This study aimed to assess the relationship between immune response adverse events (irAEs) and treatment efficacy in patients with extensive disease small cell lung cancer (ED‐SCLC).We retrospectively evaluated the clinical effects in 40 ED‐SCLC patients who had received immune‐checkpoint inhibitors (ICIs), platinum agents, and etoposide between September 2019 and September 2021. We identified and compared patients belonging to two groups: irAE and non‐irAE.p = 0.0108). However, the median overall survival (OS) was similar between irAE and non‐irAE groups . Seven (46.7%) in the irAE group and 20 (80%) in the non‐irAE group received sequential therapy. The median OS was prolonged in patients who received first‐ and second‐line therapy than in those who received first‐line therapy alone . Grade ≧ 3 irAEs occurred in five (12.5%) patients. Among them, grade 5 irAEs were observed in two patients, including exacerbation of polymyositis and pulmonary arterial embolism.Fifteen patients experienced irAEs, and 25 did not. The median progression‐free survival in patients with irAE was longer than that in patients without irAE (12.6 months [95% CI: 6.3–19.3 months] vs. 7.2 months [95% CI: 5.8–7.9 months], In this study, the development of irAEs did not affect OS in patients with ED‐SCLC who received platinum‐based agents, etoposide, or ICI therapy. We determined that managing irAEs and administering first‐ and second‐line therapies could contribute to prolonged OS. In extensive disease small cell lung cancer, immune‐related adverse events were less associated with prolonged overall survival. Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers. About two‐thirds of SCLC patients have extensive disease (ED) at diagnosis. Two clinical trials, the IMpower133 and CASPIAN studies, have demonstrated survival benefits in patients with ED‐SCLC.In the IMpower133 trial, atezolizumab alongside carboplatin and etoposide therapy demonstrated significantly extended survival  than carboplatin and etoposide therapy .Based on these findings, immune checkpoint inhibitors (ICIs), platinum agents, and etoposide are considered mainstream therapies. However, the survival benefits of adding ICIs to cytotoxic agents in ED‐SCLC are numerically lower than those in non‐small cell lung cancer (NSCLC).This study aimed to retrospectively elucidate the relationship between irAEs, clinical efficacy, and survival benefits.This retrospective study included ED‐SCLC patients who received first‐line therapy between September 2019 and September 2021 at two institutions in Hokkaido, Japan .The eligibility criteria were as follows: pathologically diagnosed ED‐SCLC or small recurrence after chemoradiation therapy. All patients underwent systematic evaluation using computed tomography (CT), brain magnetic resonance imaging (MRI), and bone scintigraphy or 18F‐fluorodeoxyglucose positron emission tomography before first‐line therapy. Patients who relapsed more than 6 months after the last cytotoxic agent administration were enrolled. IrAEs were assessed between the day of the initial ICI administration and six months after the final ICI administration.This study was approved by the Institutional Review Board of Teine Keijinkai Hospital (no. 2‐022484‐00). All procedures complied with the ethical standards of the institutional and/or national research committee and the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards. As this was a retrospective study, the requirement for informed consent was waived.t‐test for categorical and continuous variables, respectively. The chi‐square test was used to compare the proportions of patients based on their characteristics . PFS was defined as the time interval between initial treatment administration and disease progression or death from any cause. OS was defined as the time interval between the initial treatment administration and any cause of death or censored on the date of the last follow‐up. Patients without documented clinical or radiographic disease progression or those still alive were censored on the date of the last follow‐up.Statistical analyses were performed using Fisher's exact test and Welch's p‐values were two‐tailed, and the threshold for statistical significance was set at p < 0.05.PFS and OS were evaluated using the Kaplan–Meier method and compared using a two‐sided log‐rank test. Univariate analyses using Cox proportional hazards modeling were performed to determine the correlation between OS and various factors. Hazard ratio (HR) and 95% CI were estimated using the Cox proportional hazards regression model. All All statistical analyses were performed using EZR version 1.40 .A total of 88 patients were enrolled in this study between September 2019 and September 2022  patients received sequential therapy. Amrubicin was the most commonly administered drug (51.9%). Two patients received ICIs beyond PD after local therapy because the progressive lesion was in a single organ.The response and disease control rates in the overall population were 87.5 and 97.5%, respectively. The response rates for irAEs and non‐irAEs were 93.3 and 84%, respectively Table . The medp = 0.0108). The median overall survival was 27.6 months (95% CI: 15.4–NA) in irAE and 24.9 months (95% CI: 13.7–NA) in non‐irAE (p = 0.268)  than the non‐irAE group   Figure . Regardi) Figure .In the population that received subsequent therapy, excluding non‐PD patients, the median OS was 27.6 months (95% CI: 19.2–NA). Conversely, the median OS was 6.6 months (95% CI: 0.3–NA) in patients who did not receive subsequent therapy Figure .We performed univariate analysis using Cox proportional hazards modeling for OS Table . Due to Treatment‐related AEs are shown in Table The most common irAE was skin disorders (20%). Grade 4 irAEs of diabetic ketoacidosis and sclerosing cholangitis occurred in one patient (2.5%). Grade 5 irAEs of connective tissue disease exacerbation occurred in one patient (2.5%).To our knowledge, this is the first study to report that irAEs caused by durvalumab and atezolizumab affected the survival of patients with ED‐SCLC. In this retrospective observational study for ED‐SCLC, the median PFS was 12.6 months (95% CI: 6.3–19.3 months) in the irAE group, and 7.2 months (95% CI: 5.8–7.9 months) in the non‐irAE group, with there being a significant difference. In addition, the median OS was 27.6 months (95% CI: 15.4–NA) and 24.9 months (95% CI: 13.7–NA) in the irAE and non‐irAE groups, respectively. This study aimed to elucidate the relationship between irAEs and treatment efficacy. The reinvigorated immune cells due to ICI treatment attack not only tumor cells but also normal tissue, and it has been previously reported that the occurrence of irAEs could lead to a better response.2 and 12.9 months in the CASPIAN studies.In this study, the median OS of 27.6 months (range: 19.2–NA) in the population was better than clinical trials; 12.3 months in the IMpower133The longer OS observed in our study was thought to be due to the better management of irAEs and the ratio of subsequent therapies. In this study, 40 patients received a platinum‐based agent, etoposide, ICI, atezolizumab, or durvalumab, and IrAEs developed in 15 patients (37.5%). A total of 35 patients discontinued first‐line treatment because of PD and irAEs. Of these patients, 27 (77.1%) received second‐line therapy. A more significant proportion of patients received subsequent therapy in this study than in clinical trials: 50.2% in the IMpower 133 studyThese findings suggest that managing irAEs in ED‐SCLC is more difficult in practice than in previous reports.Although both irAE and non‐irAE groups underwent a median of four courses of maintenance therapy, the median PFS was longer in the irAE group than in the non‐irAE group. Regarding the onset of irAEs, the median time from the start of ICI therapy to the onset of irAEs was 4.9 months (range 0.4–20.1). It appears that in some cases the tumor continued to shrink despite discontinuation of ICI therapy for the successful management of irAEs. We believe those reasons contributed to prolonged PFS.In this study, recurrent SCLC patients were defined as patients who had received prior chemoradiotherapy for limited‐stage SCLC and had a treatment‐free interval of at least 6 months since last chemoradiotherapy, according to the protocol of the IMpower 133 study. Three patients were enrolled and grade 2 irAEs that were manageable had developed in one patient. Median OS was 29.9 months which was longer than in all patients. Although the number of patients with recurrent SCLC was small, it appear that patients with recurrent SCLC were tolerant to chemotherapy along with ICI therapy.Regarding NLR, due to lack of data about patients with ED small, we established a threshold value of 5 on the basis of previously published data.Although no phase 3 study previously evaluated the efficacy of amrubicin in combination with platinum doublet and ICI therapy in ED‐SCLC, a study reported a median OS of 11.7 months in sensitive relapse and 7.3 months in refractory relapse after treatment with atezolizumab plus carboplatin and etoposide.Our study had some limitations. First, it was a retrospective study with a small number of patients. Physicians at each institution selected eligible patients. For example, in one institution, patients who demonstrated positivity for antibodies, such as collagen vascular disease, without physical findings, such as seropositivity, did not receive ICI therapy plus chemotherapy as the first‐line treatment. In addition, due to the limited number of cases, we could not perform a multivariate analysis, and a larger cohort study is therefore needed.Second, the skills required to identify irAEs depend on the physicians. The assessment and immediate action of irAEs are important for subsequent therapy. However, there is a possibility that physicians could not immediately identify irAEs, and the ratio of patients receiving therapy was low for irAEs. These biases and differences among physicians may have affected the results of our study; however, we believe that our results are valuable in practice.In conclusion, the development of irAEs did not affect the OS in ED‐SCLC patients who received platinum agents, etoposide, or ICI therapy. Immediate management of irAEs could be attributed to the increased ratio of subsequent therapy and prolonged OS.Keiki Yokoo, Yauo Kitamura, Gen Yamada developed the concept, designed the study, interpreted the results, and drafted the manuscript. All authors collected the data and approved the results and conclusions.The authors have no conflicts of interest to declare.Figure S1. Kaplan–Meier curves of overall survival of patients with NLR <5 or NLR ≥5. ICI, immune checkpoint inhibitor; M, month; OS, overall survival; PFS, progression‐free survival.Click here for additional data file.Table S1. Univariate analyses of overall survival.Click here for additional data file.


