates from the original protocol,SD as denominator. An effect size is commonly interpreted as small (0.2), moderate (0.5) and large (0.8).Following the principle of intention to treat, all participants were included in the analyses, irrespective of missing data at any measurement points. Mixed models are not based on balanced data assumption and use all available data on each participant, thus accounting for missing data on a response variable. Under the ‘missing at random’ (MAR) assumption, these models provide unbiased estimates.2.8.1α level of .05, a power of 80%, and a correlation of .5 between measurement points, a sample size of 28–164 subjects would allow to detect moderate (Cohen's f = 0.25) to small (Cohen's f = 0.10)Power analysis was performed prior to the FABO trial based on two treatment groups (FBSFT and TAU) and three measurement points . For an 2.8.2Anthropometric data at baseline were available from all participants, including dropouts. Anthropometric data posttreatment were available for all completers. Approximately 90% of participants provided accelerometer and questionnaire data at baseline, whilst the percent decreased to 68% posttreatment.3p  > .05). The percentage of females in FBSFT and TAU groups was 61.0% and 56.4%, respectively (p = .618). Twenty‐two participants (19.3%) dropped out of the study: 12 (20.3%) from the FBSFT group and 10 (18.2%) from the TAU group (p = .771). There were no significant differences in age, sex, and BMI SDS between ‘treatment completers’ and ‘dropouts’ (p  > .05), although the latter group had higher %IOTF‐25 at baseline  (Table Participants' baseline characteristics in the FBSFT and TAU groups are presented in Tables 5) Table .3.1p < .001) and %IOTF‐25  between FBSFT and TAU , whilst changes in the TAU group were not statistically significant for BMI SDS or %IOTF‐25   of the participants in FBSFT and 13% (n = 7) in TAU, a significant difference (χ2(1) = 5.357, p = .021). Intervention effects are presented in Table Individual changes in BMI SDS of all participants in both groups are shown in Figure 3.2p = .037) between the FBSFT and TAU groups . For the percentage of time spent in MVPA we observed an overall significant reduction from pre‐ to posttreatment (p = .008), but no differences between treatment groups.There was a significant difference in changes in sleep timing  from pre‐ to posttreatment  is of similar magnitude as the findings from two recent Cochrane reviews on diet, physical activity and behavioural interventions.Narrowing the comparison to studies on standardized FBT, the pooled result of eight pioneer studies from Epstein et al. shows a BMI SDS change in FBT of −1.20 units at 6 months,In the present study, significantly more participants in the FBSFT group achieved a BMI SDS reduction of ≥0.25 (31.5% in FBSFT group compared to 13% in TAU group). Individual treatment response is an important outcome measure, in addition to mean changes.In this study, we presented BMI outcomes in terms of %IOTF‐25, in addition to BMI SDS. Changes in adiposity in children with severe obesity might be difficult to detect using BMI SDS, because large BMI differences corresponds to only small BMI SDS changes.The overall positive results in BMI outcomes with FBSFT versus TAU might be related to treatment content. In contrast to TAU, FBSFT is a structured cognitive behavioural approach that targets both children and parents.No significant differences for changes in sleep duration, physical activity, or eating behaviour during treatment were observed between the two groups. Analyses of sleep behaviour showed that changes in sleep timing were significantly different between the groups, with a small increase in mid‐sleep time from baseline to posttreatment in the FBSFT group, compared to a small decrease in the TAU group. However, the changes in mid‐sleep time from pre‐ to posttreatment were not statistically significant for either group separately. Therefore, it is unlikely that the observed changes between groups are clinically meaningful. To our knowledge, this study is the first to objectively measure sleep behaviour in school‐aged children receiving FBT for obesity.Physical activity is addressed similarly in FBSFT and TAU, therefore the nonsignificant between group difference in change was somewhat expected and in line with results from previous studies.The effects of multidisciplinary treatment for childhood obesity on eating behaviour in children with obesity has until recently been largely unknown.Altogether, the results from secondary outcomes  indicate that observed differences in change in weight outcomes cannot be explained by differences in the lifestyle behaviours measured.This study has several strengths and limitations. We used an RCT design which is given a high level of evidence for evaluation of treatment options. Because the average BMI SDS at baseline was relatively high, compared to similar RCTs,Our study also has some limitations. First, there are no data on energy intake. In the first year of the study, participants were asked to complete a 4‐day food record at baseline and posttreatment, but this was abandoned due to low compliance. It is plausible that the beneficial BMI outcomes in the FBSFT group are due to a reduced calorie intake.In conclusion, this