Glioblastoma (GBM) is an incurable form of primary astrocytic brain tumor driven by glioma stem cell (GSC) compartment closely associated with the vascular niche. GSC phenotypes are heterogeneous and range from proneural to mesenchymal-like, the latter characterised by greater invasiveness. Here we document the secretory (angiocrine) role of endothelial cells and their derived extracellular vesicles (EVs) as drivers of proneural-to-mesenchymal reprogramming of GSCs. These changes involve activation of matrix metalloproteinases (MMPs) and NFκB, and inactivation of NOTCH, while altering responsiveness to chemotherapy and driving infiltrative growth in the brain. Our findings suggest that EV-mediated angiocrine interactions impact the nature of cellular stemness in GBM with implications for disease biology and therapy. Glioma stem-like cells (GSCs) exhibit plasticity during proneural-to-mesenchymal transition. Here the authors show that extracellular vesicles from endothelial cells can promote this transition of GSCs through activation of MMPs and NFkB, and inactivation of NOTCH. The gene expression profiles of GSCs are reminiscent of the corresponding GBM subgroups7 and imply numerical preponderance8. Notably, patients with mesenchymal GBM tend to have a poorer prognosis relative to patients with proneural GBM7  is an aggressive and incurable brain tumor with pronounced vascularity. GBM is thought to arise from glioma stem-like cells (GSCs) of which at least two subtypes have been identified and described as either proneural (PN) or mesenchymal (MES)18. Interrelationships between GSCs and endothelial cells have also emerged in the context of angiogenesis20 and other processes driving tumor neovascularization23, with ongoing identification of their respective mediators17. On the other hand, endothelium is regarded as a secretory tissue, the paracrine (angiocrine) effects of which are thought to be mediated by either soluble proteins, or multimolecular carriers, such as extracellular vesicles (EVs) including exosomes24. Indeed, the endothelial secretome has recently emerged as a potent regulatory mechanism for cancer cells, their niches, and across tumor microenvironment27. However, the present understanding of the nature and scope of angiocrine influences on different subsets of cancer cells25, stem cells28 and GSCs remains relatively limited16.Interactions with the brain vasculature represent a hallmark of both neural stem cells and GSCs, epitomized by formation of endothelial stem cell nichesHere, we show that endothelial cell derived EVs (EEVs) not only harbor the ability to modify GSC behavior, but also potentiate a switch in GSC subtype. Specifically, the conditioned medium derived from endothelial cells prevents sphere formation, reduces proneural hallmarks, induces mesenchymal hallmarks, causes increased migration of proneural GSCs, and facilitates increased tumorigenicity in mice xenografted with proneural GSCs pre-treated with endothelial cell derived conditioned media relative to the controls. These findings are in harmony with the results obtained when proneural GSCs were treated with endothelial cell derived EVs (EEVs). We have identified MMPs as a key cargo component of EEVs, which when taken up by proneural cells inaugurates the mesenchymal signaling pathway, NFκB. This study sheds new light on the alteration of proneural GSCs not after an intervention, such as surgical resection, radiation or chemotherapy, but during the development of a naïve primary GBM. This change entails EEV uptake by GSCs which leads to the downregulation of the NOTCH pathway and an upregulation of NFκB.16. Indeed, co-staining of human GBM sections for NES  and CD31  enforces such a spatial association  and mesenchymal-GSCs (loose spheres) in culture. Thus, sphere cultures of several mesenchymal-GSCs (GSC83 and GSC1005) and proneural-GSCs (GSC157 and GSC1079) were incubated in the presence of endothelial cells, including brain endothelial cell line . The ability to form tumor spheres in serum free media represents a defining feature of GSCs, which is often used to interrogate their clonogenic and tumor initiating potentials24  is contact-dependent or paracrine in nature. To address this question proneural-GSC1079 spheres were incubated in conditioned media derived either from endothelial cells (CME) or their own sphere culture (CMO) for 7 days, while their responses were continuously monitored using Incucyte imaging Fig. . While C28. However, in our hands, components of the endothelial cell secretome (CME) disrupted GSC sphere formation suggesting a possibility of a paradoxical (negative) impact on cellular clonogenicity. To explore this further, we conducted time-dependent ELDA screens of clonogenic proficiency in a series of proneural GSCs  and mesenchymal GSCs , which revealed considerable subtype-dependent differences. In line with their sphere disrupting effects, CME preparations markedly suppressed and delayed clonogenicity of proneural GSCs with a considerably less pronounced impact on their mesenchymal GSC counterparts . Cancer cell stemness is often enhanced by their interactions with endothelial cellsrts Fig. . Apart frts Fig.  and 9a. 31 is largely guided by their resemblance of gene expression pattern described in TCGA and subsequent GBM classifications32  and supernatant (Sup) using optimized ultracentrifugation protocol36 . Proteomic analysis of EEVs identified several highly enriched proteins including FN1 and HSPG2, which are involved in the EV uptake and induction of cell motility38  play important roles in bidirectional tumor-stromal communicationl36 Fig. . The EEVl36 Fig.  along wiy38 Fig. .Fig. 4Ex36. To ascertain whether this is the case, we adopted the previously developed Cre–loxP reporter system where EV mediated transfer of Cre recombinase triggers the expression of fluorescent proteins in recipient cells40. In this case, ECs were transduced with Cre-mCherry expressing lentivirus, while recipient GSC157 were transduced with a DsRed/loxP/eGFP dual Cre-sensitive reporter. Indeed, 4 days after exposure to Cre-EEVs the recipient GSC157 cells turned on eGFP expression (signifying Cre-EV uptake), which was not observed in the presence of control OEVs, or Cre-EC Supernatant  and their responses were recorded using Incucyte in real time for up to 4 days  chemotherapy, with mesenchymal GSCs being more sensitive relative to their proneural counterparts15 Fig. . Therefo15 Fig. . This is41, we blocked NOTCH pathway in proneural GSC157 cells using γ-secretase inhibitor, DAPT  is an important part of their effect on proneural GSCs  on recipient cell surface18. It is therefore plausible that the homotypic cell-cell interactions of GSC157 cells, or abundance of GSC EVs (OEVs) facilitate the juxtacrine NOTCH signaling and maintenance of the proneural phenotype (with high hNICD levels). This process could be disrupted by excess of competing EEVs when cancer cells infiltrate the surrounding brain  may lead to their homing to tumor core, edge, and distant brain parenchyma, including perivascular regions, each with considerable consequences to tumor-tumor and tumor-vascular interactionsain Fig. , especiaain Fig. . To testain Fig. . To undeain Fig. . This su+) in the case of inoculates pre-treated with CME, relative to CMO  and immortalized human brain endothelial cell line (HBEC-5i) were purchased from American Type Culture Collection . Human primary brain microvascular endothelial cells (BMECs) were purchased from Cell Biologics# H-6023. HUVECs and BMECs were cultured in bullet kit media and supplements (Lonza#CC-3162). HBEC-5i were cultured in DMEM-F12 media supplemented with 10% FBS, 1% P/S and 40 μg/mL endothelial growth supplement (ECGS) (Sigma E2759). All the endothelial cells were grown as monolayers on 0.1% Gelatin (Sigma#G9391). HEK-293  and NHA  were cultured in DMEM media supplemented with 10% FBS and 1% Penicillin-streptomycin. All the cell lines purchased from ATCC or Cell Biologics were tested and validated by the respective company. Depleted media used to condition the cells and isolate their secretomes comprised DMEM-F12 and 0.125% EV-depleted FBS. GSCs, HUVECs, HBEC-5i and BMECs were cultured in depleted media for 3 days in vitro to obtain GSC specific conditioned media  and HUVEC/HBEC5i/BMEC specific conditioned media . All the conditioned media (CM) were subjected to a 2000 X g centrifugation for 20 min to sediment cell debris prior to use. For all CM experiments, GSCs were cultured in either OCM or ECM at a 1:1 ratio with FM. Most of the CM experiments were 7 days long, unless mentioned otherwise. All cells were cultured in a 37 oC incubator with 5% CO2. For co-culture assay, monolayered HUVECs were labeled with carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher#C34554) and GSCs labeled with PKH26 dye (Sigma# PKH26GL-1KT) were added in a 1:1 ratio and cells were cultured for 7 days.Patient derived GSC lines, namely, (GSC) 83, 1123, 1005, 528, 157, 84 and 1079 were isolated from GBM surgical samples, obtained in the laboratory of Dr. Ichiro Nakano. All the cell lines were grown in serum free media in suspension. The GSC culture media  comprised of DMEM-F12 basal media (GIBCO#11320033), EGF (GIBCO#PHG0311L), FGF (GIBCO#PHG0261), Heparin 0.2% (STEMCELL#07980), B27 (GIBCO#17504044), Glutamax (GIBCO#35050061) and 1% Penicillin-streptomycin (GIBCO#15070063). The gene and protein expression studies are periodically performed in houseg for 19 h to pellet EVs. Supernatant FBS was filtered through a 0.2 µm filter. This FBS was considered EV depleted. It was used to prepare starvation media for CM experiments.FBS was centrifuged at 100,000 × The following inhibitors were used, as indicated: MMP inhibitors - BB94 , AG3340 , NFkB inhibitor—Bay11-7082 , NOTCH inhibitor—DAPT . Images were obtained at 10 X every 30 min for 7 days. Cell sharpness (cell adherence) and eccentricity (change in cellular morphology) were analyzed using the standard Incucyte analysis software. Images were acquired using bright field, red and green fluorescence.The xCelligence RTCA MP instrument (ACEA Biosciences) was used to measure cell impedance (cell adherence). The E-plate VIEW 16 (ACEA Biosciences# 6324738001) was used to plate cells at a clonal density in OCM or ECM cultured for 7 days.http://bioinf.wehi.edu.au/software/elda/. Endpoint was reached once all wells contained a tumor sphere, or if no additional tumor spheres formed after 3-7 additional days of incubation post endpoint.Cells were seeded at different densities (1–40 cells/well) in 96 well plates. Up to 12 wells for 1 cell and 6 wells per 5-, 10-, 15-, 20-, 25-, 30-, 35-, and 40-cells were seeded using FACS. Cells were cultured in 100 μL of either CME , CMO , or FM for 1–6 weeks as indicated. Cells were fed every 15 days with 100 μL of respective CM/FM media. At each timepoint, the number of wells with at least one tumor sphere (clusters/spheres comprising of >20 cells) were counted. The frequency of cluster-initiating cells was calculated by an ELDA online program at n = 5). Cells were left to incubate for 3, 5, or 7 days as indicated. MTS reagent corresponding to 1/10th the total volume present in a given well was used to measure cell growth/survival in presence/absence of endothelial-cell (HUVEC) conditioned media. After 2 h of treatment, the