Patients with advanced hepatocellular carcinoma (HCC) and poor liver function lack effective systemic therapies. Low‐energy electromagnetic fields (EMFs) can influence cell biological processes via non‐thermal effects and may represent a new treatment option.This single‐site feasibility trial enrolled patients with advanced HCC, Child‐Pugh A and B, Eastern Cooperative Oncology Group 0–2. Patients underwent 90‐min amplitude‐modulated EMF exposure procedures every 2–4 weeks, using the AutEMdev (Autem Therapeutics). Patients could also receive standard care. The primary endpoints were safety and the identification of hemodynamic variability patterns. Exploratory endpoints included health‐related quality of life (HRQoL), overall survival (OS). and objective response rate (ORR) using RECIST v1.1.Sixty‐six patients with advanced HCC received 539 AutEMdev procedures . No serious adverse events occurred during procedures. Self‐limiting grade 1 somnolence occurred in 78.7% of patients. Hemodynamic variability during EMF exposure was associated with specific amplitude‐modulation frequencies. HRQoL was maintained or improved among patients remaining on treatment. Median OS was 11.3 months  overall . ORR was 24.3% .AutEMdev EMF exposure has an excellent safety profile in patients with advanced HCC. Hemodynamic alterations at personalized frequencies may represent a surrogate of anti‐tumor efficacy. NCT01686412. Ions and molecules with a high electrical dipole moment are susceptible to frequency‐dependent interactions with EMFs.Low‐energy EMFs can also cause alterations in hemodynamic regulation. Although the underlying physiology is unclear, this may offer a route toward identification of potentially beneficial anti‐cancer effects. Heart rate variability and other beat‐to‐beat parameters can be analyzed using non‐linear computing methods as indicators of alterations in hemodynamic regulation during EMF exposure.We conducted an exploratory study of the AutEMdev in patients with advanced HCC and healthy controls. The study aimed to assess safety and feasibility, and to explore hemodynamic variability parameters as a means of personalizing EMF exposure.2This was a prospective, open‐label study of the safety and feasibility of intrabuccal EMF exposure using the AutEMdev, with exploratory assessment of hemodynamic regulation alterations. The study had two sequential parts, both conducted in patients with HCC and healthy volunteers Figure . Amplitu2.1Eligible patients were aged at least 18 years and had advanced HCC, defined as unresectable locally advanced or metastatic HCC, confirmed by histology or clinical presentation according to the American Association for the Study of Liver Diseases criteria.The historical control cohort was non‐interventional and retrospective, comprising patients with unresectable HCC identified from the Hospital Sírio‐Libanês Oncology Department electronic medical records using the keyword “liver cancer.” Patients had to have a diagnosis of advanced HCC after loco‐regional therapy, have been recommended for systemic therapy, or be receiving best supportive care.2.2Participants were exposed to low‐energy radio frequency EMFs using the AutEMdev (Autem Therapeutics). The AutEMdev is an autonomous high‐precision EMF generator designed to execute and control systemic exposure to low‐energy radio frequency EMFs with a carrier frequency of 27.12 MHz and sinusoidal amplitude modulation at frequencies ranging from 10 Hz to 20 kHz. EMF exposures via AutEMdev are within the safety levels published by the International Commission on Non‐Ionizing Radiation Protection.2.3Patients with HCC were required to complete the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ–C30)2.4Patients were assigned to at least one 90‐min outpatient AutEMdev exposure procedure. Healthy volunteers were assigned to one exposure procedure only. Patients were invited to repeat procedures every 2–4 weeks if the patient or physician perceived quality of life improvement or objective tumor response. Patients could continue until withdrawal of consent or deterioration in quality of life . Patients could receive AutEMdev as a monotherapy or in combination with any conventional treatment approved by the Brazilian health regulatory agency, at any time. All patients were allowed to maintain any supportive care.Exposure procedures were conducted with continuous assistance from healthcare professionals. Participants were placed in the supine position wearing a comfortable outfit in a quiet, low‐light ambiance to allow hemodynamic parameters to stabilize. Pressure cuffs were positioned, and the spoon‐shaped AutEMdev antenna was placed over the patient's tongue Figure . After 12.5All participants underwent baseline hemodynamic recording without EMF exposure for 10 min, followed by hemodynamic recording with EMF exposure for 60 min . Adverse events were not systematically monitored at other times.Exploratory patient‐reported quality‐of‐life endpoints included the proportion of patients with improvement and worsening and the time to deterioration in global health status, functional scale, and symptom scale (defined as a ≥10% change in EORTC QLQ–C30 score from baseline). Median overall survival (OS) in the intention‐to‐treat population was an exploratory safety endpoint, and was compared with OS in the historical control cohort.No tumor assessments or clinical laboratory analyses were performed as part of the study. Routine results from radiological and laboratory assessments of patients were used  for ad hoc analyses of objective response rate using Response Evaluation Criteria in Solid Tumors (RECIST) v1.1,2.7Established hemodynamic time series algorithms were used to ensure stability and reproducibility.2.8α = 0.05). Stratification factors included Child–Pugh classification, albumin–bilirubin (ALBI) score and line of treatment.A minimum sample size of 45 patients with advanced HCC and 45 healthy controls was planned to study hemodynamic regulation. Statistical procedures were non‐parametric: Mann–Whitney U test and/or Kruskal–Wallis analysis of variance (ANOVA) for independent distributions; and Wilcoxon test and/or Friedman ANOVA for related distributions. EORTC QLQ–C30 analysis was performed according to the manual.2.9ClinicalTrials.gov before enrolment began (NCT01686412).Hospital Sírio‐Libanês was the only study site. All participants gave written informed consent before enrolment. The trial was conducted under the principles of the Declaration of Helsinki and the protocol was reviewed and approved by the institutional review board and ethics committee of Hospital Sírio‐Libanês and by the Comissão Nacional de Ética em Pesquisa. After completion of planned enrolment, the Hospital Sírio‐Libanês ethics committee approved an ongoing compassionate access program following the same protocol with an amended consent form. The trial was registered at 33.1From March 2018 to March 2019, 47 patients with advanced HCC and 51 healthy volunteers were enrolled in parts 1 and 2 of the study Figure . From ApThe study population comprised 66 patients and 51 healthy volunteers, who all underwent at least one AutEMdev exposure procedure  and 51 in healthy volunteers . All healthy volunteers tolerated the procedure. Two excluded patients were unable to tolerate decubitus during the pre‐exposure resting period (which was therefore unrelated to EMF exposure). No serious adverse events were reported during procedures. CTCAE grade 1 somnolence was the only adverse event, occurring in 52/66 patients (78.8%) and in 25/51 healthy volunteers (49.0%). This was characterized by mild but more than usual drowsiness or sleepiness, started within minutes of exposure initiation and ended spontaneously upon completion. All participants were able to resume normal activities after discharge.3.35 heartbeats) from the 26 patients in part 1 . The technique for identifying hemodynamic signals of interest was applied to 13 patients in part 1 and all 40 patients in part 2. The frequency of variation in hemodynamic signals of interest ranged from 0.9 to 5.2 mHz. Hemodynamic signals of interest occurred more frequently in patients than in healthy volunteers  and during exposure than non‐exposure in both patients and healthy volunteers  . In a detrended fluctuation analysisThe hemodynamic analysis included 134.3 h of continuous recording (5 × 10) Figure . Within 3.4p < 0.0001)  to the second, third, and fourth exposure, the majority of patients had improved or stable EORTC QLQ–C30 scores Table . An incr) Figure . Median ) Figure .3.5N = 66) was significantly longer than in the in the historical cohort  and 6.7 months  during study combination therapy (n = 18).During follow‐up, 47/66 patients 71.2%) died from cancer. The median OS of 11.3 months  with evaluable scans, the objective response rate by RECIST 1.1 was 6/19 (32%) for combination therapy and 3/18 (17%) for monotherapy, with complete responses in 1/19 (5%) and 2/18 (11%) patients, respectively (one with Child–Pugh class B) , combination with systemic treatments and previous lines of therapy. No serious adverse events were reported in patients or healthy volunteers during any EMF exposure procedure. Quality of life was maintained among patients undergoing repeated exposures every 2–4 weeks. The extended OS compared with a historical control cohort indicated that AutEMdev EMF exposure was not harmful, and may suggest potential efficacy when combined with the radiological responses observed in a minority of patients.AutEMdev EMF exposure caused subtle but reproducible alterations in hemodynamic regulation, as assessed using beat‐to‐beat heart rate variability measurements.Mild, self‐limiting somnolence was a common adverse event in individuals receiving AutEMdev EMF, consistent with previous findings.OS was similar in patients receiving AutEMdev EMF monotherapy and combination therapy, including first‐line EMF. A trend toward increased duration of sorafenib treatment was observed among patients receiving combination therapy compared with pre‐enrolment. Furthermore, complete and partial RECIST v1.1 responses were observed in patients receiving EMF as a monotherapy and in combination with tyrosine kinase inhibitors. These exploratory findings warrant future investigation of the AutEMdev using personalized treatment frequencies in a prospective randomized‐controlled efficacy study.Limitations of this study include the open‐label, single‐site design, and lack of a sham exposure procedure. This spared patients from placebo interventions and ensured consistency. Safety was only monitored during procedures, and did not include liver function tests. Few patients received PD(L)‐1 inhibitors because these were not approved in Brazil when the study began. Sources of significant potential bias included: wide eligibility criteria, selection of patients willing and able to participate; patients' freedom to repeat exposures; systemic treatment flexibility; and heterogeneity in the historical control arm. Another limitation was that the study did not aim to investigate the potential relationship between hemodynamic signals of interest and clinical efficacy or somnolence side‐effects. Strengths of the study include the blinded independent radiographic review, the evaluation of OS and the use of a validated patient‐reported outcome.In conclusion, EMF treatment with the AutEMdev had an excellent safety profile in patients with advanced HCC, as both monotherapy and combination therapy. There was evidence for maintained quality of life among patients who remained on treatment. Hemodynamic variability during exposure may provide a biological surrogate for identification of optimal amplitude‐modulation frequencies in each patient. Whether these personalized frequencies are also optimal for potential anti‐tumor effects remains to be established. This study was not designed to assess efficacy, but the positive exploratory trends warrant further investigation.Fernanda Capareli: Investigation (lead). Frederico Costa: Investigation  and Methodology . Jack Tuszynski: Methodology . Micelange Carvalho de Sousa: Investigation . Yone de Camargo Setogute: Investigation . Pablo Diego Santana: Investigation . Luciana Carvalho: Investigation . Elizabeth Santana dos Santos: Investigation . Brenda Gumz: Investigation . Jorge Sabbaga: Investigation . Tiago Bianc de Castria: Investigation . Denis Leonardo Jardim: Investigation . Daniela de Freitas: Investigation . Natally Horvat: Data curation . Regis França Otaviano: Data curation . Leonardo Testagrossa: Data curation . Tiago Costa: Data curation . Tatiana Zanesco: Investigation . Antonio Francisco Iemma: Data curation . Ghassan K Abou‐Alfa: Writing – review and editing (lead).Hospital Sírio‐Libanês sponsored this trial and provided the non‐invasive hemodynamic device. The study was part‐funded by National Institutes of Health/National Cancer Institute Cancer Center Support Grant P30 CA008748 to Natally Horvat. The AutEMdev was provided by Autem Therapeutics under a research development agreement with Hospital Sírio‐Libanês. Medical writing support was provided by Oxford PharmaGenesis, Oxford, UK, funded by Autem Therapeutics. Clinical records were reviewed by an independent auditor from Emergo  and radiological review was conducted by an independent blinded radiologist from Charité–Universitätsmedizin Berlin , funded by Autem Therapeutics.TBC: Honoraria, consultancy fees or research funding from AstraZeneca, Bayer, Eli Lilly, Genentech/Roche, Bristol Myers Squibb, Merck and Merck Sharp & Dohme. FC, AFI, and JA: Stock in and honoraria from Autem Therapeutics and patents relating to the AutEMdev. YdCS: Honoraria from Bayer, Merck and Novartis and independent contractor at Hospital Sirio‐Libanês. GKA‐A: Institutional research support from Arcus, Astra Zeneca, BioNtech, BMS, Celgene, Flatiron, Genentech/Roche, Genoscience, Incyte, Polaris, Puma, QED, Silenseed, Yiviva; consulting fees from Adicet, Alnylam, Astra Zeneca, Autem, Beigene, Berry Genomics, Boehringer Ingelheim, Celgene, Cend, CytomX, Eisai, Eli Lilly, Exelixis, Flatiron, Genentech/Roche, Genoscience, Helio, Helsinn, Incyte, Ipsen, Merck, Nerviano, Newbridge, Novartis, QED, Redhill, Rafael, Servier, Silenseed, Sobi, Vector, Yiviva; registered patent: PCT/US2014/031545 filed on March 24, 2014, and priority application Serial No.: 61/804,907; Filed: March 25, 2013. DLJ honoraria, consultancy fees or research funding from AstraZeneca, Bayer, Janssen, Astellas, Genentech/Roche, Bristol Myers Squibb, Merck and Merck Sharp & Dohme.Data S1.Click here for additional data file.