study demonstrates that among children and adolescents with severe obesity, FBSFT delivered in a public healthcare setting has overall better treatment effects on BMI‐related outcomes, compared to 1 year of TAU. However, changes in the measured lifestyle behaviours were minimal, thus indicating that observed differences in weight outcomes cannot be explained by differences in the included lifestyle behaviours. Considering these findings, expanding access to FBSFT for children and adolescents with severe obesity is an important next step in the treatment of childhood obesity. Alternatively, it may be beneficial to include FBSFT in a stepped approach offered to individuals who do not respond to standard lifestyle treatment.The authors declare no conflict of interest.Hanna F. Skjåkødegård, Yngvild S. Danielsen, Mathieu Roelants, Rachel P. K. Conlon, Denise E. Wilfley and Petur B. Juliusson conceived and designed the study. Hanna F. Skjåkødegård, Yngvild S. Danielsen and Petur B. Juliusson collected the data. Sven J. G. Olsson developed the tailor‐made software for processing physical activity data. Hanna F. Skjåkødegård, Yngvild S. Danielsen, Bente Frisk and Sigurd W. Hystad performed statistical analyses. Hanna F. Skjåkødegård wrote the paper in consultation with Yngvild S. Danielsen, Mathieu Roelants, Bente Frisk, Sigurd W. Hystad, Sven J. G. Olsson, Rachel P. K. Conlon, Denise E. Wilfley and Petur B. Juliusson. All authors discussed the results and contributed to the final manuscript.TABLE S1 Comparison of baseline characteristics between participants who completed the study and dropoutsClick here for additional data file.


To explore the appropriate dosage of levothyroxine treatment for congenital hypothyroidism patients with different thyroid stimulating hormone (TSH) levels.A total of 116 patients, who were regularly followed-up in our endocrine clinic from January 2010 to December 2020, were divided into four groups based on their thyroid function  < 6.6 pmol/L, and group D: TSH > 4.6 mIU/L and < 20 mIU/L with FT4 > 6.6 pmol/L). The initial dosage of levothyroxine was individualized for each patient based on their TSH level and then adjusted according to their thyroid function at every follow-up time point. The levothyroxine dosage at each time point was compared between the groups, and thyroid function and physical and neurological development after treatment were also compared.p < 0.05), development was within the normal range for all groups.After individualized dosage adjustment, all patients achieved normal thyroid function. Although there were statistical differences in neurological development between the four groups (An individualized levothyroxine dosage can provide the same therapeutic effect compared to the recommended dosage. This strategy may also reduce the risk of a drug overdose. Congenital hypothyroidism (CH) is a common endocrine disease with an incidence of 1:2000 to 1:4000 in newborns; it is one of the most common preventable causes of intellectual disability worldwide [Consensus statement on the diagnosis and management of congenital hypothyroidism issued by the Subspecialty Group of Endocrinologic, Hereditary and Metabolic Diseases, The Society of Pediatrics, Chinese Medical Association [Patients who were regularly followed-up in our endocrine clinic between January 2010 and December 2020 were enrolled in this study. All legal guardians were informed and signed the informed consent. This study was approved by the research ethics committee of The First People’s Hospital of Yunnan Province. The inclusion criteria were as follows: (1) patients who were diagnosed with CH based on the ociation , (2) a gPatients were categorized into four groups based on their thyroid function at the time of diagnosis. The patient groups included group A: TSH level > 100 mIU/L, group B: TSH ≥ 20 and < 100, group C: TSH > 4.6 mIU/L and < 20 mIU/L with free thyroxine (FT4) < 6.6 pmol/L, and group D: TSH > 4.6 mIU/L and < 20 mIU/L with FT4 > 6.6 pmol/L .Group A patients, whose TSH level was ≥100 mIU/L, received 10 μg/kg·d as the initial levothyroxine dosage, which was at the lower limit of the recommended dosage. Group B patients, who had a moderately elevated TSH level (20 mIU/L ≤ TSH < 100 mIU/L), were administered a dosage of 5–8 μg/kg·d. Groups C and D, who had a mildly elevated TSH level (4.6 mIU/L < TSH < 20 mIU/L), were administered 3–4 μg/kg·d levothyroxine. However, as the specification of levothyroxine is 50 μg per tablet, for dividing convenience, it is usually prescribed as “1 ½ Tab or 1 2/3 Tab … .”Maternal thyroid function was tested through antenatal care, and the clinicians would inquire about the results once their babies were confirmed. The initial levothyroxine dosage information for the four groups was collected. TSH levels of the four groups were analyzed 1 month after treatment to determine if they had reached the normal range. In addition, physical and neurological development were evaluated and compared between groups at the age of 1, 2, and 3 years.Blood samples of patients were collected, and serum was isolated and transferred to our laboratory for thyroid function tests. A time-resolved fluorometry kit  was adopted for the TSH test following the manufacturer’s instructions; while T4, T3, FT4, and FT3 were tested using a time-resolved immunofluorescence assay (TRFIA) kit  and analyzed using an immunofluorescence analyzer , strictly following the manufacturer’s instructions.Growth monitoring was conducted by qualified clinicians at the age of 1, 2, and 3 years; height, weight, and head circumference were also documented. The Gesell Development Scale score was adopted for neurological development assessment and performed at the age of 1, 2, and 3 years, including evaluating gross and fine motor development, adaptability, and sociability.P-value < 0.05 was considered statistically significant.Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and assessed by t-test, whereas skewed-distribution variables were summarized as medians and interquartile ranges and assessed using the Mann-Whitney U test. Categorical variables were demonstrated by frequencies and proportions (%) and analyzed by Pearson’s chi-square test or Fisher’s exact test. SPSS 26.0 software  was employed for statistical processing, and a two-tailed p > 0.05). However, the differences in abnormal maternal thyroid function rate  and the initial TSH screening levels among the four groups were significant  ; all levels gradually increased and then stabilized at the normal range at the follow-up time points  Fig. a. The FTnts Fig. b.Fig. 2Tp > 0.05). While a statistical difference in neurological development among the four groups was observed (p < 0.05), the Gesell Developmental Scores were all within the normal range  in group A, one patient (3.8%) in group B, four patients (23.5%) in group C, and three patients (8.3%) in group D who did not reach the normal TSH level. Furthermore, there were eight patients (21.6%) in group A, one patient (3.8%) in group B, and one patient (2.7%) in group D who had an FT4 level beyond the upper limit of normal values at 1 month following treatment. At the follow-up time, the dosage of levothyroxine for each patient was adjusted according to thyroid function, and their thyroid function eventually returned to a normal range. Our study suggests that to reduce the risk of levothyroxine overdose, individualized levothyroxine dosage should be adjusted. Currently, there is no exact dosage for patients with different TSH levels; however, for most patients with mildly elevated TSH, low-dose thyroxine could restore their TSH level to normal 1 month after treatment. It is critical to follow up regularly during treatment to monitor thyroid function and adjust the thyroxine dosage to maintain thyroid function within the normal range.n = 45) treated with a high dose of levothyroxine (10–15 μg/kg·d) developed hyperthyroidism [Research performed by Soliman et al. (2012) revealed that 25% of the patients  could also cause FT4 levels to return to normal within 16 d .The patients enrolled in our study were diagnosed at around 1 month of life; this delay was due to regional factors and cultural disparities. Our hospital’s province is a remote and backward area in China, and resources are limited. After the initial screening was positive, many parents were notified via text message. However, many family members did not pay much attention to this message because of their limited medical knowledge, and their babies were usually asymptomatic. Thus, the parents and patients did not return for follow-up in time. Chinese women have the custom of confinement and are reluctant to go outside, unless necessary, during confinement.p > 0.05); however, we could not determine whether these results were due to the small sample size. Therefore, further prospective, multicenter control studies are required. Although there was a statistical difference in the Gesell Development Scale score between the groups (p < 0.05), they were all within the normal range. As reported in the literature, if CH patients are treated within 1 month of birth, they can gain a normal IQ; the longer hypothyroidism goes undetected and untreated, the lower the IQ achieved [All patients were treated within 2 months of birth in the present study. The levothyroxine dosage was adjusted to maintain their thyroid function at each follow-up, which remained within the normal range. There was no difference in physical and neurological development between the treated groups (achieved , 18–20.In our study, even for patients with significantly elevated TSH levels, the initial individualized dosage of levothyroxine received was still lower than recommended; yet, they eventually achieved normal thyroid function with no physical or neurological developmental impairment. In summary, the key factor in the successful treatment of CH is early detection and intervention, rather than a high levothyroxine dosage.The present study has limitations, including that it was retrospective and the sample size was small, which may lead to unreliable conclusions. Furthermore, we only enrolled term infants, whereas the prevalence of CH is higher in preterm infants than in term neonates. Thus, our study was not universally representative. Given the importance of precise levothyroxine treatment, further prospective studies are required.Compared to the recommended dosage, an individualized levothyroxine dosage can provide the same therapeutic effect. And such strategy might reduce the risk of a drug overdose.