absorbance at 490 nm was read using a plate-reader. The readings were normalized to the MTS readings obtained using CMO alone and CME alone without the presence of cells. MTS for temozolomide  was done after 24 h incubation with TMZ.500 cells/well of a given cell-line were seeded in 96 well plates with 100 μL of either CME  or CMO  media to afford 5 technical replicates  was injected i.v. 30 m prior to euthanizing the mouse. Brains from mice were harvested and put in cold PBS. The brains were sectioned at 200 μm using a vibratome (Leica VT 1200 s). The tissues were placed in a µ-Dish 35 mm, high Glass Bottom dish  and taken for high resolution confocal microscopy . An end point of a mouse with intracranial injection was established based on the first sign of neurological symptoms such as circling or dehydration. No decline in well being was allowed. All mice were maintained at MUHC RI and McGill University animal care facility, monitored daily, under 12 h of light/12 h of dark cycle. All procedures comprising animals were conducted in accordance with the guidelines of the Canadian Council of Animal Care and the Animal Utilization Protocols, approved by the Institutional Animal Care Committee at the Research Institute of the McGill University Health Center and McGill University.Orthotopic injections were carried out by injecting 2 μl of 25,000 GSCs stereotactically into the brains of 3 month old NOD.Cg-PrkdcSCID Il2rgtm1Wjl/SzJ (NSG) mice (Charles River Labs). For staining blood vessels, 54. Briefly, aortas of 2 month old C57bl/6 (Charles River Labs) mice were collected and ~1 mm rings were cut and placed in the BME matrix  in a 12-well dish. After the ring solidified in the BME, aortic ring media containing DMEM/F12 , 1X Penstrep , 2% FBS  and ECGS  were added to cultures. After 3d, when the endothelial cell sprouts developed, GFP labeled GSCs were incorporated in BME and placed at the edge of the well containing aortic rings. Images were taken 3 days after co-culture initiation.Aortic ring sprouts were prepared as described previouslyGSCs were grown as monolayers in 12-well dishes coated with poly-l-ornithine  and laminin . A scratch was generated by running a P1000 sterile pipette tip across the confluent GSC culture. Respective media was added in each well and images were recorded over time using Incucyte S3 live analysis system (EssenBio).3. Briefly, the respective conditioned media were cleared of cells at 300 × g for 5 min, and dead cells/debris was further eliminated by centrifugation at 2000 × g for 20 min, after which the supernatant was concentrated by spinning at 3500 × g for 20 min using Amicon Ultra-15 Centrifugal Filter Units −100 KDa-  to a final volume of 1 ml. The 1 ml Concentrate was subjected to ultracentrifugation at 110,000 × g for 1 h using a tabletop ultracentrifuge (Beckman TLA100.2 rotor). The pelleted EVs were washed with sterile PBS  at 110,000 × g for 1 h in the ultracentrifuge suspended in sterile PBS or RIPA buffer and analysed.EVs were purified as previously describedParticle number and size were investigated using NS500 nanoparticle tracking analysis system . 1:1000 aliquots were loaded into the chamber. Three recordings of 30 s each were obtained under automatic detection and processing settings at 37 °C with camera level at 15, detection threshold of 5 and blur size set by auto by NTA software (version 3.0).g for 1 h 30 m. After overnight incubation, respective media was changed. Three days following spinoculation, cells were washed three times in sterile PBS and cells exhibiting red fluorescence were sorted by FACS in the case of both, GSCs and HUVECs.pLV-CMV-LoxP-DsRed-LoxP-eGFP  lentiviral vector was transduced into the PN-GSC157 cells. pLM-CMV-R-Cre  lentiviral vector was transduced into HUVECs. Briefly, cells were plated at 10,000 cells per well in a 24 well dish. Based on titration, 10μl of the prepared virus was added to the cells and subjected to spinoculation at 800 × oC. Protein concentrations were assessed using the Pierce Micro BCATM Protein Assay . Using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), lysates were resolved. Transfer of the resolved proteins onto polyvinylidene difluoride membranes  was performed at 30 V overnight. Membranes were blocked for 1 h in 5% milk or 5% BSA and probed with primary antibodies and respective horseradish peroxidise (HRP)-conjugated secondary anti-mouse , anti-rabbit  or anti-goat  antibodies. Chemiluminescence  was visualized using ChemiDoc MP system (Biorad). Primary antibodies used included: rabbit anti-CD63 , rabbit anti-CD9 , rabbit anti-CD81 , rabbit anti-BIP , rabbit anti-NOTCH1 , rabbit anti-SOX2 , rabbit anti-NES , rabbit anti-TGM2 , rabbit anti-NICD , goat anti-VIM , rabbit anti-P65 , rabbit anti-pP65  and mouse anti-βactin .Total cell proteins and EV proteins were isolated using RIPA buffer  containing protease inhibitor . After incubation on ice for 30 m, the lysates were centrifuged at 15,000 x g for 5 m at 4oC for 15 min in unmasking solution  for antigen retrieval. Primary antibodies: rabbit anti-CD31  and mouse anti-NES  were incubated overnight at 4oC. The tissues were washed for 5 min three times in PBS. The respective fluorescent secondary antibodies, purchased from Invitrogen, were incubated with tissues for 1 h at 37 oC. Secondary antibodies: goat anti-rabbit IgG Alexa Fluor 488  and donkey anti-goat IgG Alexa Fluor Plus 594 . Tissues were washed for 10 min three times, then the slides were cover slipped using DAPI mounting reagent .All brain tissues were preserved in 4% PFA after dissections. Tissues were passed through the tissue processor (Leica TP 1050) and embedded in paraffin blocks from which 5 μm thick sections were cut using a microtome . Tissues were de-waxed and re-hydrated in a series of 5 min steps involving Xylene and 95%- >50% ethanol. Tissues were further processed by heating at 95GSC157 cultures were treated with CMO, CME, OEVs and EEVs for 7 days, then stained for CD44  and IgG control. For the CFSE experiment, purified EVs were stained with CFSE  prior to incubation with cells. Cells were treated with CFSE labeled EVs for 24 h followed by FACS analysis. The cells were subjected to flow cytometry (BD LSR Fortessa) and data was analysed using the FlowJo software 10.7.1. For ImageStream, GSC157 cells were incubated with OEVs pre-stained with PKH26  and EEVs pre-stained with DiD . 24 h later, NucBlue  was added and GSCs were subjected to Image Stream analysis (Amnis ImageStream Mark II).3. Briefly, data was collected using the Thermo Orbitrap Fusion mass spectrometer operating at 120,000 resolution (FWHM in MS1) with HCD sequencing . Data was analyzed using Prism 9. The raw data were converted into *.mgf format (Mascot generic format) for searching using the Mascot 2.6.2 search engine (Matrix Science) against Human Uniprot sequences (2020). The database search results were loaded onto Scaffold Q + Scaffold_4.9.0 (Proteome Sciences) for statistical treatment and data visualization. Analysis for biological pathways found in highly enriched proteins was performed using DAVID: https://david.ncifcrf.gov platform.Proteomics was done as previously describedProtein lysates (10-15 μg) were processed using the MMP analysis kit  as per the manufacturer’s instructions.oC with agitation. Preparations were then subjected to ultracentrifugation, 110,000 × g for 1 h to pellet the treated EEVs. GSC157 was then incubated with the KCl treated EEVs and analysed as indicated.EEVs were prevented from internalization by exposing the proneural-GSC157 cells to heparin (10 μg/ml) for 1 h prior to EEV treatment. EEVs were stripped off the outer proteins by treating 1.5 ml of EEVs with 1.5 ml of 2 M KCl solution for 30 min at 4 t test analysis was used to compare two groups while one way ANOVA was used for comparison of more than two groups of data sets using GraphPad Prism v9. XCelligence data are plotted as average and standard deviation of well replicates. Every experiment was independently repeated > /= 3 times  and each experiment had > /= 5 technical repeats. The results were considered not significant (NS) when P > 0.05, or significant when P < 0.05 (*); P < 0.01 (**); P < 0.001 (***) and P < 0.0001 (****). All graphs were plotted to show the standard deviation of replicates.Standard unbiased Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting Summary


The association between out-of-hospital cardiac arrest patient survival and advanced life support response time remained controversial. We aimed to test the hypothesis that for adult, non-traumatic, out-of-hospital cardiac arrest patients, a shorter advanced life support response time is associated with a better chance of survival. We analyzed Utstein-based registry data on adult, non-traumatic, out-of-hospital cardiac arrest patients in Taipei from 2011 to 2015.Patients without complete data, witnessed by emergency medical technicians, or with response times of ≥ 15 minutes, were excluded. We used logistic regression with an exposure of advanced life support response time. Primary and secondary outcomes were survival to hospital discharge and favorable neurological outcomes , respectively. Subgroup analyses were based on presenting rhythms of out-of-hospital cardiac arrest, bystander cardiopulmonary resuscitation, and witness status.A total of 4,278 cases were included in the final analysis. The median advanced life support response time was 9 minutes. For every minute delayed in advanced life support response time, the chance of survival to hospital discharge would reduce by 7% and chance of favorable neurological outcome by 9%. Subgroup analysis showed that a longer advanced life support response time was negatively associated with the chance of survival to hospital discharge among out-of-hospital cardiac arrest patients with shockable rhythm and pulse electrical activity groups.In non-traumatic, adult, out-of-hospital cardiac arrest patients in Taipei, a longer advanced life support response time was associated with declining odds of survival to hospital discharge and favorable neurologic outcomes, especially in patients presenting with shockable rhythm and pulse electrical activity. Out-of-hospital cardiac arrest (OHCA) is a major disease worldwide, with a high mortality rate. Taiwan has approximately 9,815 cases of OHCA annually, with a survival rate of approximately 9.8% . SeveralThe effect of advanced life support (ALS) treatments in prehospital settings remains controversial. Several studies reported that earlier ALS intervention is associated with increasing survival rate –16, whilRegarding the EMS system in Taipei, previous studies showed that the intervention of ALS and the number of ALS personnel are associated with better outcomes in OHCA patients , 23–27; We conducted a 5-year retrospective cohort study using prospectively collected Utstein-based registry data from the Taipei EMS to investigate the association between the response time of ALS care and OHCA patient outcomes. All methods were performed in accordance with the study protocol which was approved by the Institutional Review Board (IRB) of the National Taiwan University Hospital (201606007RIND). Informed consent was waived due to the anonymized database and retrospective nature of the study, which was also approved by IRB of the National Taiwan University Hospital. The preliminary version of the abstract had been published at the European Resuscitation Council annual conference 2019 in Slovenia before we develop it into a full-length article.The Utstein-based OHCA registry system from the Taipei EMS was initially developed for OHCA quality control . The reg2, with 2.6 million registered residents. The majority of the population is Taiwanese. Taipei City has an EMS system based on the fire service with a two-tiered response team, including a basic life support (BLS) team and an ALS team. Taipei City has 45 prehospital BLS stations with 1,279 emergency medical technician (EMT) intermediate staff, who have completed at least 320 hours of training; and four ALS stations with 141 EMT paramedics, who have completed at least 1280 hours of training and need to conduct OHCA re-training every year  Q1–Q3). We performed chi-squared or Fisher’s exact tests to assess the associations between the categorical variables and outcomes. For continuous variables, we conducted non-parametric Mann–Whitney rank sum tests for analyses. All variables previously determined to be associated with the outcomes were included in the multivariable logistic regression analysis to prevent overfitting. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and two-tailed p-values < 0.05 were considered statistically significant.We conducted a subgroup analysis using the new Utstein template, with methods suggested by the International Liaison Committee on Resuscitation (ILCOR) in 2014, to explore the effect of ALS response time among different subgroups of patients with OHCA. For this analysis, we stratified the data based on presenting rhythms of OHCA, including shockable rhythm (pVT/Vf), pulseless electrical activity (PEA), and asystole . We alsoOf the 16,062 OHCA cases treated between 2010 and 2015, 7,571 cases were adult, non-trauma OHCA without EMT witness with resuscitation attempted and 4,278 cases with ALS dispatch were included in the final analysis.  The propThe restricted cubic spline curve in In this large observational study in Taipei, we found that every minute of delay in ALS response time was associated with a 7% reduction in survival to hospital discharge and a 9% reduction in favorable neurological outcome in adult, non-traumatic, OHCA patients. A swift response by ALS not only benefited all OHCA patients, but also significantly benefited the subgroups of patients initially presenting shockable rhythms or PEA. An ALS response time of less than 8 minutes was associated with better outcomes in OHCA patients, while over 11 minutes was associated with a diminished chance of survival. We previously announced the preliminary results of this study [Our findings are similar to those of Grunau et al. , with neThere are several reasonable explanations for early ALS team response time improving the rate of survival to hospital discharge and neurological outcome among OHCA patients. ALS treatment includes airway management and drug administration. Network meta-analysis of randomized control trials reveal that ET tube placement and supraglottic airway (SGA) do increase the rate of ROSC compared to BVM ; the sucSome studies have opposed ALS treatment. The OPALS study compared the before and after of implementation of ALS paramedics into EMS in Canada. The results revealed no improvement in the rate of survival to hospital discharge and functional outcome in OHCA patients after ALS intervention . AlthougOur study had several limitations. First, this was a retrospective observational study. While selection bias inherent to our study design cannot be eliminated, we believe it was mitigated by our population-based approach. While we adjusted for common OHCA confounders, additional unmeasured or unmeasurable confounders may have been present, such as in-hospital care, which may have influenced OHCA survival , 57. SecIn non-traumatic, adult, OHCA patients in Taipei, a longer ALS response time was associated with worse odds of survival to hospital discharge and favorable neurological outcomes, especially in patients presenting with shockable rhythm and PEA.Our study further suggests the optimal ALS response time for the EMS system.S1 TableALS: advanced life support. BLS: basic life support. CPR: cardiopulmonary resuscitation. EMS: emergency medical service. EMT: emergency medical technician. OHCA: out-of-hospital cardiac arrest. OR: odds ratio. pVT: pulseless ventricular tachycardia. PEA: pulseless electrical activity. STHD: survival to hospital discharge. VF: ventricular fibrillation.(DOCX)Click here for additional data file.S2 Tablea: cannot performed due to N = 1 in group CPC1-2.ALS: advanced life support. BLS: basic life support. CPC: cerebral performance category. CPR: cardiopulmonary resuscitation. EMS: emergency medical service. EMT: emergency medical technician. OHCA: out-of-hospital cardiac arrest. OR: odds ratio. pVT: pulseless ventricular tachycardia. PEA: pulseless electrical activity. VF: ventricular fibrillation. (DOCX)Click here for additional data file.


SC was measured at four timepoints under three noise interference conditions each : after screening , HA- fitting (t1), additional auditory training (t2), and at 70-day follow-up (t3). Repeated-measure analyses of covariance investigated the effects of HAs (t0–t1), auditory training (t1–t2), and the stability of the combined effect (t2–t3) on SC per noise interference level and HL subgroup. Correlational analyses examined associations between SC, age, and psychological indices. Results: Patients showed mildly elevated tinnitus-related distress, which was negatively associated with SC in patients with mild but not moderate HL. At 0 dB, the intervention lastingly improved SC for patients with mild and moderate HL; at 55 dB, for patients with mild HL only. These effects were mainly driven by HAs. Conclusions: The here-investigated treatment demonstrates some SC-benefit under conditions of no or little noise interference. The auditory training component warrants further investigation regarding non-audiological treatment outcomes.Background: Patients with chronic tinnitus and mild-to-moderate hearing loss (HL) can experience difficulties with speech comprehension (SC). The present study investigated SC benefits of a two-component hearing therapy. Methods: One-hundred-seventy-seven gender-stratified patients underwent binaural DSL Tinnitus has been defined as “the conscious awareness of a tonal or composite noise for which there is no identifiable corresponding external acoustic source” . PrevaleSimilarly, research investigating the effects of auditory training on HL or SC suggests only small improvements across outcome domains such as speech intelligibility, cognition , or selfN = 177 patients with chronic tinnitus and mild-to-moderate HL  = 3.25, p < 0.05, p²η = 0.03, small effect) and moderate HL , both of which appeared to be driven by the time × age interaction effects of the HAs .At 0 dB noise interference, the intervention yielded significant time × age interaction effects for patients with mild  but not moderate HL. Post hoc analyses revealed no significant change but a weak trend (p = 0.06) for the t0–t1 effect of HAs.At 55 dB noise interference, the intervention yielded a significant main effect of time for patients with mild  and moderate HL .To further investigate the time × age interaction effect on patients’ SC in silence, correlational analyses revealed an association between higher age and higher HA-related improvement in patients with mild .We further conducted two (patients with mild vs. moderate HL) × two  vs. old) additional rmANCOVAs which found that, for patients with both mild and moderate HL, significant medium- and large-sized main effects of time emerged for older but not younger individuals. Post hoc analyses revealed significant improvements between t1: Ftime  = 8.05, 1: Ftime  = 14.32,r = −0.29, p < 0.05) but not younger patients  showed an association between higher HA-related improvement and higher TFI-measured TRD.Moreover, correlational analyses for younger vs. older patients with mild vs. moderate HL showed that in patients with mild, but not moderate HL, older (© hearing therapy for patients with mild (n = 124) and moderate (n = 53) HL on SC at four timepoints: after screening , after HA fitting (t1), after additional auditory training (t2), and at 70-day follow-up (t3) for three noise interference conditions . The intervention comprised binaural DSLchild-algorithm-based HA fitting and 14 days of daily, CD-enhanced auditory training exercises.The present study investigated the effects of TerzoCompared to patients with mild HL, patients with moderate HL were significantly older and had higher SC difficulties in silence and under noise interference—yet comparable, mildly elevated, levels of TRD. These findings are in agreement with previous studies that reported positive associations between age and HL  as well In patients with mild HL, lower SC was associated with (a) higher TFI-measured TRD at 0 dB noise interference; (b) higher anxiety and psychological distress, as well as higher TQ-THI- and TFI-measured TRD at 55 dB noise interference in this subgroup only; and (c) higher TFI-measured TRD at 65 dB noise interference in this subgroup only.Against the backdrop of overall mildly elevated TRD levels , the herInterestingly, an association between TRD and SC emerged only in patients with mild but not moderate hearing loss. This finding elucidates the importance of conjointly considering the degree of HL and psychological distress when operationalising SC success or planning HA fittings . Future Higher HA-related improvements of SC were associated with (a) older age and lower perceived stress at 0 dB noise interference, and (b), in this subgroup only, higher TFI-measured TRD at 55 dB noise interference.In line with existing recommendations , this fiDespite the overall low levels of psychological distress, the association between the beneficial effects of HAs on SC and lower perceived stress suggests the influence of psychological factors on the effectiveness of audiological interventions ,59 and vIn patients with mild HL, higher auditory training-related improvements were associated with higher TRD and perceived stress.© HA fitting (cf. below), its effect was associated with higher levels of psychological distress at baseline. Against the backdrop of our predecessor paper, which reported overall psychological benefits following hearing therapy [Although the auditory training did not significantly improve patients’ SC beyond the effects of the Terzo therapy , the pre therapy ,63, the Moreover, higher stability of the combined effect was associated with lower TRD, perceived stress, anxiety, depressivity, and psychological distress at the baseline, again highlighting the potential importance of psychological contributors to the effects of the here-investigated hearing therapy on SC .