Upper tract urothelial carcinoma (UTUC) is the malignant transformation of urothelial cells, from the renal calyces to the ureteral orifices. While the benefits of minimally invasive nephroureterectomy over their open counterpart have been established, the optimal technique remains a debate. We aimed to assess current evidence in the literature and compare outcomes between robotic‐assisted (RANU) and laparoscopic nephroureterectomy (LNU).Review Manager 5.A systematic review of the literature was performed for studies comparing RANU and LNU for bladder cancer. Outcome measurements were recurrence rates , positive margins, positive lymph node yield and perioperative outcomes. Meta‐analysis was performed using p = 0.008); however, these results were inconsistent on sensitivity analysis and should therefore be interpreted with caution. No significant difference was observed for other outcomes.Our results demonstrate a significantly higher mortality rate in patients undergoing laparoscopic nephroureterectomy when compared with the robotic‐assisted approach for the treatment of UTUC (1.8% vs. 1.1%, The ideal approach to minimally invasive radical nephroureterectomy remains undetermined. Future research, ideally prospective randomised studies, should focus on long‐term outcomes, in particular recurrence, recurrence‐free survival, overall survival and the correlation between surgical technique and survival. The available data are primarily retrospective and includes only small, single‐institution sample sizes with limited follow‐up.22.1This study is a systematic review of retrospective cohort studies and follows PRISMA reporting guidelines.2.2The following databases were searched as part of the systematic review in March 2022: Medline, EMBASE and Web of Science. The following search terms were used: “robotic‐assisted*,” “laparoscopic*,” “nephroureterectomy*,” “urothelial carcinoma.” The symbol “*” was used to allow variations on a word stem to be included in the search results. Furthermore, the following MeSH  were used: (robotic‐assisted[MeSH] OR laparoscopic[MeSH]) AND . The last date of search was 3 March 2022. The grey literature was also searched to further identify ongoing works of literature.2.3Randomised controlled trialsCohort studiesCase–control studiesStudy design:Patients with urothelial carcinomas undergoing nephroureterectomyParticipantsComparison of robotic‐assisted versus laparoscopic approachIntervention:Primary: Recurrence , positive surgical margins, lymph node yield, severe morbidity (Clavien–Dindo >2)Secondary: Quantitative measure of overall morbidity, operative time, intraoperative complications, transfusions, postoperative length of stay, pathological outcomes, metastasis, mortality, recurrence‐free survival, cancer‐specific survival, correlation between surgical technique and survivalBasic participant characteristics: age, sex, primary tumour location, T‐stage and gradeOutcomes:Studies in English assessing the outcomes of robotic‐assisted versus laparoscopic nephroureterectomies were assessed for eligibility based on the following inclusion criteria:2.4Case reportsCase seriesConference abstractsStudy design:Patients without urothelial malignancyParticipantsNo comparison of RANU and LNUHand‐assisted laparoscopic nephroureterectomyIntervention:Qualitative measures onlyUnclear outcomesOutcome:2.5The primary aim of this study was to quantitatively assess recurrence, positive surgical margins, lymph node yield and severe morbidity (Clavien–Dindo >2)Primary outcomes:The secondary aims of this study were to quantitatively assess overall morbidity, operative time, intraoperative complications, transfusions, postoperative length of stay, pathological outcomes, metastasis, mortality, recurrence‐free survival, cancer‐specific survival and correlation between surgical technique and survival.Secondary outcome:Basic participant characteristics, including age, sex, primary tumour location, T‐stage and grade2.6A database was created using the reference managing software EndNote X9™. Two researchers reviewed outputs from the searches independently of each other.Initially, duplicates were removed. Study titles were then screened and assessed for potential relevance. The abstracts of selected potential studies were then read and assessed for eligibility for inclusion, based on the inclusion/exclusion criteria detailed above. Rejected studies were grouped together in the database by their reason for exclusion. The full texts of the abstracts deemed eligible for inclusion were then further analysed using the same criteria. Conflicts between the two reviewers were resolved following an open discussion and final decision by the senior author.In order to extract and store data efficiently, the Cochrane Collaboration screening and data extraction tool, Covidence, was used. Data were collected by two reviewers independently, using the following headings: Study details, study design, population, intervention, comparison groups and outcomes. Conflicts between the two reviewers were resolved following an open discussion and final decision by the senior author.A critical appraisal of the methodological quality and risk of bias of the included studies was performed. The critical appraisal was completed by two reviewers independently. Quality assessment of nonrandomised controlled trials (non‐RCTs) was performed according to Newcastle–Ottawa Scale (NOS). Furthermore, the certainty of evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) tool for grading quality of evidence.2.7I‐squared statistics, with >50% being considered as considerable heterogeneity. A random effects model was applied in cases of considerable heterogeneity (>50%) and a fixed effects model applied otherwise. Statistical significance was attributed to p value <0.05. Sensitivity analysis was performed using Revman Statistical Software . Consistency of results was determined by excluding studies based on size, chronology and methodological quality.Statistical analysis was performed using Revman Statistical Software . Binary outcome data were reported as odd ratios (OR) and 95% confidence interval (95% CI) were estimated using the Mantel–Haenszel method. For continuous data, mean differences and 95% CI were estimated using inverse variance weighting. Outcome measures (mean + standard deviation and median + interquartile range) were recorded. If needed, outcome variables (mean and SD) were estimated from the median and range using formula described by Hozo et al.2.8Our systematic review was registered on PROSPERO in February 2022 (ID: 374275).33.1The literature search described above yielded a total of 4466 results Figure . FollowiFrom these 43 full texts, 33 were excluded for the following reasons: 13 were an incorrect study design; 10 did not analyse patients with urothelial malignancy; 10 did not compare outcomes of RANU and LNU. A total of 10 studies met the inclusion criteria, all of which were included in the quantitative analysis.3.2All 10 of the included studies were retrospective cohort studies.3.3The total number of participants from the 10 included studies was 29 987. Overall, 9175 patients underwent robot‐assisted radical nephroureterectomy, with the remaining 20 812 patients undergoing laparoscopic radical nephroureterectomy. The baseline characteristics of participants are outlined in Table 3.4n = 2212) of tumours were located within the kidney, 31.2% (n = 1056) were within the ureter and the remaining 3.5% (n = 119) were involved both the kidney and ureter.Tumour location was reported in six of our included studies.3.5p = 0.53), with no heterogeneity reported across the included studies (I2 = 0%)  in the RANU group and 9.6% (295/3063) in the LNU group. A meta‐analysis of the included studies using the M‐H fixed‐effects model showed no significant difference between the groups for this outcome , with no heterogeneity between the studies (I2 = 0%)  between the two groups. The rate was 8.3% 345/4139) in the RANU group and 8.4% (1178/14036) in the LNU group. A meta‐analysis of the included studies using the M‐H fixed‐effects model showed no significant difference between the two groups for this outcome , with considerable heterogeneity between studies (I2 = 0%)  in the RANU group and 32.7% (5545/16983) in the LNU group. A meta‐analysis of the included studies using the M‐H random‐effects model revealed no significant difference between the two groups in terms of overall morbidity . Considerable heterogeneity was found (I2 = 99%) . A meta‐analysis performed using the random‐effects model revealed a reduced operative time in the LNU group; however, these results were not statistically significant , with considerable heterogeneity between included studies (I2 = 69%) . There was considerable heterogeneity between studies (I2 = 100%)  Figure  (very lo3.11p = 0.008), with marginal heterogeneity between studies (I2 = 50%)  in the RANU group and 1.8% (288/16240) in the LNU group. A meta‐analysis of the included studies using the M‐H fixed‐effects model revealed a significant difference between the two groups in regards to mortality rate . There was considerable heterogeneity between studies (I2 = 99%)  Figure  (very lo3.13p = 0.11), chronology  and methodological quality . These inconsistencies warrant caution when interpreting the meta‐analysis results for perioperative mortality.A sensitivity analysis for the rate of perioperative mortality was performed to evaluate the stability of the results. Mortality rates proved inconsistent when studies were excluded based on size  in their RANU group, compared with 54.5% (n = 12) in the LNU cohort.n = 9) and 24.3% (n = 25), respectively.Unfortunately, due to a lack of data available, we were unable to quantify and meta‐analyse several of our primary and secondary endpoints, including recurrence, lymph node yield, need for transfusion, several pathological outcomes and long‐term survival outcomes. Two of our included studies reported recurrence rates.4I2 = 50%) and inconsistent results on sensitivity analysis. Ambani et al. were the only study to provide an individual breakdown of cause of death, reporting hematemesis as the aetiology of the single death in their robotic‐assisted cohort.Our results demonstrate no clear advantage of one minimally invasive technique over another in treatment of UTUC via nephroureterectomy. With the exception of perioperative mortality, the robotic‐assisted approach provides no clear benefit over the pure laparoscopic approach; however, mortality benefit should be interpreted with caution due to moderate heterogeneity between studies I2 = 50% aA large prospective controlled study, ideally randomised, comparing laparoscopic nephroureterectomy and robotic‐assisted nephroureterectomy in the treatment of adult patients with upper tract urothelial carcinoma.Clear description of method of bladder cuff excision, with a subgroup analysis of oncological outcomes for each method.Long‐term data, incorporating a comparison of recurrence, overall survival, disease‐free survival, overall mortality and disease‐specific mortality.Quality of life data, via a standardised patient‐reported outcome measures (PROMs) questionnaire.Long‐term, prospective studies comparing these outcomes between the robotic‐assisted and laparoscopic approaches have yet to be performed and will undoubtedly provide invaluable information into the optimal surgical approach in the treatment of patients with UTUC. While the benefits of MIS are well established, it is imperative that further research is performed to identify the ideal method to provide patients with the best perioperative and long‐term outcomes possible. Future research, ideally prospective randomised studies, should focus on long‐term outcomes, in particular recurrence, recurrence‐free survival, overall survival and the correlation between surgical technique and survival. In order to achieve the above, the authors have made the following recommendations for future studies comparing the laparoscopic and robotic‐assisted approaches to nephroureterectomy:Our study had several limitations. While the evidence regarding RANU in the treatment of UTUC has increased substantially in recent years, it still remains sparse and of generally low quality. Similarly, a lack of prospective, randomised trials, as well as long‐term data on recurrence, prevented us from analysing this outcome and drawing conclusions on the superiority of one minimally invasive technique over another. Despite these limitations, our review demonstrates no clear benefit of RANU or LNU in terms of perioperative outcomes and provides a good foundation for future research to focus on long‐term outcomes comparing these two techniques.5The ideal approach to minimally invasive radical nephroureterectomy remains undetermined. Future research, ideally prospective randomised studies, should focus on long‐term outcomes, in particular recurrence, recurrence‐free survival, overall survival and the correlation between surgical technique and survival.The authors report no conflicts of interest.Data extraction: Niall J. O'Sullivan, Ailish Naughton, and Hugo C. Temperley. Data synthesis: Niall J. O'Sullivan and Ailish Naughton. Manuscript preparation: Niall J. O'Sullivan, Ailish Naughton, Hugo C. Temperley, and Rowan G. Casey. Manuscript writing: NOS, AN, RC. Manuscript review/editing: Niall J. O'Sullivan and Rowan G. Casey.Data S1. Supporting InformationClick here for additional data file.