Apis laboriosa is the largest honeybee that lives mainly on cliff faces, with strong migratory ability. In this study, we firstly sequenced and assembled two complete mitochondrial genomes of A. laboriosa isolated from two distant locations in China (Chongqing and Shangri‐La regions). Combined with the published mitochondrial genome of A. laboriosa from Nepal, comparative genomic analyses were conducted to gain insight into the genetic diversity of giant honeybees from different geographical distributions. The mitochondrial genomes of A. laboriosa from Chongqing and Shangri‐La regions were 15,579 and 15,683 bp in length, respectively, both larger than that from Nepal with the length of 15,510 bp. Three mitochondrial genomes all harbor 37 common genes and present the same AT bias and the frequency of codon usage. However, the fragments including COX1, SSUrRNA, LSUrRNA, and the AT‐rich region of the mitochondrial genome from Shangri‐La region demonstrate distinctive insertions and deletions compared to those from Chongqing and Nepal regions. Phylogenetic trees of mitochondrial genomes show that A. laboriosa from Chongqing is most closely related to that from Nepal, rather than to Shangri‐La. Genetic distance between Shangri‐La and Chongqing or Nepal was even larger than that between the various subspecies of Apis mellifera. Overall, these results unmark that A. laboriosa in different geographical distributions can exhibit high genetic diversity at the mitochondrial genomic level, and therein, A. laboriosa from Shangri‐La may be the subspecies. All these studies will contribute to our understanding of the geographical distribution and genetic differentiation of black giant honeybee in Asian region. Apis laboriosa with different geographical distributions have rich genetic diversity at the mitochondrial level, where Apis laboriosa from Shangri‐La even reached the subspecies differentiation level. Meanwhile, our study also found that geographical distance is not the main reason for genetic differentiation, which provides a reference for further expanding research on the geographical distribution of Apis laboriosa and analyzing their mechanisms of genetic differentiation.Overall, this study demonstrates that  The details of the primer sequences used in this study were summarized in Table 2O. The following PCR procedure was used: a predenaturation step at 94°C for 3 min, followed by 45 cycles of 10 s at 98°C, annealing for 15 s at 58–60°C, then 5 min at 68 °C, and final extension for 10 min at 68°C. After the DNA electrophoresis, the PCR target products were retrieved and sent to Sangon Biotech for DNA sequencing.One worker from each colony was randomly selected and total DNA was extracted from the head and chest muscles by CTAB (cetyltrimethylammonium bromide) method . The calibration of gene annotation was performed using Geneious v4.8.4 [32] . The two mitochondrial genomes obtained in this study were deposited to GenBank database with accession OP764682 and OP764683.SeqMan was used to assemble the sequencing results and then achieved complete mitochondrial genomes were annotated using MITOS , Apis mellifera , Apis laboriosa (NC_036155), Apis dorsata (NC_037709), Apis andreniformis (NC_039709), Apis florea (KC170303), and Bombus ignitus (DQ870926). After alignment with the complete mitochondrial genome sequences and 13 concatenated nucleotide sequences of PCGs (protein‐coding genes), phylogenetic trees were constructed using IQTREE, MEGA, and MrBayes 3.2 software , 84.8% (Shangri‐La), and 85% , respectively, indicative of obvious AT preference. All three mitochondrial genomes harboring a total of 37 genes, including 13 protein‐coding genes , 22 tRNA genes, 2 rRNA genes, and 1 AT‐rich region  of 13 protein‐coding genes (PCGs) in the mitochondrial genomes of 1 Figure , indicat3.3COX1, the small subunits of rRNA (SSUrRNA), large subunits of rRNA (LSUrRNA), and the AT‐rich region based on the alignment of multiple sequences  was smaller than Shangri‐La and Chongqing (0.0201), or Shangri‐La and Nepal (0.0200), supporting the same conclusion that the A. laboriosa from Chongqing and Nepal have a closer relationship , as shown in Figure A. laboriosa from Shangri‐La may be a geographical subspecies.Two phylogenetic trees based on the mitochondrial genomes and 13 protein‐coding genes were constructed using three methods including IQTREE, MEGA, and MrBayes Figure . Both trp Figure . Signifi4A. laboriosa, is usually found in the Himalayas and Hengduan Mountains at an altitude of about 1200–3500 m  is significantly smaller than the distance between Chongqing and Nepal (2312 km), but the number of SNPs and indel loci between the m Smith, . HoweverA. laboriosa in different geographical distributions exhibits high genetic diversity at the mitochondrial genomic level, and therein A. laboriosa from Shangri‐La may approach the subspecies level of differentiation. Although more evidence is still needed to support these inferences.In conclusion, our results unmark that Xiang‐You Tang: Writing – original draft . Yu‐Xin Yao: Formal analysis . Yao‐Hui Li: Formal analysis . Hua‐Li Song: Methodology . Rui Luo: Methodology . Peng Shi: Resources . Ze‐Yang Zhou: Supervision . Jin‐Shan Xu: Project administration .We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.