© hearing therapy yielded a medium-sized, significant time × age interaction effect at 0 dB noise interference. This effect was driven by an age-dependent effect of HAs.Terzo© hearing therapy resulted in a small-sized, significant main effect of time at 55 dB noise interference that was driven by a trend effect of HAs.Exploratory additional analyses suggested a main effect of HAs for older but not younger patients with mild HL. Moreover, TerzoThis finding is in agreement with the literature emphasizing age in the process of HA fitting  and suggIn patients with moderate HL, higher SC was associated with higher TQ-measured TRD at 0 dB noise interference.Although preliminary, the tinnitus-related or broader emotional distress might modulate executive control towards heightened processing of external stimuli in the context of moderate hearing loss, for example by means of altered executive control or allocation of attentional resources ,66,67. IHigher HA-related improvements were associated with (a) lower TQ-measured TRD at 0 dB noise interference, and (b) lower THI-measured TRD at 55 dB noise interference.In line with previous studies investigating psychological influences on successful HA use ,69, psyc© hearing therapy yielded a large-sized, significant time × age interaction effect at 0 dB noise interference, which was driven by an age-dependent effect of HAs. Additional exploratory analyses again suggested the main effect of HAs for older but not younger patients at 0 dB noise interference.TerzoThe current study has important limitations, most notably the absence of control group analyses and the non-stratification of included patients by psychological distress. Importantly, owing to the study’s inclusion and exclusion criteria, the current results cannot be generalised to individuals with severe hearing loss.© HA fitting on SC for patients with chronic tinnitus and mild or moderate HL under conditions of little or no noise interference. At medium-level noise interference, HAs benefit patients’ SC in context of mild but not moderate HL. Psychological variables appear to interact with the degree of peripherally mediated HL in influencing both patients’ SC and HA-related benefits, however inconclusively so. Overall, low psychological distress rates prohibited further examination of affective–cognitive influences; thus, interactions of emotional distress and intervention benefits (including the duration of HA-use) remain to be investigated in future studies.In summary, the results of the present study revealed benefits of Terzo


The intestinal microbiota is essential in absorbing nutrients and defending against pathogens and is associated with various diseases, including obesity, type 2 diabetes, and hypertension. As an alternative medicine, Traditional Chinese Medicine (TCM) has long been used in disease treatment and healthcare, partly because it may mediate gut microbiota. However, the specific effects of TCM on the abundance and interactions of microbiota remain unknown. Moreover, using TCM ingredients and data detailing changes in the abundance of gut microorganisms, we developed bioinformatic methods that decipher the impact of TCM on microorganism interactions.The dynamics of gut microorganisms affected by TCM treatments is explored using a mouse model, which provided the abundance of 70 microorganisms over time. The Granger causality analysis was used to measure microorganism interactions. Novel “serial connection” and “diverging connection” models were used to identify molecular mechanisms underlying the impact of TCM on gut microorganism interactions, based on microorganism proteins, TCM chemical ingredients, and KEGG reaction equations.Codonopsis pilosula (Dangshen), Cassia twig (Gui Zhi), Radices saussureae (Mu Xiang), and Sijunzi Decoction did not cause an increase in the abundance of harmful microorganisms. Most TCMs decreased the abundance of Bifidobacterium pseudolongum, suggesting a Bifidobacterium pseudolongum supplement should be used during TCM treatment. The Granger causality analysis indicated that TCM treatment changes more than half the interactions between the 70 microorganisms, and “serial connection” and “diverging connection” models suggested that changes in interactions may be related to the reaction number connecting species proteins and TCM ingredients. From a species diversity perspective, a TCM decoction is better than a single herb for healthcare. The Sijunzi Decoction only significantly increased the abundance of Bifidobacterium pseudolongum and did not cause a decrease in the abundance of other species but was found to improve the alpha diversity with the lowest replacement rate.Because most of the nine TCMs are medicinal and edible plants, we expect the methods and results presented can be used to optimize and integrate microbiota and TCMs into healthcare processes. Moreover, as a control study, these results can be combined with future disease mouse models to link variations in species abundance with particular diseases. TCM medp < 0.01 and accept the hypothesis when x2 causes x1 at the level of p < 0.01. For the python function “grangercausalitytests,” there are four kinds of tests for the null hypothesis: two tests are based on the F distribution, “params_ftest” and “ssr_ftest,” and two are based on the χ2 distribution, “ssr_chi2test” and “lrtest.”The interactions of microorganisms contribute to the stability of the microbiome. The strength of this interaction can be measured by the Granger causality approach, which identifies causality between different time series Granger, , can be To benefit healthcare and intestinal disease TCM treatment, we used the bio-statistic and Granger causality methods to explore the impact of TCM on gut microorganism abundance and interactions using nine TCMs and standard mouse gut microorganism sequencing data. Moreover, by novel serial and diverging connection models, we also deduced possible reaction pathways underlying the impact of the interactions.Codonopsis pilosula , Poria cocos , Rhizoma dioscoreae , Radices saussureae , Rhizoma Zingiberis , Cassia twig , Magnolia officinalis , White Atractylodes rhizome , Poria cocos , Liquiritiae glycyrrhizae , Astragalus mongholicus , and Chinese angelica .The TCMs were obtained from Kangmei Pharmaceutical Industry Co., Ltd, with batch numbers (BN) as follows: Codonopsis pilosula may affect small intestinal propulsion movement , Poria cocos (Fuling), Rhizoma dioscoreae (Shan Yao), Rhizoma zingiberis (Ganjiang), Liquiritiae glycyrrhizae (Gan Cao), Astragalus mongholicus (Huangqi), and Chinese angelica (Dang Gui), are medicinal and edible plants. TCM samples listed in Initially, we weighed seven single dried Chinese herbs and two compounds used in intestinal treatment  C57BL/6J mice aged 6 weeks. The mice were housed in the SPF animal breeding room of KMHD under License No. SYXK (Yue) 2019-0205, at a temperature of 23–25°C, moisture 54–57% and a 12-h light/dark cycle. This experimental study was approved by the Ethics Committee of KMHD under Approval No. IACUC-0210831-1.After feeding for a week, 198 mice were randomly grouped into 11 groups, with each group consisting of 18 mice. Among the 11 groups, nine groups were treated with different TCMs by gastric infusion , and theFecal collection process: after alcohol disinfection of each cage, three mice in each cage acted freely and defecated freely. Using sterile forceps, fresh feces were collected, placed into 2EP tubes, and quickly placed at −80°C. The fecal collection was performed before intragastric administration. The intragastric administration was suspended during the 3rd week (days 15 to 21) of the experiment.merged_abundance_table_species70.csv, we list the relative abundance of 70 species at 0, 3, 14, 21, and 28-day time points under the 11 different treatments, where the sum of each column is 100.High-quality genomic DNA was extracted from the mouse feces. The DNA that passed quality control was then used to construct a library using the TruSeq DNA HT Sample Prep Kit. Paired-end sequencing (2 × 150 bp) was carried out using the Illumina HiSeq X10 platform. After removing the host (mouse) and low-quality sequences, the relative abundance was calculated using MetaPhlAn 3.0  includes 11,075 reactions and represents a primary source to relate TCM with microorganisms. Chemical compositions and keywords of chemical reactions related to Cassia twig (Gui Zhi) and Sijunzi Decoction are listed in KEGG (Kyoto Encyclopedia of Genes and Genomes)  normalizing each column of merged_abundance_table_species70.csv, from the 11 TCM-free samples, we estimated the mean abundance values and variances for all 70 microorganisms. Then, we calculated the mean relative abundance for each microorganism species under each TCM treatment on days 3, 14, 21, and 28. If the mean is in the 95% confidence interval, treatment does not significantly change the abundance of the species at the p1 < 0.05 level according to the two-tail test. A mean located on the left side of the 95% confidence interval indicates that treatment significantly decreases the abundance of a species, whereas a mean on the right side of the confidence interval indicates that treatment increases the abundance of a species, with p2 < 0.025 using the one-tail test.Most microorganisms with increasing abundance following treatment with the nine TCMs have positive health functions for humans. There were 11 TCM-free samples on day 0. After p2 < 0.005 with the one-tail test, no microorganisms treated with the nine TCMs showed a decrease; however, setting p2 < 0.05 with the one-tail test revealed that Dang Gui Bu Xue Decoction, Cassia twig (Gui Zhi), and Sijunzi Decoction did not cause a significant decrease in the abundance of all microorganisms, whereas the other TCMs caused a reduction in the abundance of Bifidobacterium pseudolongum. This observation suggests that people taking TCM for healthcare should also take probiotics (including Bifidobacterium pseudolongum) to neutralize the mild adverse effects because this species is enriched with specific enzymes that degrade complex plant carbohydrates and host glycans  benefits to human health, including Lactobacillus_johnsonii  content in the caecum, Enterorhabdus_caecimuris , and the results are presented in Rhizoma dioscoreae (Shan Yao) was found to have five biomarkers: Bacteroides_caccae, Blautia_coccoides, Ruthenibacterium_lactatiformans, Clostridium_innocuum, and Lactobacillus_intestinalis. Rhizoma zingiberis (Ganjiang) was found to have Intestinimonas_butyriciproducens and Escherichia_coli as biomarkers. Codonopsis pilosula (Dangshen), Sijunzi, and Magnolia officinalis (Houpo) were found to have only one biomarker. Compared with Rhizoma zingiberis (Ganjiang), where the abundance of the two biomarkers increased.The LDA Effect Size (LEfSe) , a χ2 distribution test, to calculate the p-values of the Granger causality. We found many possible causality relationships under the 11 treatments when p < 10−4. For example, we showed that the Granger causality network for mouse gut microorganisms treated by Sijunzi Decoction has a p < 10−4 (Codonopsis pilosula (Dangshen), Poria cocos (Fuling), Rhizoma zingiberis (Ganjiang), Cassia twig (Gui Zhi), Magnolia officinalis (Houpo), Radices saussureae (Mu Xiang), saline control, Rhizoma dioscoreae (Shan Yao), and the Sijunzi Decoction have 372, 414, 426, 428, 467, 588, 443, 543, 431, 410 and 392 pairs of significantly correlated species, respectively.Taking the python package statsmodels.tsa.stattools, importing the grangercausalitytests function and using gc_res = grangercausalitytests , the Granger causality p < 10−4 . Supplemp < 10−4. Thus, correlations between the 70 species were found to change considerably