The coexistence of species with similar ecological niches is one of the core interests of community ecology research. However, how functional feeding traits, including bill size and leg length, determine the niche of mixed flocks of shorebird species has seldomly been studied, as well as, microhabitat variables affect the spatial patterns of availability and the quality of patches for wintering. From October 2016 to March 2017 at Shengjin Lake, Anhui Province, China, we recorded 226 scan samples from the different microhabitats and 93 focal animal videos of four common shorebird species: common greenshank, spotted redshank, Kentish plover, and little ringed plover. We found that the species participating in the mixed groups were different in each microhabitat. The results of the overlap index for microhabitats and foraging techniques between the species were consistent with the morphological characteristics of these species. Kentish and little ringed plovers had the highest Pianka's niche overlap index values of 0.95 and 0.98 for microhabitats and foraging techniques, respectively, whereas common greenshank and spotted redshank had values of 0.78 and 0.89, respectively. Common greenshank and spotted redshank used four foraging techniques: a single probe (PR), multiple probes (MPR), a single peck (PE), and multiple pecks (MPE). Kentish and little ringed plovers only used PE and MPE. The mean bill size, mean leg length, and mean foraging frequency were significantly associated with water depth. The mean bill size and mean leg length were both significantly correlated with the mean foraging frequency of shorebirds. The vegetated area was the most important variable for grouping among shorebirds. We concluded that the four species showed differences in their preferred microhabitats and foraging patterns. Interspecific morphological differences, including bill and leg lengths, resulted in niche differentiation. Thus, effective resource allocation by regional species was realized, and a dynamic balance was achieved by the mixed foraging species. The information on foraging behavior and habitat requirements could be useful in the management of water levels in natural areas and conservation of a diversity of wintering shorebirds. The four species  showed differences in their preferred microhabitats and foraging patterns. Interspecfic differences, including bill and leg lengths, resulted in niche differentiation. The rainy and dry seasons last from April to October and from November to March, respectively. Annual water levels fluctuate, increasing and dropping during the rainy and dry seasons, respectively. In the dry season, large mudflats in the lake serve as foraging habitats for wintering shorebirds. The lake is separated by sluice gates, and the water level is artificially controlled, which causes a particularly rapid rise and descent of water levels leading to flooding or a rapid exposure of the mud flats. The foraging and distribution of shorebirds is strongly influenced by these rapid changes which result in the formation of many habits of varying depth and size.Shengjin Lake  is a river‐connected lake, with an area of 13,300 hectares. It is situated on the East Asian–Australasian flyway and is a wetland of international importance  vegetated area, vegetation that grows on mud‐ or sandflats, with a height not exceeding the bird's body height; (H2) dry mud, which birds can walk across and not sink; (H3) soft mud, which is covered by surface water with a depth of 0–0.5 cm, where birds can sink; (H4) shallow water, with a water depth of 0.5–3 cm; (H5) intermediate water depth, with a water depth of 3–6 cm; (H6) deep water, with a water depth of 6–9 cm; and (H7) the water body, with a depth of >9 cm.2.3Tringa nebularia), spotted redshank (Tringa erythropus), Kentish plover , and little ringed plover (Charadrius dubius)  Figure . These sWe rapidly recorded total number of individuals, foraging individual number, and individuals' instantaneous foraging techniques for each species in mixed‐flock shorebird groups by sequentially scanning habitats with a ZEISS video telescope . Scanned videos that included clearly identifiable individuals and instantaneous foraging techniques were collected every 30 min in the three sites that consisted of seven microhabitat types (H1–H7). The instantaneous foraging techniques of birds were classified into the following types by using the PIPI Player  at a playback speed of 0.5× to observe them: a single peck ; multiple pecks ; a single probe ; and multiple probes  ; a foraging bout length ) were recorded by focal animal sampling methods until their behavior changed or when they moved out of sight. A ZEISS video telescope  was used to observe shorebirds and record videos of their foraging activities from a tent. The length of each observation was 5 min, and 93 focal animal videos were recorded, including 30 common greenshank, 22 spotted redshank, 20 Kentish plover, and 21 little ringed plover, respectively File . For anaThe different species fed in bare mudflats, or in water depths that suited their morphological traits. In the video material, the water depth was calculated based on the length of the submerged part of the tarsal plantar. Common greenshank and spotted redshank birds have similar bill sizes  and leg lengths , as do Kentish and little ringed plovers  Zhao, .2.4Pearson's correlation was used to analyze the relationships between the mean bill size, mean leg length, foraging time ratio, foraging frequency, a foraging bout length, and the water depth of the foraging habitat Kober, . A KolmoA multiple linear regression (MLR) was applied to predict the effects of the different microhabitat selection by the different species of shorebirds. The total and foraging individual number of birds were used as the response variables, and the type of microhabitat selected, as well as the foraging techniques, were the fixed predictor variables when there was a relationship with the response variables /sqrt(sum((pij)2)*sum((pik)2))) was calculated using Pianka's index  was used as the grouping variable, all habitat types (H1–7) and foraging techniques  were used as predicted variables. A significance value of All analyses were conducted using SPSS version 23. The significance level of all statistical tests was ≤0.05, and results were stated as the mean ± standard deviation.33.1Common greenshank and spotted redshank birds foraged in six and five of the seven microhabitats, respectively. Common greenshanks did not forage in the vegetated area (H1). Spotted redshanks foraged in dry mud or habitats with water depths >0.5 cm (H4–H7). Kentish and little ringed plovers did not forage in shoal areas with water depths >3 cm (H5–H7). The occurrence of mixed‐species flocks varied based on the habitat type, and five mixed flocks were found: H1: Kentish plover and little ringed plover; H2: all four species mixed; H3: three species, excluding spotted redshank; H4: three species, excluding little ringed plover; H5–H7: common greenshank and spotted redshank , as well as in soft mud . Kentish plover and little ringed plover only differed in the vegetated area   as well as Pianka's index and leg length .Kentish and little ringed plovers were most similar in their microhabitat preference, and hence the overlap index of microhabitats used by them was the highest. However, the overlap index of Kentish plovers and spotted redshank was 0.15, and that of Kentish plovers and common greenshank was 0.36 (Table R2 = .527)  (File A MLR was employed to predict the selection of foraging microhabitats by the shorebird species. All of the foraging techniques and microhabitat types (H1‐7) excluding the Deep water (H6) were used in the model. The variables Shallow water (H4), PE, Soft mud (H3), Vegetated area (H1), Water body (H7), Intermediate water depth (H5), and Dry mud (H2) predicted the total individual number, and the Shallow water (H4) was the most important (adjusted 00) File .3.2Z = 2.238, p = .000).Common greenshank and spotted redshank used four foraging techniques, showed similarities in morphological characteristics, and their overlap index was 0.89 Table . All speThe little ringed plover and Kentish plover used PE and MPE foraging techniques, and the overlap index of foraging techniques between them was the highest (0.98) Table . MoreoveZ test showed significant differences between the species in terms of foraging frequency , a foraging bout length , and water depth  but not in the foraging time ratio . Between the species, the foraging frequency by common greenshank (15.91 frequency/5 min) was lower than that of spotted redshank (23.88) , whereas a foraging bout length (1.54 s vs. 0.93 s) was higher . Foraging frequency (19.28 ± 10.17 vs. 39.87 ± 9.85 frequency/5 min)  and a foraging bout length (1.3 ± 0.9 s vs. 0.52 ± 0.22 s)  of the larger‐sized group of birds (common greenshank and spotted redshank) was lower than that of the smaller‐sized group (Kentish plover and little ringed plover), however, the water depth (3.7 ± 2.3 cm vs. 0.2 ± 0.2 cm) was greater  (File The results of the Kolmogorov–Smirnov 01) File .r = .993, p = .007), mean leg length , and mean foraging frequency  were significantly associated with the water depth of the bird's foraging habitats. Kentish plover's a foraging bout length was positively correlated with water depth ; these two variables were negatively correlated for little ringed plover ; however, the foraging frequency for both was positively correlated with water depth .The mean bill size  and mean leg length  were significantly correlated with the mean foraging frequency for shorebirds. The foraging frequency was significantly correlated with a foraging bout length . Additionally, the foraging frequency was only correlated  for little ringed plover and common greenshank.The mean bill size (R2 = .513)   and Deep water (H6) were used in the model. PE, PR, Vegetated area (H1), and MPR variables completely predicted foraging individual number, and the PE variable was the most important (adjusted 00) File .3.3Three typical functions were generated from discriminant analysis Table . The vegThe large‐sized bird group (common greenshank and spotted redshank) had higher scores than the small‐sized group (little ringed plover and Kentish plover), suggesting that the large birds chose different microhabitats and foraging techniques Figure . The ori4In this study, it was found that four shorebird species at Shengjin Lake overlapped in their selection of foraging microhabitat and in their use of foraging techniques, to a certain extent. This study demonstrated niche separation of shorebirds at a wintering site.The mechanism for mixing among these species of shorebirds was reflected in their spatial coexistence, which was largely related to their characteristics, as has been shown in previous studies ; investigation ; methodology ; writing – original draft . Ruilin Zhang: Data curation ; investigation ; methodology ; writing – original draft . Lizhi Zhou: Conceptualization ; funding acquisition ; supervision ; writing – review and editing . Lei Cheng: Software . Yiwei Bao: Software . Yunwei Song: Investigation .File S1.Click here for additional data file.File S2.Click here for additional data file.


Daily and event‐driven oral pre‐exposure prophylaxis (PrEP) reduce the risk of HIV acquisition. PrEP use can vary over time, yet little is known about the trajectories of PrEP use irrespective of the chosen PrEP regimens among men who have sex with men (MSM).Using data from a mobile, web‐based diary application collected daily from 17 August 2015 until 6 May 2018, we analysed PrEP use and sexual behaviour in two large cohorts, AMPrEP  and Be‐PrEP‐ared . In both cohorts, participants could choose between daily and event‐driven oral PrEP every 3 months. We used group‐based trajectory modelling to identify trajectories of PrEP use over time and their determinants. In addition, we estimated the incidence rate of chlamydia, gonorrhoea and syphilis within these trajectories.n = 322 AMPrEP; n = 194 Be‐PrEP‐ared), of whom 24% chose event‐driven PrEP at PrEP initiation. Participants contributed 225,015 days of follow‐up (median = 508 days [IQR = 429−511]). Four distinct PrEP use trajectories were identified: ≤2 tablets per week , 4 tablets per week , “almost daily” (31%) and “always daily” (41%). Compared to participants with “low frequency” PrEP use, participants with “variable”  and “almost daily” PrEP use were more often AMPrEP participants . “Almost daily” PrEP users were more often employed  and were younger compared to participants with “low frequency” PrEP use. In addition, the number of days on which anal sex occurred was lower among participants with “low frequency” PrEP use compared to the other groups . Compared to “low frequency” PrEP users, the incidence rates of chlamydia and gonorrhoea were higher for participants with “almost daily” and “always daily” PrEP use.We included 516 MSM (We uncovered four distinct PrEP use trajectories, pointing to different patterns of PrEP use in practice beyond the two‐regimen dichotomy. These trajectories were related to sexual behaviour and rates of sexually transmitted infection. Tailoring PrEP care according to different PrEP use patterns could be an important strategy to improve efficient PrEP delivery. AFor daily PrEP, variation in use is limited to stopping and restarting a daily PrEP regimen. For event‐driven PrEP, there has been a large variation observed in the use of this regimen . The Be‐Identifying more refined patterns of use, and what types of behaviours and outcomes are associated with these patterns, may help inform PrEP programmes by allowing more tailored care and more efficient PrEP delivery. In this study, we established PrEP use trajectories and their determinants among MSM participating in the Be‐PrEP‐ared and AMPrEP studies. In addition, we assessed the incidence rate (IR) of syphilis, gonorrhoea and chlamydia within these trajectories.22.1n = 5) were included in both studies, they were excluded from these analyses.For this study, we pooled pseudonymized data from the Be‐PrEP‐ared 2015–2018) and AMPrEP (2015–2020) cohort studies. Full procedures of both studies have been published previously , 8. Brie and AMPrAt PrEP initiation, participants could choose between daily and event‐driven PrEP, and could switch between regimens at every 3‐monthly study visit. Study staff explained both regimens. During follow‐up visits, these regimens were sometimes discussed, depending on the needs of the participant .Since follow‐up for the Be‐PrEP‐ared study was shorter than for AMPrEP, we included data for the first 73 weeks after PrEP initiation for all participants (i.e. the time at which the majority of Be‐PrEP‐ared participants reported daily data). The study period for this analysis included visits from 17 August 2015 until 6 May 2018.2.2For the Be‐PrEP‐ared study, a personal web‐based diary application (app), and later an additional smartphone app, were used to collect daily information on PrEP use. For the AMPrEP study, an app was used to collect daily information on PrEP use , 15. On Participants were screened for syphilis, chlamydia and gonorrhoea at 3‐monthly study visits , 11. Res2.3To exclude participants with overly unreliable data, we included those who reported data on PrEP use for at least 10% of all days during the entire 73‐week follow‐up period after the date of PrEP initiation (i.e. roughly the fifth percentile of % reporting data). To assess potential bias from non‐response, we compared the distribution of baseline socio‐demographic and sexual behaviour variables of participants who reported data in the apps at least 10% of days during follow‐up to those who did not.Follow‐up started at PrEP initiation (i.e. baseline) and continued until PrEP discontinuation (as indicated by the participant) or 73 weeks after PrEP initiation, whichever occurred first.We used group‐based trajectory models to identify groups of participants who followed distinct individual‐level trajectories of PrEP use . Group‐bk = 1, 2, …, 6) and determined the optimal number of groups from the model resulting in the lowest Bayesian Information Criteria, highest entropy values, and all trajectories having a marginal prevalence of >10% . PrEP use per day during the 73 weeks is visualized in Figure 3.2p<0.001, Table p = 0.011). Participants with “low frequency” PrEP use were least often employed (p = 0.016) and had the lowest total number of reported anal sex days over the study period (p<0.001).Participants with “always daily” PrEP use had the least missing data in the apps compared to the other trajectories p<0.00, Table 1p = 0.040) had higher odds of being “variable” PrEP users compared to “low frequency” PrEP users. Additionally, we found that AMPrEP participants (p = 0.009) and those employed (p = 0.001) had higher odds of being “almost daily” PrEP users compared to “low frequency” PrEP users, while older participants had lower odds of being “almost daily” PrEP users . Compared to “low frequency” PrEP users, participants in all other trajectories had more days on which they reported anal sex . There was no evidence of interaction between any of the covariates and the study . IR was 12.2/100PY (95% CI = 9.6−15.4) for syphilis, 47.8/100PY (95% CI = 42.4−53.9) for chlamydia and 51.9/100PY (95% CI = 46.2−58.2) for gonorrhoea. Compared to “low frequency” PrEP users, the IR of chlamydia was higher for participants with “almost daily”  and “always daily” PrEP use  , these individuals were predominately daily PrEP users. “Low frequency” and “variable” PrEP use groups were mainly event‐driven PrEP users, yet 17% and 40%, respectively, of them were in fact daily PrEP users at baseline. These observations demonstrate that PrEP users have varying patterns of PrEP use and these patterns do not exactly correspond to simply “daily” or “event‐driven” PrEP. These profiles are also linked to varying degrees of sexual behaviour and risk of STI, thereby giving rise to differing PrEP needs.There were differences in the distribution of PrEP use profiles between studies. Participants using PrEP “variable” and “almost daily” were more likely to be AMPrEP participants. It should be noted that Be‐PrEP‐ared participants were actively reminded to fill in the daily diary, contrary to AMPrEP participants, and as a result, individuals participating in Be‐PrEP‐ared were less likely to have missing daily data. We assume that missing data for weeks with incompletely filled data represent no PrEP use, and hence it becomes difficult to distinguish AMPrEP participants using PrEP “variable” or “almost daily” who truly forgot a single tablet during the week from those who forgot to record their PrEP use once during the week. Alternatively, age and certain behaviours associated with STI and HIV infection were different between studies, while these characteristics are known to influence PrEP use , 15. DesThere was substantial variation in the median numbers of days per week on which PrEP was taken in participants with “low frequency” and “variable” PrEP use. Most participants with “low frequency” PrEP use had 2 or less days of PrEP use within the week. This result is somewhat surprising as the recommended schedule for event‐driven PrEP usually covers a span of at least 3 days . This level of PrEP intake would suggest inadequate adherence to the event‐driven PrEP regimen, which was uncommon in both AMPrEP  and Be‐PAs more information has become available about non‐daily PrEP use and the World Health Organization (WHO) and Centers for Disease Control (CDC) have recommended to include event‐driven PrEP for cis‐gender men in PrEP programmes , 5, commWe found that participants with “almost daily” PrEP use were younger than participants with “low frequency” PrEP use (mean ≤2 PrEP days/week from the group‐based trajectory model). Although hypothetical, older participants might have found it easier to plan event‐driven PrEP use in accordance with their sex life . ParticiThis study has other limitations. First, some participants never or rarely used the daily diary application and were, therefore, excluded from the analysis. As certain determinants, most importantly the choice of PrEP regimen at baseline, differed between included and excluded participants, this may have induced selection bias. Second, the assumptions of imputing missing data depended on whether an individual filled in the app at least once during a given week. This method could bias trajectories towards higher levels of PrEP use if PrEP users no longer took PrEP during entire weeks without app use. Third, most study participants were white and highly educated and may not reflect the broader population using PrEP. Fourth, when Be‐PrEP‐ared and AMPrEP began enrolling participants, event‐driven PrEP was less well known and not yet included in WHO or CDC guidelines , 5. This5We identified four distinct PrEP use trajectories, which represented average PrEP use ranging from 2 days per week or less to daily or almost daily use. These trajectories were associated with sexual behaviour. To accommodate different schedules of PrEP use and ensure its effectiveness, it would be more appropriate to shift away from defining and presenting PrEP use as two separate regimens. We recommend to focus, rather, on how to more effectively use PrEP when the need for it changes over time. Future research should investigate how PrEP care could be adapted according to different usage patterns, for example, by evaluating whether the frequency of STI screening among participants with low levels of PrEP use can be reduced.The study medication for the Be‐PrEP‐ared study of Antwerp and the Amsterdam PrEP study was provided by Gilead Sciences based on unconditional grants. EH received advisory board fees from Gilead Sciences and speaker fees from Janssen‐Cilag, both paid to her institute. UD received unrestricted research grants and speaker's fees from Gilead Sciences, paid to his institute. HJCdV received grants from Medigene, and advisory board and speaker fees from Gilead Sciences, Medigene, Abbvie, Janssen‐Cilag and Willpharma, paid to his institute. MP received unrestricted research grants and speaker's fees from Gilead Sciences, Roche, Abbvie and MSD, paid to her institute. MSvdL served on an Advisory Board of MSD, paid to his institute. All other authors declare no competing interests.BV, TR, EH, MP, ML, MSvdL, HJCdV and UD conceptualized and designed this study and obtained funding. VWJ, AB, MSvdL, TS and TR were involved in the data analysis. VWJ, TR, AB, MSvdL, UD, HZ, LC, HZ, MvdE, HJCdV, CK, IDB, TS, BV, MP, ML and EH were involved with the interpretation of the data. VWJ drafted the manuscript. All authors read and approved the final manuscript.The Tale of Two Cities project was funded by ZonMW (grant number: 522008009). TR is a postdoctoral fellow of the Research Foundation—Flanders. The Be‐PrEP‐ared study was funded by the Applied Biomedical Research (TBM) Program of the Belgium Research Agency (IWT). CT/NG diagnostic kits for Real Time PCR used during the Be‐PrEP‐ared study were donated by Abbott. The AMPrEP project received funding as part of the H‐Team Initiative from ZonMw (grant number 522002003), the National Institute for Public Health and the Environment and GGD research funds. The H‐Team initiative is being supported by the Aidsfonds Netherlands (grant number 2013169), Stichting Amsterdam Diner Foundation, Gilead Sciences Europe Ltd (grant number PA‐HIV‐PREP‐16‐0024), Gilead Sciences , Janssen Pharmaceuticals (reference number PHNL/JAN/0714/0005b/1912fde), M.A.C AIDS Fund, and ViiV Healthcare .Supporting InformationClick here for additional data file.