by TCMs.Compared with the blank control group, the newly added causality pairs included 374, 383, 400, 454, 552, 423, 513, 410, 371, and 347 for the last 10 treatments, and 332, 329, 344, 359, 336, 352, 351, 351, 333, and 327 causality pairs vanished. In p < 0.00000001, a stricter cutoff, for blank control, Dang Gui Bu Xue Decoction, Codonopsis pilosula (Dangshen), Poria cocos (Fuling), Rhizoma zingiberis (Ganjiang), Cassia twig (Gui Zhi), Magnolia officinalis (Houpo), Radices saussureae (Mu Xiang), saline control, Rhizoma dioscoreae (Shan Yao), and Sijunzi Decoction, there remained 36, 30, 44, 45, 73, 22, 58, 61, 31, 64, and 45 pairs of significantly correlated species, respectively. Compared with the blank group, the number of newly added causality pairs was 29, 44, 45, 73, 22, 57, 61, 31, 63, and 45 for the last 10 treatments, and 35, 36, 36, 36, 36, 35, 36, 36, 35, and 36 causality pairs vanished, respectively. These results further confirmed that TCMs greatly changed the species correlations.After setting lrtest to Moreover, the biomarkers shown in Poria cocos (Fuling) and Cassia twig (Gui Zhi), a decrease in the alpha diversity values were observed because their diversity at point “5” was significantly lower than that at point “1”. The remaining TCMs showed no significant change in their alpha diversity. From the perspective of species diversity, TCM decoctions were found to have a greater effect when compared with single herbs.A diversity index for a dataset is a quantity reflecting how many different species exist and how evenly individuals are distributed among those species. The diversity index increases with increasing species number and increasing evenness. Alpha diversity measures, in one sample, the number of different species and their different abundance, whereas beta diversity focuses on a group of samples and compares species compositions between different samples. For each sample of each treatment in our study, an alpha diversity index was calculated . Here, wAs shown in Codonopsis pilosula (Dangshen) treatment group was 0.2443439. A value of 0.2672811 was determined for Poria cocos (Fuling) treatment, 0.3333333 for Rhizoma zingiberis (Ganjiang) treatment, 0.2918660 for Cassia twig (Gui Zhi) treatment, 0.3302752 for Magnolia officinalis (Houpo) treatment, 0.3594470 for Radices saussureae (Mu Xiang) treatment, 0.2127660 for the saline control treatment, 0.4285714 for Rhizoma dioscoreae (Shan Yao) treatment and 0.1842105 for Sijunzi Decoction treatment. The Sijunzi Decoction, saline, and blank treatments have the lowest replacement rates, whereas Ganjiang, Houpo, Mu Xiang and Shan Yao have the highest replacement rates. Interestingly, the Sijunzi Decoction was found to only increase the abundance of Bifidobacterium_pseudolongum significantly, with no decrease in any other species abundance. Thus, this treatment improved the alpha diversity with the lowest replacement rate.Beta diversity measures differences between the species composition of samples, with larger beta diversity values indicating a larger replacement rate  and two microorganisms (Adlercreutzia equolifaciens and Bifidobacterium pseudolongum) were used as examples to clarify the novel mechanism exploration method as follows. First, the two microorganisms present very different correlation strengths under the two TCM treatments. Second, specific reactions involving the chemical compositions of the two TCMs and metabolisms of the two microorganisms were retrieved. Finally, by using novel serial and diverging connection reaction path models, contrast analysis was performed to deduce the possible mechanism of TCM that changes the microorganism correlation strength.Two TCMs (Adlercreutzia equolifaciens and Bifidobacterium pseudolongum presents very different correlations: treatment with Cassia twig (Gui Zhi) gave a Granger causality lrtest p-value of 0.000064934, whereas treatment with the Sijunzi Decoction yielded a Granger causality lrtest p-value of 0.93943. We typically study three models to explore the relationship among the three objects (a TCM and two microorganisms): serial connection, diverging connection, and converging connection. Here, the “converging connection” is not applicable because microorganisms cannot have an impact on the TCM.The interaction between Cassia twig (Gui Zhi) and Sijunzi Decoction, respectively, and the metabolites of Adlercreutzia_equolifaciens and Bifidobacterium_pseudolongum are available from KEGG (data not shown due to size). “Serial connection” means three equations are involved in Adlercreutzia_equolifaciens → Bifidobacterium_pseudolongum → chemical composition of TCM, or Bifidobacterium_pseudolongum → Adlercreutzia_equolifaciens → chemical composition of TCM, where the TCM may increase the inter-species correlation by driving the chemical reaction based on Le Chatelier's principle/the equilibrium law.For convenience, in the following study, in Cassia twig, taking the following as an example: {rn:R00237} acetyl-CoA + pyruvate ↔ (3S)-citramalyl-CoA (A. equolifaciens) → {rn:R02955} acetate + (3S)-citramalyl-CoA (A. equolifaciens) ↔ Acetyl-CoA (B. pseudolongum) + Citramalate → {rn:R10474} Acetyl-CoA (B. pseudolongum) + cinnamyl alcohol (Cassia twig) ↔ CoA + Cinnamyl acetate. Clearly, there are three equations involved in A. equolifaciens → B. pseudolongum → Cassia twig. Here, the metabolite “(3S)-citramalyl-CoA” of A. equolifaciens helps to produce the metabolite “acetyl-CoA” of B. pseudolongum, and the chemical composition of “cinnamyl alcohol” from Cassia twig can consume “acetyl-CoA” of B. pseudolongum. Hence, this serial connection may increase the correlation between A. equolifaciens and B. pseudolongum.For A. equolifaciens → B. pseudolongum → Cassia twig. In B. pseudolongum → A. equolifaciens → Cassia twig. In A. equolifaciens → B. pseudolongum → Sijunzi Decoction, and in B. pseudolongum → A. equolifaciens → Sijunzi Decoction. Clearly, for Adlercreutzia_equolifaciens and Bifidobacterium_pseudolongum, the number of serial connections driven by Cassia twig  is much larger than that driven by the Sijunzi Decoction . This observation may partially explain the stronger Granger causality of A. equolifaciens and B. pseudolongum under Cassia twig than that under the Sijunzi Decoction.In A. equolifaciens and B. pseudolongum through the same or different reaction equations. As shown in Cassia twig (Gui Zhi) connects with both A. equolifaciens and B. pseudolongum through 75 shared chemical reactions, whereas Cassia twig (Gui Zhi) has five specific equations with A. equolifaciens and 15 specific equations with B. pseudolongum. Data in A. equolifaciens and B. pseudolongum through 11 shared chemical reactions, without any specific reactions. The more shared chemical reactions may explain, in part, the higher correlation between A. equolifaciens and B. pseudolongum under Cassia twig treatment than that found under Sijunzi Decoction treatment.The “diverging connection” indicated that the compositions of TCM may react with both metabolites of assia twig (Gui Zhi) and Sijunzi Decoction. Moreover, among the 70 microorganisms annotated in our mice model, only 28 microorganisms were annotated in KEGG. Given more detailed data, the novel serial and diverging connection models should provide additional insights into how TCM affects microorganism abundance and inter-species correlations.Mouse gut experiments and sequencing technology afforded a systematic analysis of the abundance of gut microorganisms over time with and without TCM treatments. The study also provided an opportunity to explore how TCM affects microorganism abundance and their inter-species correlations. Ingredient and reaction pathway analysis was used to explain the impact of TCM on gut microorganisms, and we focused our analysis on two TCMs, CCNP0002573.The datasets presented in this study can be found in the Fecal Metagenomic sequencing reads from CNGB Nucleotide Sequence Archive under accession number The animal study was reviewed and approved by the Ethics Committee of KMHD on the use of animal subjects under Approval No. IACUC-0210831-1. Written informed consent was obtained from the owners for the participation of their animals in this study.LH, XF, HY, and YZ designed the project. YC, XS, YD, QQ, and DZ conducted the experiments. DZ, YZ, and XB analyzed the data. DZ and XB developed the algorithm and software. YZ, DZ, and LH wrote and revised the paper. All authors contributed to the article and approved the submitted version.This paper was supported by Science Technology and Innovation Committee of Shenzhen Municipality under Grant No. JCYJ20160331190123578 and the Natural Science Foundation of Hebei Province of China under Grant No. A2019208336.Authors YZ, DZ, XB, and XF were employed by Kangmeihuada GeneTech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This article provides the context for the ambition outlined in the the National Institute for Health and Care Excellence (NICE) 2021–2026 strategy to ‘lead globally on the potential to include environmental impact data in its guidance to reduce the carbon footprint of health and care’. Anthropogenic environmental changes pose a catastrophic risk to human health, with potential to widen national and global health inequalities. Recognising the fact that NICE guidance influences the way health and care is delivered and its consequent environmental impact, NICE has included environmental sustainability among its strategic priorities. This article outlines the work underway to meet this sustainability agenda at NICE. In recent years, the National Institute for Health and Care Excellence (NICE) has continued to expand its engagement with environmental sustainability and work to promote environmental sustainability in the health system.NICE is identifying and supporting implementation of measures to assess and reduce the environmental impacts of NICE guidance and advice. A number of NICE products reference information on the environmental impact of health interventions to support more sustainable healthcare, including a medicine optimisation sustainability report, a patient decision aid for asthma inhalers and a medical technologies guideline.Upcoming work will include formalising how environmental sustainability considerations are integrated into health technology evaluation and other NICE processes, developing the inhaler decision aid to enhance its accessibility and usefulness and running a programme of deliberative public engagement on sustainability.NICE is also working to reduce its own organisational footprint. In 2022, NICE has been working to meet and surpass the ‘Greening Government Commitments’.Anthropogenic environmental changes, including air and water pollution, climate change and biodiversity loss, pose potentially catastrophic risks to human health.The scale of the existential threat of climate change led the Intergovernmental Panel on Climate Change in 2018 to recommend that greenhouse gas emissions (GHGs) must reach net zero by 2050 to prevent a catastrophic temperature rise of 1.5°C.,Recognising that NICE guidance affects how the NHS operates and consequently its environmental impact, NICE laid out ambitions to support environmental sustainability in the NICE strategy 2021–2026.In the 2021–2026 NICE strategy, NICE outlines its ambition to ‘lead globally