Citrus reticulata ‘Chachi’ (CRC), a series of activity assessments were performed on spleen deficiency mice. The constituents in CRC with different storage years were analyzed by LC‐Q‐Orbitrap/MS. A total of 53 compounds were identified, and CRC stored for more than 5 years showed higher flavonoid content, especially that of polymethoxyflavones. Anti‐spleen deficiency bioactivity analysis among various CRC with different storage years showed aged CRC (stored for more than 3 years) could significantly alleviate fatigue and depression behaviors much better, increase D‐xylose and gastrin secretion, and upregulate the expression of the linking protein occludin in the colon walls. Results from 16S rDNA sequencing showed that aged CRC could downregulate the abundance of Enterococcus, Gemmata, Citrobacter, Escherichia_Shigella, and Klebsiella, which were significantly overrepresented in the model group. Bacteroides, Muribaculum, Alloprevotella, Paraprevotella, Alistipes, Eisenbergiella, and Colidextribacter were downregulated in the model group but enriched in the CRC groups. At last, the spectrum‐effect relationship analysis indicated that flavonoids such as citrusin III, homoeriodictyol, hesperidin, nobiletin, and isosinensetin in aged CRC showed the highest correlation with better activity in ameliorating spleen deficiency by regulating gut microbiota. Flavonoids contribute most to discriminating aged CRC and could disclose the basis of “the longer storage time, the better bioactivity” of aged Guang Chenpi.To further explore the mechanism of “the longer storage time, the better bioactivity” of aged Guang Chenpi, the dry pericarp of  We found that aged Guang Chenpi could rescue spleen deficiency by ameliorating gut microbiota dysfunction and fatigue and depression behaviors, upregulating D‐xylose and gastrin secretion, and increasing the integrity of the colon tissue better than a year‐old sample. And these rescuing effects were associated with citrusin III, homoeriodictyol, hesperidin, nobiletin, and isosinensetin in samples with different storage years. Our findings will serve as a reference for the comprehensive evaluation of the mechanism of “the longer storage time, the better bioactivity” in the processing of Guang Chenpi. It is recorded in the Chinese Pharmacopeia (2020 edition) to exert bioactivities such as regulating qi and strengthening the spleen, drying dampness, and eliminating phlegm  was collected from the Xinhui area in the Guangdong Province and identified by Professor Yang Quan at Guangdong Pharmaceutical University, Guangzhou, China. The storage period ranged from 1 to 36 years.Guang Chenpi (Smashed CRC samples (passed through a 40‐mesh sieve) were boiled with water and filtered twice. The filtrates were combined and concentrated to 1 g/mL. The positive control drug, Shen Ling Bai Zhu powder, was dissolved in water to yield a concentration of 1.5 g/mL  and a Waters Symmetry C18 column . The mobile phases were aqueous 0.1% formic acid (A) and acetonitrile (B). The gradient elution was as follows: 0–10 min, 10%–25% B; 10–20 min, 25%–60% B; 20–30 min, 60%–95% B. The flow rate was 1 mL/min, the column temperature was 30°C, and the injection volume was 3 μL. Mass spectrometry (MS) was performed using a Q Exactive Focus mass spectrometer  combined with an electrospray ion source and positive and negative ion switching scanning modes. The scanning range was 150–2000 m/z, the full MS primary resolution was 35,000, and the secondary resolution was 17,500. The ion‐spray voltage was 3.5 kV, the ion‐source temperature was 320°C, the sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 15 arb, and the ion‐transport tube temperature was 320°C. The collision energy gradient was 20, 40, and 60 eV. Identification of compounds was done using Compound Discoverer software (version 3.0), and the original data were matched with mzCloud and the Chinese Medicine Compounds (OTCML) database.2.3An equal number of 6–7‐week‐old male and female SPF‐grade Swiss mice weighting 20–25 g were purchased from Guangdong Medical Laboratory Animal Center (SCXK2018‐0002). All mice were housed at a constant temperature (26 ± 2°C) and standard humidity and subjected to appropriate light/dark conditions. Mice were allowed free access to water. All procedures were approved by the Institutional Animal Use Committee .n = 8 per group). Except for those in the control group, all other mice were administered aqueous senna extract intragastrically (i.g. 0.2 mL/20 g). Mice in the control group received a similar volume of normal saline by gavage for 14 days. Mice in each group were forced to swim once daily in a water tank for 14 consecutive days. The water temperature was 25°C, and the tank depth was 30 cm. Moreover, mice in each group were fasted every other day for 14 days , whereas those in the CRC groups received a corresponding dose of CRC extract (i.g. 2 g/kg) and Shen Ling Bai Zhu powder for 14 days. The administered dose is calculated as 6–9 g for one person per day, according to the Chinese Pharmacopeia (2020 edition).All mice were randomly divided into either a control group (Control), a spleen deficiency model group (Model), a CRC group with different storage years , or a positive control group (SLBZ)  was performed using a KW‐OPF open‐field activity box  after 10 days of drug administration. Mice were gently placed in an open box with black walls and a bottom. The test was conducted in absolute silence and without any direct light source. Behavioral changes within 5 min, including the total distance and the time spent in the central area (25 × 25 cm), were recorded using a synchronous camera system  of D‐xylose solution. Serum from mice was collected after 30 min. Mice were anesthetized using 1.25% avertin, and their whole blood was collected after removing their eyeballs. The blood was allowed to stand for 4 h at 4°C. The upper layer of serum was collected for analysis. Serum D‐xylose  and gastrin  levels were determined using the phloroglucinol method according to the manufacturer's instructions.For the determination of serum D‐xylose levels, mice were fasted for 12 h before the 2.6Mice were administered a mixture of activated carbon and 10% sodium carboxymethyl cellulose. After 1 h, they were anesthetized using 1.25% avertin. Blood was collected, the pylorus was ligated, and the mesentery was stripped. The intestine from the pylorus to the ileocecal junction was cut and gently placed on the tray in a straight line. The total length of the small intestine was measured, and the distance from the pylorus to the furthest point to which the carbon had advanced in the intestine was recorded . The V4 regions of 16S rDNA were amplified using 341F (5′‐CCTACGGGNGGCWGCAG‐3′), 805R (5′‐GACTACHVGGGTATCTAATCC‐3′). Samples were sequenced on the NovaSeq PE250 platform according to the standard protocols. Sequencing data were processed using the Quantitative Insights Into Microbial Ecology  software.2.8Colon tissues were immersed in 4% paraformaldehyde to fix for dehydration and then paraffin‐embedded and sectioned for hematoxylin and eosin (H&E) staining. Pathological changes were observed using the OLYMPUS/BX fluorescence microscope .2.9The total protein of colon tissue was extracted by adding a RIPA lysis solution containing a phosphatase inhibitor and protease inhibitor (1:100). The total protein concentration was determined using a bicinchoninic acid assay kit (BCA) . After quantification, 40 μg protein was used for the electrophoretic separation on a 10% separation gel. Next, the protein was transferred to a polyvinylidene fluoride membrane. After blocking, they were incubated at 4°C with the primary antibody occludin  for 12 h. After washing three times with TBST solution (10 min/wash), the secondary antibody  was added and further incubated at 26°C for 1 h. An enhanced chemiluminescence chromogenic solution was added for imaging, and the gray value of the bands was measured using ImageJ software.2.10GRA is a method to study the degree of similarity or dissimilarity of development trends among factors. It is used to measure the degree of association between different factors and to evaluate the degree of correlation between the components and pharmacological effects of traditional Chinese medicine  with Tukey's post hoc test. 33.1i.g. administration of aqueous senna leaf extract and to physical exhaustion by forced swimming , suggesting that the spontaneous movement of model mice had decreased , which was indicative of fatigue  . This finding indicated that Guang Chenpi extracts with different storage years could rescue spleen deficiency‐induced fatigue and that the fatigue‐ameliorating effect increased with an increase in storage years.Guang Chenpi is the dry pericarp from the fruit of CRC obtained from the Guangdong province in China. “Chen Jiu Zhe Liang” means “longer storage years and better activities.” An extended storage period is a representative approach used in traditional Chinese medicine  Figure . Treatme3.2p < .01). The intestinal propulsive rate was lower than that in mice with spleen deficiency, but the difference was not significant. After the administration of Guang Chenpi extracts, intestinal propulsive rates were nearly identical to those of the control mice, indicating that Guang Chenpi could promote the digestive ability of mice with spleen deficiency. D‐xylose is a pentose that is not detected in the blood. D‐xylose levels in the blood can be used to indirectly evaluate the absorption function of intestinal mucosa after administering a certain dose of D‐xylose solution , suggesting absorption dysfunction in the intestine. Guang Chenpi samples with different storage years could increase D‐xylose levels, especially when samples stored for longer than 7 years were used. GAS secreted by the G cells in the gastric antrum is an important index that indicates the physiological function of the gastrointestinal tract  and rescued after the administration of Guang Chenpi samples. However, there were no significant differences in outcomes after the use of within‐year and aged samples.We further evaluated the amelioration of digestive dysfunction, gastric residual rate, and gastric emptying rate after administration of different Guang Chenpi samples. As shown in Figure The ameliorative effect increased with an increase in storage years compared with that of the within‐year sample. In addition, there were no significant differences among all indices, except D‐xylose and GAS levels, when Guang Chenpi extracts from different storage years were used. Additionally, aged Guang Chenpi samples could influence the digestive system by ameliorating gastric and intestinal function much more than samples aged for only 1–5 years.3.3p < .05). Occludin expression was significantly higher in the CRC‐36Y group than in the model group (p < .05); however, there were no significant differences in expression between the CRC‐1Y and model groups. In addition, H&E staining of colon tissues from the model group showed a thinner intestinal wall, which could be rescued by treatment with Guang Chenpi  effect size (LEfSe) was used to determine the differential microbial composition among groups. According to a cladogram that records the hierarchical taxonomic structure of the gut microbiota, there are notable differences in the phylogenetic distributions among the phyla and genera among the groups Figure . By usins Figure .Enterobacter, Enterococcus, Citrobacter, and Escherichia‐Shigella was significantly lower in the model group compared with that in the CRC group, whereas that of Alloprevotella and Tannerellaceae_unclassified increased after CRC treatment in mice with spleen deficiency . These data showed that aged Guang Chenpi extracts could restore intestinal wall integrity as well as enteric microbial richness and diversity in mice with spleen deficiency.The relative abundance of the genera y Figure . Some ofp Figure . There w3.4To compare the chemical constituents among Guang Chenpi of different storage years and their association with bioactivities, LC‐Q‐Exactive Orbitrap/MS was used to analyze the chemical constituents of Guang Chenpi water extract. The procedure is shown in Figure + and [M−H]− and