on the potential to include environmental impact data in its guidance to reduce the carbon footprint of health and care’.Since this recent strategic commitment to support a sustainable health system, NICE has been working to identify and implement actions that it can take to support environmental sustainability across the health and care system. This requires collaboration to ensure coherence across different areas of NICE’s work, including overseeing methodological development work, and to share progress on NICE’s work to support sustainability.NICE has previously demonstrated how implementation of its medicines optimisation guidelines on the safe and effective use of medicines in health and social care settings can also produce benefits from an environmental point of view.The responsibility to raise wider system awareness of environmental impacts of medication use extends to work NICE has been undertaking around use of inhalers for respiratory conditions. Inhalers are responsible for 3% of the NHS’s carbon footprint. A NICE patient decision aid included a question to enable patients to consider environmental impact when making a choice about which asthma inhaler they use.In guidance published in January 2022 (MTG65), ‘Sedaconda ACD-S for sedation with volatile anaesthetics in intensive care’, the medical technologies advisory committee considered environmental impact (based on data provided by the manufacturer), alongside clinical and cost effectiveness evidence. Sedaconda ACD-S is a system to capture and reduce the release of anaesthetic gases, which is important because volatile anaesthetic drugs are potent GHGs.In addition to supporting system sustainability across health and care, NICE is also working to reduce the environmental impact of its own activities given its reputational and leadership role globally. NICE adheres to the ‘Greening Government Commitments’ to:reduce carbon emissionsminimise wastereduce water usemake sustainable choices about procurementsupport biodiversity and nature recoveryadapt to climate change andreduce the environmental impacts of digital workingNICE has an internal action-focused group engaging staff and working to reduce NICE’s environmental footprint in line with these commitments.In line with its strategic commitment, NICE has commissioned an academic centre to explore possible approaches to incorporating environmental sustainability considerations in NICE’s methods and processes, including health technology evaluation. The academic centre has identified data sources and methods that can be used. This exploration has provided insights which may inform development of a framework for presenting and quantifying environmental impact information. Work is also underway to prepare for the implementation of such a framework, including deliberative public engagement to gain insights into public perceptions and preferences.In response to the consultation at the scoping stage for the BTS/SIGN/NICE asthma guideline, which is due for publication in November 2022, stakeholders requested that NICE address environmental sustainability considerations. The importance of selecting the right inhaler device for each patient will be included in the guideline, and where devices are equally acceptable, environmental factors will be taken into account. The developers intend to recruit an expert on environmental issues related to respiratory care to support the committee.During 2022, NICE is also undertaking focused projects to incorporate environmental considerations within specific activities. An important piece is a programme of deliberative public engagement, NICE Listens, which NICE will use to explore views on NICE’s role and priorities in the environmental sustainability space.In its 2021–2026 strategy, NICE has acknowledged the importance of assessing environmental impact in its guidance and the responsibility it has as a health leader to promote environmental sustainability. This article has highlighted how NICE is engaging with this agenda and contributing to the positive changes that are underway in the UK health system.S.W. is an unpaid Associate of the Centre for Sustainable Healthcare.K.S. and S.W. are employed by the National Institute for Health and Care Excellence.In his previous employments, K.S.' employers have received funding from a variety of sources, including pharmaceutical companies and the Association of the British Pharmaceutical Industry.M.S. has nothing to declare.


Cancer cells exhibit unique metabolic properties, including a high requirement for methionine. However, the mechanism that explains how cancer cells suffer from the absence of methionine more significantly than healthy cells remains elusive. Methionine is essential for epigenetic reprogramming of cells, both at the DNA level and for post-translation histone code modification. The post-translational modifications of histone variants (hPTMs) in normal and cancer cells were characterized by mass-spectrometry in the absence and presence of methionine gamma-lyase (MGL), a bacterial enzyme that degrades methionine and inhibits the growth of cancer cells. Results indicate a complex pattern of PTMs on histone variants and striking differences between normal and cancer cells that might help in the understanding of the molecular mechanisms triggered by methionine depletion and in the fine-tuning of MGL-based cancer therapy.Methionine is an essential amino acid involved in the formation of polyamines and a precursor metabolite for DNA and protein methylation. The dependence of cancer cells on methionine has triggered extensive investigations aimed at its targeting for cancer therapy, including the exploitation as a therapeutic tool of methionine γ-lyase (MGL), a bacterial enzyme that degrades methionine, capable of inhibiting cancer cells growth due to methionine starvation. We have exploited the high-resolution power of mass spectrometry to compare the effects of reduced availability of the methyl donor SAM, induced by MGL treatment, on the post-translational modifications of the histone tails in normal Hs27 and cancer HT-29 cells. In the absence of MGL, our analysis detected a three-fold higher relative abundance of trimethylated K25 of H1.4 in HT-29 than Hs27 cells, and a complex pattern of methylated, unmethylated and acetylated peptides in H2 and H3.3. In the presence of MGL, in HT-29, the peptide H2A1_4_11 is predominantly unmodified with mono-methylated K5 increasing upon treatment, whereas in Hs27 cells, H2A1_4_11 is monomethylated at K5 and K9 with these marks decreasing upon treatment. The time dependence of the effects of MGL-mediated methionine depletion on PTMs of histone variants in HT-29 cancer cells was also monitored. Overall, our present data on histone variants H1, H2A, H2B as well as H3.3 integrated with our previous studies on histones H3 and H4, shed light on the epigenetic modifications associated with methionine starvation and associated cancer cell death. Malignant cells exhibit proliferative advantages when compared to non-malignant cells due to an altered metabolism that supports their rapid growth. The Warburg effect that describes the preference of cancer cells to metabolize glucose anaerobically rather than aerobically, even under normoxia, is a typical example. In addition, cancer cells often present an increased demand for selected amino acids (AAs), resulting in a dependency on exogenous sources or upregulation of their de novo synthesis . The expA notable example of metabolic vulnerability in cancer cells is the elevated requirement for methionine ,4. The hMethionine is involved in many relevant biological processes, such as protein synthesis, glutathione formation, polyamine synthesis and methyl group donation in methylation reactions. Thus, low methionine availability widely perturbs cellular mechanisms . In partIn yeast cells, the imbalance in SAM/SAH (S-adenosylhomocysteine) ratio alters histone methylation, increasing or reducing viability . Yeast cSensing methionine availability is thus crucial for cellular, epigenetic, and genomic integrity, but the mechanisms for methionine perception are different throughout different organisms, despite a conserved role of methionine as a growth signal.In mammalian, the molecular mechanisms are still elusive. It was reported that methionine is sensed indirectly through SAM , which bA few recent studies have started to address the epigenetic changes occurring in cancer cells exposed to methionine restriction ,29,30,31Our laboratory carried out an extensive characterization of the differential histone methylome, acetylome and proteome of normal and cancer cells upon MGL-induced methionine deprivation by mass spectrometry-based proteomics . MGL treThe chromatin of individual eukaryotic cells is wrapped around histone octamers to form the nucleosome core particles. An octamer contains two of each core histone  with 145–147 base pairs of DNA wound around it. A histone known as linker histone H1 is bound to the outside of the nucleosome core particle, forming a full nucleosome, and stabilizing higher-order chromatin structures.Histone variants diverge in sequence and impart effects on many DNA-templated processes and are deposited throughout the cell cycle often by variant-specific histone chaperones. Replacement of canonical histones by variants affects nucleosome structure and accessibility of the histone tail. Variants of histones H2A, H2B, H3, and H1 are well-described in literature, while there is little evolutionary divergence in H4.Distinct amino-terminal modifications of the histone tails characterize a specific “histone code” that determines dynamic transitions between transcriptionally active or transcriptionally silent chromatin states.Histone amino-terminal modifications include acetylation, methylation, phosphorylation, ubiquitinylation, sumoylation, ADP ribosylation, and deamination . In partThis process increases the information potential of the genetic code and provides the cell with a rapid response to solicitations/stresses that require modulation of transcription. However, the histone code and its role together with the relevance of PTMs on histone variants are still not completely deciphered.To our knowledge, our present study describes for the first time how MGL-mediated methionine depletion affects post-translational modifications deposited on histone variants (hPTMs) in normal and cancer cells. Our effort was to help better understand the complex epigenetic cell alterations due to methionine restriction but also to define new biomarkers to better calibrate anticancer therapy based on enzymes able to cause methionine deprivation.E. coli BL21(DE3) cells containing the plasmid with MGL gene from C. freundii were grown. Enzyme was purified, and protein concentration was determined according to the protocol previously described [escribed ,40,41. P2O → methanethiol + NH3 + α-ketobutyrateL-methionine + H−1 cm−1) at 37 °C. The rate of α-ketobutyric acid production was measured in a coupled assay at 30 °C. The reaction mixture contained 100 mM potassium phosphate, 100 mM pyridoxal 5′ phosphate (PLP), 5 mM DTT, 0.2 mM NADH, pH 7.2. The amount of the enzyme that catalyzes the formation of 1.0 μmol min−1 of α-ketobutyrate at pH 8.0, 37 °C, was defined as one unit of enzyme activity. Kinetic parameters of MGL mutants in the α,γ-elimination reaction with L-methionine were previously reported to be kcat = 7.9 s−1 ± 0.3; KM = 0.8 mM ± 0.1; kcat/KM = 9.7 × 103 M−1s−1 [MGL catalytic activity was determined using L-methionine as a substrate and measuring the rate of α-ketobutyrate production in the coupled reaction with D-2-hydroxyisocaproate dehydrogenase (HO-HxoDH) by monitoring the decrease of NADH absorption at 340 nm , pH 7.4, and sterilized through Millipore GV 0.22 µm filters and stored at −80 °C. The methionine concentration in the medium for cell growth was quantified to be 400 μM by the afore mentioned coupled activity assay with HO-HxoDH. This concentration corresponds to half of the Knditions . The confter 1 h .v/v) fetal bovine serum, in a humidified CO2 (5%) incubator at 37 °C.The effects of methionine depletion caused by MGL treatment were tested on two human cell lines provided by ATCC: the human colon adenocarcinoma cell line HT-29  and the human skin fibroblast cell line Hs27 . Cell culture conditions have been previously reported [5 cells were seeded in 12-well plates, with 2 mL of DMEM complete medium and, after treatment, cell nuclei were isolated, and histones were extracted with 0.2 M H2SO4 for 2 h and precipitated with 33% trichloroacetic acid (TCA) overnight. The histone protein pellets were dissolved in 30 μL of 50 mM ammonium bicarbonate, pH 8.0. To ensure complete derivatization, two rounds of propionylation were performed mixing propionic anhydride with acetonitrile in a ratio of 1:3 (v/v) and adding to histone solutions in the ratio of 1:4 (v/v) for 20 min, at room temperature. After digestion, to derivatize peptide N-termini, the derivatization procedure was repeated twice.Histone acid extraction and analysis by mass spectrometry were performed following our previously detailed protocol ,42. 2.5 Samples were desalted prior to LC-MS analysis by using C18 Stage-tips. They were then resuspended in 10 µL of 0.1% TFA and loaded onto a Dionex RSLC Ultimate 300 , coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). Chromatographic separation was performed with a two-column system, consisting of a C-18 trap cartridge  and a picofrit analytical column  packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin.5 and an HCD collision energy of 30.To analyze the histones, peptides were separated using a 60 min gradient from 4%-30% buffer B  at a flow rate of 300 nl/min. Data were acquired using a data-independent acquisition (DIA) method. In particular, the full MS scan (300–1100 m/z) was acquired in the Orbitrap with a resolution of 120,000. MS/MS was performed by setting consecutive isolation windows of 50 m/z with an AGC target of 2 × 10t-test heteroscedastic with two-tails (significant if p < 0.05).Histone peptide data were analyzed using EpiProfile 2.0 software . The rawT-test. Significance was assumed if the obtained p-value was < 0.05. Prior to T-test calculation, protein abundances were log2 transformed, normalized by the average value of each sample and missing values were imputed using a normal distribution 2 standard deviations lower than the mean. The distribution of data points of replicates was assumed to be normal, but this was not formally tested.To assess statistical significance, we used a two-tails heteroscedastic parametric https://chorusproject.org/) at the project number 1712, accessed on 4 March 2021.Histone raw files are available for download at the repository Chorus  . On the In cancer cells the mono-methylation at K33 in H1.2 decreases after treatment, while the unmodified peptide increases. In addition, acetylation at K31 in H1.4 increases in treated cancer cells .The abundance of the peptide corresponding to the N-terminal portion (1–35) of H1.4 versus H1.5 seems to be unaffected by the treatment in normal cells with H1.4 predominant over H1.5. On the contrary, in cancer cells H1.4 is predominant in untreated cells with respect to H1.5 but becomes equal upon treatment .In normal cells, upon MGL50 treatment, the peptide corresponding to the 54–84 fragment of H1.1 increases with a decrease in the peptide characteristic of H1.5. In cancer cells, the increase in H1.5 is at the expense of H1.4 with no changes in H1.1 abundance .H2 histone variants—In cancer cells, H2A1_4_11 is predominantly unmodified with the mono-methylation at K5 that increases upon treatment. On the contrary, in normal cells, H2A1_4_11 is monomethylated at K5 and K9 with these marks that consistently decrease upon treatment. The abundance of the unmodified peptide is similar in both normal and cancer cells with a change in the opposite direction: in Hs27 increases upon treatment, while in HT-29 decreases .For the H2AJ_4_11 peptide, in cancer cells, K5me1 is the most abundant mark and increases upon treatment whereas K9 mono-methylation and acetylation marks decrease. In Hs27 cells, the most abundant marks in the untreated cells are K5me1 > K9ac > K9me1 > unmodified. Upon treatment K5me is the dominant mark associated with K9ac and the unmodified peptide and K9me1 almost disappears .In H2AX_4_11 after treatment, K5me1 increases in normal and cancer cells, while K9me1 decreases .In HT-29 cells, H2A1_1_11 peptide K5 is acetylated, and treatment causes a 10% decrease. In normal cells, the unmodified peptide is as abundant as the acetylated one and the treatment increases acetylation on K5 .H2A1_12_17 K15ac is a dominant mark in cancer cells and almost absent in normal cells .H2A3_12_17 K15me1 is significantly dominating in untreated cancer cells with a significant fraction of unmodified peptide. K13me1 abundance increases upon treatment. In normal cells, the dominant mark is K13me1 when untreated and K15me1 when treated.H2A_1_88 H2AY.SAKAGVIFPVGR (macroH2A) skyrockets in normal cells upon treatment , while tIn H2B1A, K86ac is the predominant mark in cancer cells independently of treatment, while in normal cells a consistent fraction is unmodified, and acetylation is strongly affected by treatment. However, statistical significance is not reached by these data .Abundance of the variants affecting the N-terminal peptide of H2B is different in untreated normal and cancer cells and changes in response to MGL-mediated methionine depletion. H2B_1_29 1C.PEPAKSAPAPKKGSKKAVTKAQKKDGKKR and H2B_1_29 1H.PDPAKSAPAPKKGSKKAVTKAQKKDGKKR are more abundant in normal than in cancer cells upon treatment .H3.3 histone variants—MGL-mediated methionine depletion does not induce statistically significant alterations on hPTMs in H3.3 histone variants .H1 histone variants—24 and 48 h MGL treatments were applied to HT-29 cells in order to investigate how the deposition of marks on variants changed during progressive and sustained enzyme-mediated methionine starvation . InteresH2 histone variants—Variation in abundance of methylation at K5 and K15 of H2A1 displays opposite direction after 48 h treatment: the first one decreases and the second one increases. Little but significant variations were observed for H2A1K13ac and H2AZK7ac upon 24 h of methionine starvation, and for H2A3K15ac after 48 h . H2AJ unIn HT-29 cells upon treatment, the double acetylation at H2AZK4acK7ac and the triple acetylation at H2AZK4acK7acK11ac decrease after 48 h; on the contrary, the double acetylation at H2AZK7acK15ac decreases after 24 h .We evaluated also the time course of alterations induced by MGL50 on hPTMs in H2B variants, upon 24 h of MGL-mediated methionine depletion. In this case, H2B1A unmod increases and H2B1AK86ac decreases compared to control. After 48 h, the relative abundance of H2B1B unmodified peptide decreases and the acetylation at Y83 increases .H3 histone variants—hPTMs on the H3.3 histone variants when HT-29 cells were treated for 24 and 48 h are shown in MGL50 treatment caused a higher acetylation at K27 and a lower one at S28 already after 24 h than the control. Subsequently, the S28ac is reduced, but not significantly .The treatment for 48 h induced a reduction of K27me1K36me2, K27me1K36me1 and K27me3S28acK36me1 relative abundance .The K27me3K36me1 and K27me1K36me3 increase already upon 24 h methionine depletion, but only the second one decreases significantly upon 48 h .In the last years, significant effort has been exerted to decipher the histone code and to establish a relationship between hPTMs and stress exposition. Histone code could be employed to manage genome/epigenome alterations and to establish a genetic/epigenetic crosstalk beyond DNA sequences. hPTMs play a pivotal role in many biological processes and represent the crucial mechanism by which histones exert regulatory control over different processes and therefore determine cellular identity and metabolism ,49,50. HThis study deepens and integrates our knowledge of hPTMs in HT-29 and Hs27 cells in absence and in presence of methionine depletion. In our previous work, we focused on histone 3 and 4 specific marks . In thisIn HT-29 and Hs27cells epigenetic modification occurs mainly at lysine residues. Literature studies suggest that lysine acetylation is usually related to DNA damage response, cell-cycle control, chromatin architecture, RNA splicing, and transcription ,52. IndeIn particular, in untreated cancer cells we observed several mono-, di- and tri-methylation  on H1.4, H2A1, H2A3 and H3.3 histone variants, and a different pattern of mono-acetylation . Non-malignant cells show mono-methylation on H2A  and tri- or double-methylation on H3.3 histone variants. Nowadays, the relevance of PTMs modifications is well understood, but the complexity of the epigenetic signature and its impact on cellular context are unknown.We carried out a detailed analysis of the histone PTMs induced by methionine starvation. Methionine is an essential amino acid and is involved in many biological processes, such as glutathione formation, polyamine synthesis, and methyl group donation. Our previous results highlighted that malignant cell growth was significantly impaired in methionine-depleted conditions, while normal cells growth was unaffected . These eOur findings suggest that the changes of significant hPTM marks are different after MGL50 treatment in comparison with normal cells. These marks are abundant in cancer cells only after MGL50 administration. The most important alterations occur on H2A and H3.3 variants. The precise role of the histone modifications remains largely unclear and there is little information about the specific cellular mechanisms influenced by the deposition of such marks either in normal and cancer cells.Using information deriving from in-silico tools such as the “HISTome2: The HISTone Infobase”  we triedAmong the changes highlighted during methionine starvation induced by MGL treatment on the HT29 tumor cell line , changesAs described in previous studies , our masThe analytical power of mass spectrometry was applied to the characterization of the PTMs of histone tails, the region more affected by epigenetic changes, including those caused by methionine depletion in cell cultures. Significant differences were observed between normal and cancer cells in the PTM pattern both in the absence and presence of MGL, an enzyme that is able to decrease the available methionine, suggesting diversified metabolic adaptations. Future studies are needed to examine the consequences of these profile changes in normal and cancer cells at the proteomic level.