were unambiguously or tentatively identified through alignment with accurate molecular weights within the mass accuracy of <10 ppm. Detailed information on the chemical compounds and their distribution in Guang Chenpi samples with different storage years is shown in Table A total of 53 compounds were identified from Guang Chenpi samples. Quasi‐molecular ions exhibited equivalence to [M+H]The flavone and PMFs in Guang Chenpi exert several effects such as antioxidant, digestion‐promoting, cough‐ and asthma‐relieving, and anticancer . The mean coefficient of the 29 peaks was >0.8, indicating a strong correlation, and the mean correlation coefficient of the other 13 peaks was between 0.7 and 0.8, indicating a moderate correlation Table . The strSpectrum‐effect analysis revealed that besides the uncommon peaks, the common peaks from different samples, especially homoeriodictyol, hesperidin, isosinensetin, and nobiletin, correlated well with spleen deficiency amelioration. Although the levels of these compounds were mostly unchanged, they correlated most with activity differences associated with storage years. Previous studies have shown that hesperidin and nobiletin can ameliorate brain, liver, and heart dysfunction.Although interesting results were obtained after multivariate statistical analysis in this study, some problems remain to be resolved. From the aspect of identification of chemical compounds, the high‐throughput method should be used to determine differences in chemical constituents induced by storage years. Our future study will involve qualitative and quantitative analyses of the primary compounds. Moreover, the specific mechanisms of action of new compounds will be elucidated, which will provide more clarity on the mechanism of action of “Chen Jiu Zhe Liang” in Guang Chenpi.4In this study, LC‐Q‐Exactive Orbitrap/MS profiles and in vivo anti‐spleen deficiency effects were combined to determine the spectrum‐effect relationship of Guang Chenpi with different storage years. Aged Guang Chenpi showed better effects in ameliorating spleen deficiency by regulating gut microbiota than those of within‐year samples. GRA revealed that flavonoids such as citrusin III, homoeriodictyol, hesperidin, isosinensetin, and nobiletin were the main active constituents associated with alleviating spleen deficiency. The exact pharmacological mechanism of the main compounds will be studied subsequently to explain the theory of “Chen Jiu Zhe Liang.”Xiaoming Sun: Conceptualization (lead); data curation (lead); methodology (lead); validation (lead); writing – original draft (lead). Haidan Deng: Data curation ; investigation ; methodology ; validation . Baojun Shan: Data curation ; investigation ; methodology ; validation . Yunqi Shan: Methodology (supporting); project administration (supporting); writing – review and editing (supporting). Jiaying Huang: Methodology (supporting). Xinshu Feng: Methodology (supporting). Xiaomin Tang: Methodology (supporting); project administration (supporting). Yuewei Ge: Investigation (supporting); methodology (supporting); project administration (supporting); software (supporting). Peiran Liao: Investigation (supporting); validation (supporting). Quan Yang: Conceptualization (lead); funding acquisition (lead); investigation (lead); methodology (lead); supervision (lead); validation (lead); writing – review and editing (lead).This work is supported by the Key Project at the Central Government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302) and University Characteristic Innovation Project of Guangdong Province (2019KTSCX072).All authors agree to submit this article and declare no conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Appendix S1Click here for additional data file.


Arntl, Clock, Nr1d1, Cry1, Cry2, Per1, and Per2) and metabolism‐ and inflammation‐related genes  in the male murine lung disrupted by reversed feeding (RF). In mice, RF disrupted the diurnal expression pattern of core clock genes. It decreased Nfe2l2 and Pparg and increased Rela and Cxcl5 expression in lung tissue. There were elevated levels of IL‐6, TNF‐alpha, total cells, macrophages, and lymphocyte counts in bronchoalveolar lavage (BAL) with a significant increase in vascular congestion and cellular infiltrates in male mouse lung tissue. Administration of PZ regained the diurnal clock gene expression, increased Nfe2l2 and Pparg expression, and reduced Rela, Cxcl5 expression and IL‐6, TNF‐alpha, and cellularity in BAL. PZ administration at 7 p.m. was more efficient than at 7 a.m.The circadian rhythm system regulates lung function as well as local and systemic inflammations. The alteration of this rhythm might be induced by a change in the eating rhythm. Peroxisome proliferator‐activated receptor gamma (PPARG) is a key molecule involved in circadian rhythm regulation, lung functions, and metabolic processes. We described the effect of the PPARG agonist pioglitazone (PZ) on the diurnal mRNA expression profile of core circadian clock genes ( Diurnal variation of the clinical signs, symptoms, and exacerbations also occurs in pulmonary diseases . PZ increased PPARG expression and inhibits local and systemic inflammation during acute lung injury  and PZ on the expression of the core clock, inflammatory and metabolic genes in the male murine lung.22.1We placed male BALB/c mice in single cages to avoid aggression and kept them following a light–dark cycle of a 12:12 pattern . From 4 weeks of age, the mice were housed individually, with unlimited access to food and water. The Ethics Committee of Poltava State Medical University approved the study. At 8 weeks of age, the mice were randomly divided into daytime feeding (RF) and nighttime feeding (NF) groups, with water provided ad libitum. Normally, the mice have increased activity and feed in the dark period. Thus, the daytime feeding is called reversed or inverted feeding. The RF group received food from 7 a.m. to 7 p.m., whereas the NF group received food from 7 p.m. to 7 a.m.  and midnight (HALO 17). The lungs were extracted, promptly frozen, and preserved at a temperature of −80°C for future use.2.2For RNA extraction, lung tissue samples were collected from six mice at each time point. The RNeasy kit (QIAGEN), Cat. No. 74104, was used to extract total RNA from the lung tissue.We generated single‐strand DNAs by reverse transcribing approximately 1 μg of total RNA from each sample using the QuantiTect® Reverse Transcription Kit (QIAGEN), Cat. No. 205313.For SYBR Green‐based analysis, the QuantiTect® SYBR Green PCR Kit (QIAGEN), Cat. No. 204143, was used to amplify the cDNA equivalent of 50 ng of total RNA from each sample in the CFX96TM RealTime PCR Detection System (BIO‐RAD).−ΔCt, and all values were normalized to the expression of the housekeeping gene β‐actin, which had weak circadian variation in the lung , TNF‐alpha (Cat. No. MTA00B), and TGF‐beta 1 (Cat. No. DY1679) in the BAL fluid using kits from R&D Systems (Minneapolis MN). The absorbance was determined by LabLine‐026 (Labline).2.5To perform histological analysis, the left lobe of each animal's lung was removed and inflated with 10% formalin after sacrifice. Paraffin‐embedded lung tissue 3 μm sections were stained with Hematoxylin and Eosin  for examination by histopathology. Morphological analyses were carried out using a light microscope Axio Lab.A1 (Carl Zeiss) and Zen 2.5 lite (blue edition) software. A blinded pathologist reviewed all lung sections using a special scoring system  staining technique  and quantified PAS‐positive cells through a semiquantitative approach  and used the Ashcroft scoring system was used for fibrosis quantification  was applied for the analysis of data using GraphPad Prism 5.0 software . The two‐way ANOVA and post hoc Bonferroni tests were employed to compare gene expression and cytokines concentration. The one‐way ANOVA test with post hoc tests and 2 × 2 table statistics, including Fisher's exact test, were used to analyze histopathological data and compare proportions. The study relied on the null hypothesis that RF, PZ, and the time of its administration did not influence the expression of clock, inflammatory, and metabolic genes in mouse lungs.33.1Per1, Per2, Cry1, Cry2, Clock, and Arntl mRNA at midnight than at noon in the lung tissues of NF mice. The level of Nr1d1 mRNA was increased at noon. The core clock gene expression did not change after the oral intake of PZ at 7 a.m. At midnight, Per1 mRNA was increased after PZ administration at 7 p.m. (p < 0.001).The levels of core clock genes' mRNAs during NF and after RF and PZ administration are shown in Figure Per1, Per2, Clock, and Arntl mRNA were decreased at midnight and increased at noon. At midnight, Cry1 and Cry2 mRNA were decreased in comparison with NF (p < 0.001). Also, we observed an increased Nr1d1 mRNA at midnight with a decrease at noon (p < 0.001). There were no changes in core clock genes’ expressions after PZ at 7 a.m., excluding a slight decrease in Clock mRNA at noon. The treatment with PZ at 7 p.m. led to increased Per1, Per2, Cry1, Cry2, and Arntl mRNA at midnight. We also found PZ treatment at 7 p.m. reduced Per1, Per2, Cry1, Clock, and Arntl mRNA as well as induced Nr1d1 mRNA at noon. Taken together, these findings reflect a normalization of the core clock mRNA profile under the influence of PZ treatment at 7 p.m.RF influenced the core clock genes’ expression profile. 3.2Nfe2l2, Pparg, and Cxcl5 at midnight in comparison with noon expression. In contrast, Rela mRNA was significantly reduced at midnight .3.5In RF‐untreated animals, we found a significant elevation of vascular congestion and interstitial cellular infiltrates Figure . PZ inta4Clock, Npas 2, Arntl, Per1/Per2, Cry1/Cry2, and Nr1d1/2), and Nr1/2/3. The protein products of these genes take part in the regulation of more than 80% of genes expressed throughout the body  and metabolism‐related genes . The RF reversed the profile of Nfe2l2 gene expression with the prevalence of noon expression over midnight expression accompanied by the loss of diurnal variation in Pparg expression. The transcriptional factor NRF2 contributed to the regulation of lung inflammation  may affect the values for total clock gene expression in the tissue.Further studies are needed to investigate chronic airway diseases and their interactions with the lung peripheral clock as well as the therapeutic potency of PPARG agonists. Additional experiments are needed with several time points (not only noon and midnight) to estimate circadian parameters  as well as with a model of prolongated (14 and more days) RF.5Nfe2l2 and Pparg, and exaggerating Rela and Cxcl5 expression accompanied by the elevation of IL‐6, TNF‐alpha concentrations as well as cellularity in BAL.We concluded that the RF disrupted the lung diurnal expression of core clock genes, alleviating Nfe2l2 and Pparg mRNAs, and alleviated Rela, Cxcl5 mRNAs, and IL‐6, TNF‐alpha, and cellularity in BAL. PZ treatment had a significantly higher chronopharmacological activity at 7 p.m. than at 7 a.m.PZ treatment repaired the diurnal clock gene expression, increased Oksana Shlykova contributed to investigation, methodology, and visualization. Olga Izmailova contributed to investigation, methodology, data curation, and statistical analysis. Alina Kabaliei and Vitalina Palchyk contributed to investigation and visualization. Viktoriya Shynkevych contributed to investigation, visualization, and writing—original draft. Igor Kaidashev contributed to project administration, methodology, data curation, visualization, writing—original draft and writing—review and editing.The study was supported by grants No. 0120U101166 “The study of the pathogenetic role of the circadian molecular clock in the development of metabolic diseases and systemic inflammation and the development of treatment methods aimed at these processes” and No. 0122U201686 “Development of methods for treatment and prevention of pulmonary fibrosis by activation of PPAR‐gamma receptors” funded by the Ministry of Public Health of Ukraine.The authors declared no competing interests.All experimental procedures were ethically approved by the Committee on Bioethics and Ethical Issues of Poltava State Medical University.


We report an atypical granular cell tumour (GCT) presenting in a 6‐year‐old boy. GCTs are of neural origin and most cases arise in patients between the ages of 40 and 60. There are few reported cases in children, in whom malignant presentations are exceptionally rare. This patient presented with a 1 year history of a slowly enlarging nodule on the right anterior abdomen. Examination revealed a firm, nodular dermal skin lesion, which was fully excised. Histology revealed an atypical GCT. We report a rare case of atypical granular cell tumour presenting in a 6‐year‐old boy. We report the presentation, excision, and histological findings of this unique case. Examination revealed a firm, nodular, dermal skin lesion measuring 20x11 mm. The skin overlying was erythematous , suggestive of atypical GCT. The nodule was fully excised with a 3 mm margin, including a cuff of Scarpa's fascia to which the lesion had adhered. Subsequent histology confirmed a well‐circumscribed but non‐encapsulated dermal tumour. The morphological impression of GCT was supported through strong S‐100 staining on immunohistochemistry. There were 7 mitoses per 10 high power fields (x200) and the mib proliferation index was approximately 5% , high nuclear to cytoplasmic ratio, and pleomorphism.The tumour extended deep to the scarpa's fascia, illustrating these tumours can behave in an invasive manner even if otherwise categorised as benign. Wide local excision and long‐term monitoring is recommended, as local recurrence is 2%–18%.This case report highlights the clinical features of an unusual paediatric presentation of an atypical GCT. Granular cell tumour should be amongst the differential for solitary subcutaneous nodule and this case demonstrates the importance of histological sampling. There is need for follow‐up guidelines for GCT.All authors declare no conflicts of interest.Yuma Kitahara: Project administration ; Writing – original draft (Lead); Writing – review & editing . Catherine Elizabeth Hook: Writing – review & editing . Kana Miyagi: Writing – review & editing . Nigel P. Burrows: Conceptualization (Lead); Project administration ; Writing – review & editing .Not applicable.


E. Mahé has undertaken paid activities as consultant, advisor or speaker for AbbVie, Amgen, Celgene, Janssen, Leo Pharma, Lilly and Novartis. A. Geldhof, M. Jazra, P. Bergmans and A. Azzabi are employees of Janssen and may own company stock/stock options. M.M.B. Seyger received grants from/was involved in clinical trials with AbbVie, Amgen, Celgene, Eli Lilly, Janssen, Leo Pharma and Pfizer; and served as a consultant for AbbVie, Eli Lilly, Janssen, Leo Pharma, Novartis, Pfizer and UCB, with fees paid directly to her institution.This study complied with ethics requirements as specified by the Independent Ethics Committee/Institutional Review Board and by local regulations. Each participant signed a participation agreement/informed consent form in line with local regulations and trial sponsor policy before data collection.Dear Editor,The European Medicines Agency approved ustekinumab for treating adolescent psoriasis patients in 2015,This 42‐month progress report of a real‐world, postapproval study (NCT03218488) comprised 70 patients enrolled between 25 October 2017 and the progress report data cut‐off (8 January 2021). Included patients were ≥12 to <18 years of age with a confirmed diagnosis of moderate‐to‐severe psoriasis who were initiated on ustekinumab treatment within 3 months before, or 2 months after, the first assessment. Patients enrolled in an interventional clinical trial were excluded. After baseline data collection, follow‐up visits were conducted every 3 months for the first year and at least every 6 months thereafter, for a maximum of 6 years or until the patient reached 18 years of age, early withdrawal or study closure. Serious infections, malignancies and adverse events related to autoimmunity were predefined serious adverse events of special interest (SAESI).n = 6; due to weight increase, n = 4; to improve both efficacy and safety, n = 1; for other reasons, n = 1). Nine patients (12.9%) stopped or interrupted ustekinumab therapy due to lack of efficacy (n = 2), lack of efficacy and adverse events , AEs  or did not attend further visits (n = 3).Most patients (76.8%) started ustekinumab at a dose of 45 mg (0.30–1.41 mg/kg); 12 patients (17.1%) required a dose increase , fatigue  and pyrexia  being the most frequently reported. Table A total of 184 AEs were reported by 39 patients (55.7%), with headache  patients Figure . FatigueThirteen serious AEs (SAEs) were reported in nine patients (12.9%), of which two were considered of special interest. The first SAESI was a case of appendicitis (serious infection) that was considered possibly related to ustekinumab; it was classified as severe and resulted in the interruption of ustekinumab treatment. The second SAESI was a case of severe worsening of pre‐existing plaque psoriasis (due to the relationship between plaque psoriasis worsening and autoimmunity), considered unrelated to ustekinumab. Both SAESIs resolved. The remaining SAEs were considered unrelated to ustekinumab treatment. No cases of malignancy or death were reported.The percentage of patients reporting at least one AE during ustekinumab treatment was lower in this study compared with the similar population in the CADMUS trial (55.7% vs 89.3%). This may reflect more limited recall of AE‐related information at patient visits which, for this study and the CADMUS trial, were every 6 and 3 months, respectively. This may have led to less complete capture of information, underreporting of AEs, or introduction of additional biases.In conclusion, this study identified no new safety concerns related to ustekinumab treatment in adolescent patients, with findings being generally consistent with the known safety profile for ustekinumab. After study completion in 2032, the results will provide a better understanding of the long‐term efficacy and safety profile of ustekinumab in adolescents with moderate‐to‐severe psoriasis.This article was sponsored by Janssen. Medical writing and editorial support were funded by Janssen.


Two loop diuretics, torsemide and frusemide, can affect the urinary system and consequently the cardiordiovascular haemodynamics in different ways.This study compared a number of echocardiographic parameters and systemic arterial blood pressure (ABP) changes following administration of furosemide or torsemide.Five shelter dogs underwent transthoracic two‐dimensional M‐mode echocardiography to obtain the following measurements: left ventricular internal dimension at end‐systole (LVIDs), left ventricular internal dimension at end‐diastole (LVIDd), fractional shortening (FS), heart rate (HR) and the distance between the mitral valve socket and the ventricle wall . Arterial blood pressure was measured using the oscillometric method. Measurements recorded before treatment (baseline data) were compared to those after the dogs received furosemide (5 mg/kg) or torsemide (0.5 mg/kg).Torsemide significantly reduced blood pressure 1 h after administration, but this was not seen with furosemide. Fractional shortening, LVIDd and SEP decreased following both treatments, but there were no significant differences between the treatment groups. Torsemide increased heart rate above that seen in the furosemide groups.The results of this study indicate that 1 h after administration, torsemide increases heart rate and decreases blood pressure when compared to furosemide, but both drugs have similar effects on measured cardiovascular indices. Cardiovascular effects of torsemide and frusemide were compared in dogs. Torsemide was more potent in decreasing blood pressure. Cardiovascular indices were the same for these two drugs. Each dog was also vaccinated against rabies.Echocardiography was performed on the five dogs before receiving any treatment to establish baseline values and then 1 h after receiving a single dose of furosemide at a dose of 5 mg/kg. Echocardiography was repeated 1 week later, 1 h after the dogs were given a single dose of torsemide at a dose of 0.5 mg/kg. During this week of wash out period, dogs did not receive any medications.To perform echocardiography, the right side of the chest was completely shaved between the 3rd to 6th intercostal spaces. Initially, by using the two‐dimensional B‐mode method, echocardiographic images were obtained from the right approach in the longitudinal axis. After identifying and evaluating the cardiac structures, transverse images were obtained by 90° transducer rotation, and the following measurements were obtained using M‐mode: left ventricular internal dimension at end‐systole (LVIDs), the left ventricular internal dimension at end‐diastole (LVIDd), fractional shortening (FS), heart rate (HR), and septal to E point (SEP). To measure LVIDs, the distance between the two peaks was measured in the M‐mode images, and the distance between the two near‐dips was measured to obtain the LVIDd. FS was calculated as ([LVIDd−LVIDs]/LVIDd) × 100  and a cuff size that was approximately 40% the circumference of the cuff site  were compared to posttreatment data using one‐way analysis of variance (one‐way ANOVA) and differences were considered significant when 3p = 0.047), SEP (p = 0.007) and LVIDd (p = 0.009). No significant changes were seen in HR, BP or LVIDs.The baseline and posttreatment cardiac and echocardiographic data are shown in Tables p = 0.031), SEP (p = 0.005) and LVIDd (p = 0.001), but there was also a significant increase in HR (p = 0.016) and a significant decrease in BP (p = 0.022).Treatment with torsemide also resulted in a significant decrease in fractional shortening (p = 0.84), SEP (p = 0.84), LVIDd (p = 0.28) or LVIDs (p = 0.45), but the HR was significantly higher following torsemide treatment (p = 0.007) and the BP was significantly lower.When the posttreatment effects of furosemide and torsemide were directly compared, there was no significant difference between the FS  decreases and generally leads to a reduction in the FS. However, if this moderate increase in the heart rate can actually affect FS directly, needs to be investigated in a bigger study group. In terms of the echocardiographic parameters, decreased LVIDd is followed by diminished diastolic end points due to decreased blood volume and reduced cardiac preload. It is indicated that fractional shortening is influenced by many parameters including preload, afterload and contractility, all of which may act independently or in combination to affect it (Gugjoo et al., 5The results of this study indicate that the use of torsemide leads to a greater reduction in blood pressure than furosemide, while both drugs have similar impacts on the other measured cardiovascular parameters. In this study, only a single dose of each diuretic in a few dogs was used and a long‐term study using different dosages of these diuretics in a larger group is required to perform a more comprehensive comparison between the effects of torsemide and frusemide. Moreover, absorption of frusemide and torsemide may be affected by poor gastrointestinal perfusion due to heart failure and findings of this study cannot be applied to clinical patients.Farzaneh Hosseini: investigation; supervision; writing – review & editing. Morteza Hosseininejad: funding acquisition; investigation; methodology; project administration; supervision; writing – original draft; writing – review & editing.The authors declare no conflicts of interest.guide for the care and use of laboratory animals (Chetboul et al., Because the medications used in this study revealed to be safe for the dogs in previous studies, and based on the https://publons.com/publon/10.1002/vms3.1129The peer review history for this article is available at