The challenges of treating central nervous system (CNS) tumors in young children are many. These include age-specific tumor characteristics, limited treatment options, and susceptibility of the developing CNS to cytotoxic therapy. The aim of this study was to analyze the long-term survival, health-related, and educational/occupational outcomes of this vulnerable patient population.Retrospective study of 128 children diagnosed with a CNS tumor under 5 years of age at a single center in Switzerland between 1990 and 2019.Median age at diagnosis was 1.81 years . Median follow-up time of surviving patients was 8.39 years . The main tumor subtypes were pediatric low-grade glioma (36%), pediatric high-grade glioma (11%), ependymoma (16%), medulloblastoma (11%), other embryonal tumors (7%), germ cell tumors (3%), choroid plexus tumors (6%), and others (9%). The 5-year overall survival (OS) was 78.8%  for the whole cohort. Eighty-seven percent of survivors > 5 years had any tumor- or treatment-related sequelae with 61% neurological complications, 30% endocrine sequelae, 17% hearing impairment, and 56% visual impairment at last follow-up. Most patients (72%) attended regular school or worked in a skilled job at last follow-up.Young children diagnosed with a CNS tumor experience a range of complications after treatment, many of which are long-lasting and potentially debilitating. Our findings highlight the vulnerabilities of this population, the need for long-term support and strategies for rehabilitation, specifically tailored for young children.The online version contains supplementary material available at 10.1007/s11060-022-03963-3. CNS tumors are the most common pediatric solid cancers. The average annual incidence rate is 6.18 per 100,000 in 0–4-year-olds in the U.S. –4. DevelYoung children (< 5 years of age) are particularly prone to long-term sequelae of cytotoxic therapies . Due to Health care professionals often face the dilemma between augmenting treatment intensity for optimal disease control and minimizing acute toxicity as well as the risk of long-term sequelae . ClinicaIn this retrospective study, young children aged 0–5 years with a newly diagnosed primary CNS tumor and treated at the University Children’s Hospital of Zurich between January 1990 and December 2019 were identified. Date of diagnosis was defined as either date of histological confirmation of tissue sample or, if not available, date of diagnostic imaging.The design of the study was approved by the Ethics Committee of the Canton of Zurich. A general research consent was implemented at our institution in 2015. The need for informed consent was waived for deceased patients, patients diagnosed prior to 2015 and lost to follow-up. Patients with documented refusal to participate in research were excluded.The baseline clinical characteristics included age at diagnosis, sex, tumor characteristics , hydrocephalus at diagnosis, underlying genetic predisposition and treatment details. Extent of resection was determined based on neurosurgical reports and postsurgical MRI when available; if a residual tumor was described in the neurosurgical report and/or postsurgical MRI, the tumor was considered partially resected. Progression-free survival (PFS) was calculated from date of diagnosis to disease progression leading to change in treatment or death in patients without such a progression, and overall survival (OS) from date of diagnosis to death. In the absence of progression and for patients alive at last follow-up, PFS and OS were censored at the last documented date that the patient was seen by a physician (last follow-up).The long-term health-related outcome information was extracted from the medical charts of all patients and included neurologic status, endocrine function, hearing loss, visual acuity, secondary malignancies, and cerebral vasculopathy. Ototoxicity was graded according to Chang after review of available audiograms . NeuroloAt our pediatric institution, patients are followed up until 20 years of age and information on schooling and employment is regularly documented at long-term follow-up clinic visits. Academic achievement was categorized into two groups. The first group contains patients who attend regular school or work in a skilled job and the second group includes patients who attend an assisted or modified school program e.g. with smaller student numbers per class and additional assistance or who work in an unskilled or assisted job. A skilled job was defined as student with graduation after vocational training with a Federal Diploma . An unskilled job includes only training on-site without graduation. Preschool-aged children were excluded from this analysis.Descriptive analyses were used to summarize the study population. Kaplan–Meier survival curves were generated to estimate OS probability and progression-free survival probability. Log-rank test was performed for comparison between different subgroups.To quantify the association between different treatment modalities and long-term outcomes fisher’s exact test was used.R version 4.0.3 and RStudio (v1.3) were used for statistical analysis. The following additional packages were used: Beeswarm, ggplot2, ggpubr, openxlsx, plotly, plotrix, plyr, survival, survminer, tableone, tidyverse, viridis.We identified 164 children under 5 years of age and diagnosed with a primary CNS tumor between 1990 and 2019 at the University Children’s Hospital of Zurich, the largest pediatric oncology center in Switzerland. Thirty-six patients were excluded: 28 patients due to lack of sufficient information and 8 patients due to refusal to participate in research. Thus, 128 patients were included in the final analysis (Table S1).The main tumor subtypes were pediatric low-grade glioma (pLGG) , pediatric high-grade glioma (pHGG) n = 14, 10.9%) including five diffuse intrinsic pontine gliomas (DIPG), ependymoma , medulloblastoma , other embryonal tumors , germ cell tumors , choroid plexus tumors , and others   and choroid plexus tumors (n = 8) were diagnosed in children of younger age with the eldest child being 1.09 years and 1.98 years old, respectively. The other tumor subtypes showed a more balanced distribution from 0 to 5 years of age , followed by the cerebral hemispheres (26%). Most tumors found in the optic pathway and hypothalamus/midline region were pediatric low-grade gliomas n = 15, 71%), 3 of those patients were diagnosed with neurofibromatosis 1 (NF1). Tumors located in the thalamus and brainstem were mostly pediatric low-grade  or high-grade gliomas   underwent surgery, in 41% gross total resection (GTR) was achieved (Table S1). Fifty percent of patients had an uneventful postoperative course, for 9% there was no detailed information available. Seven patients (6.3%) had an ischemic event perioperatively, 5 (4.5%) presented with hemorrhages in the perisurgical period, with one being hemodynamically relevant. Ten patients (8.9%) developed hygroma or craniospinal fluid (CSF) leakage, 11 (9.8%) had nerve lesions with subsequent (transient) paresis in the corresponding area and 18 (16%) had other postoperative complications such as epilepsy, hemisyndromes, and endocrine deficiencies. Some patients suffered multiple complications.Sixty-three percent of patients received chemotherapy, most of them enrolled on or treated as per tumor-specific protocols  , 15.Radiotherapy was performed in 39.8% of patients (n = 51), either upfront (n = 27) or at progression (n = 24), two of those 24 were re-irradiated after further progression. The majority (56.9%) underwent proton radiation (introduced in Switzerland in 1996), 37.3% photon radiation, and 5.9% both modalities. Almost 30% underwent craniospinal radiation.Median follow-up time of surviving patients was 8.39 years [range 0.74–23.65 years]. Median progression-free survival (PFS) of the whole cohort was 3.3 years . The 5- and 10-year OS probability was 78.8% resp. 76.1% for the whole cohort  Fig. A with a Most of the deceased patients died within the first 5 years after diagnosis. Of those patients who died, the majority  died of tumor progression/relapse. One patient initially diagnosed with medulloblastoma died after diagnosis of radiation-induced high-grade glioma. The cause of death was undocumented or unclear for two patients.Twenty-six patients (20.3%) presented with endocrine deficiencies at last follow-up, where 21 (81%) received hormonal replacement therapy. Most (85%) presented with hypopituitarism. Hypopituitarism was associated with radiotherapy  and chemotherapy  (Table S3). Other endocrine alterations included obesity or precocious puberty.Fourteen patients were over 18 years of age at last follow-up. As far as documented none of them conceived a child. Four patients  had ovarian insufficiency and two required estradiol replacement. For the male patients (n = 6), spermiograms were not available.Visual impairment is multifactorial and prevalent also in the general population. Focusing on the subset of patients with optic pathway glioma and tumor-associated visual impairment, all patients  with optic pathway glioma showed some degree of visual impairment at last follow-up and four patients were blind in at least one eye.Sixteen patients 12.5%) had hearing impairment at last follow-up (≧ Chang Grade 2b)  presented with a moyamoya vasculopathy during follow-up. Both had been diagnosed with posterior fossa ependymoma and had received focal proton radiation at below 4 years of age, with a dose to the tumor bed of 54 Gy and 59.4 Gy, respectively.Nine patients (7%) experienced a stroke during follow-up time, in four patients after surgical resection of the tumor and in 2 patients stroke was deemed related to radiation-induced cerebrovascular disease (2.5 years and 13 years post completion of radiation).In summary, among survivors followed for more than 5 years (n = 77), 87% present with any tumor- or treatment-related sequelae, 61% had any neurological deficit, 30% presented with endocrine sequelae and 81% of them with need for hormone replacement, 17% with hearing impairment, and 56% with visual impairment at last follow-up.Thirty-seven patients had not yet reached school age at time of last follow-up. Information on schooling was not available for 6 patients. Of the remaining 85 patients, 71.8% were able to attend regular school and/or work in a skilled job, whereas 28.2% were schooled in a modified program or working in an unskilled or assisted job Fig. . Of the Among survivors of childhood cancer, the cumulative burden of chronic health conditions is highest in patients diagnosed with CNS malignancies . ProvidiWith a 5-year OS of 78.8%, the survival outcome of our cohort is comparable to previous studies, albeit differences regarding patient characteristics and therapy between studies , 22–24. Other studies reported a higher mortality rate in children diagnosed under 1 year of age when compared to older children , 26, 27.Survival rates of children diagnosed with a CNS tumor seem to have increased over the last years , 24, whiA study summarizing the outcomes of a cohort of 20 survivors, previously diagnosed with a brain tumor in the first year of life, reported 70% with neurological dysfunction, 25% with endocrine dysfunction, 15% with hearing impairment, and 45% with visual impairment . HearingHypopituitarism was associated with radiotherapy, as previously described –35. A reWe found that most (71.8%) of our school-aged patients or older at the time of analysis attended regular school or were employed in a skilled job. This is higher than what a previous study reported including children diagnosed with a CNS tumor in the first year of life . A recenThe size and heterogeneity of our study population, as well as limitations associated with the retrospective nature of the study, need to be considered when interpreting our findings. Assessment of long-term outcome has not followed a standard procedure in terms of frequency and tools used, limiting interpatient comparisons. The classification of CNS tumors has undergone several important revisions over the decades covered in this study. The main tumor entities have been reclassified and subdivided into new subgroups, reflecting the heterogeneity in tumor biology, especially in pediatric brain tumors in young children , 43. ThoYoung children are at a high risk for long-term morbidity after diagnosis of a CNS tumor. Encouragingly, though a vast proportion of survivors experience health-related sequelae, most were integrated in regular schools. Our study highlights the importance of long-term support strategies, tailored to young children. These include early screening for visual and hearing impairment, as well as endocrinopathy and neuropsychology assessments, to offer appropriate support. Advances in treatment modalities, including targeted anti-tumor therapies and improvement in high-precision radiation techniques, will hopefully lead to a further reduction in treatment burden and better long-term outcomes in these children.Supplementary file1 (DOCX 607 KB)Below is the link to the electronic supplementary material.