Extracellular vesicles (EVs) are a heterogeneous population of membrane‐enclosed nanoparticles released by cells. They play a role in intercellular communication and are involved in numerous physiological and pathological processes. Cells release subpopulations of EVs with distinct composition and inherent biological function which overlap in size. Current size‐based isolation methods are, therefore, not optimal to discriminate between functional EV subpopulations. In addition, EVs overlap in size with several other biological nanoparticles, such as lipoproteins and viruses. Proteomic analysis has allowed for more detailed study of EV composition, and EV isolation approaches based on this could provide a promising alternative for purification based on size. Elucidating EV heterogeneity and the characteristics and role of EV subpopulations will advance our understanding of EV biology and the role of EVs in health and disease. Here, we discuss current knowledge of EV composition, EV heterogeneity and advances in affinity based EV isolation tools. AF4asymmetric flow field fractionationAFMatomic force microscopyAgo2argonaut‐2AlixALG‐2 interacting protein XAPOapoptotic bodiesAPPamyloid precursor proteinARF6ADP‐ribosylation factor 6C6Schondroitin 6 sulfateCSFcerebral spinal fluidddPCRdigital droplet PCREpCAMepithelial cell adhesion moleculeERKextracellular signal‐regulated kinaseESCRTendosomal sorting complexes required for transport machineryEVextracellular vesicleEXOexosomeFCSfetal calf serumGFPgreen fluorescent proteinGPIglycosylphosphatidylinositolHDLhigh‐density lipoproteinHMC‐1human mast cell 1HPLChigh performance liquid chromatographyHSP70heat shock protein 70IL‐1βinterleukin 1 betaILVintraluminal vesicleISEVinternational society for extracellular vesiclesLDLlow‐density lipoproteinL‐EVlarge EVLPlipoproteinMHCmajor histocompatibility complexmiRNAmicro RNAMSCmesenchymal stem cellMVmicrovesicleMVBmultivesicular bodyNCAMneural cell adhesion moleculeNKnatural killerNTAnanoparticle tracking analysisPEGpolyethylene glycolRFPred fluorescent proteinSECsize exclusion chromatographySELEXsystematic evolution of ligands by exponential enrichmentS‐EVsmall EVSTEDstimulated emission depletionTEMtransmission electron microscopyTENPOtrack‐etched magnetic nanoporeTFACtangential flow for analyte captureTFFtangential flow filtrationTNFαtumour necrosis factor alphaTRPStunable resistive pulse sensingTSG101tumour susceptibility gene 101UCultracentrifugationVLDLvery low‐density lipoproteinXNAxenobiotic nucleic acid1Cells release a variety of biomolecules and nanoscale particles into the extracellular environment as part of normal physiological processes. Extracellular vesicles (EVs) are nanoscale lipid bilayer enclosed particles ranging in size from 20 to 10,000 nm in diameter. EVs are involved in processes such as cell‐to‐cell communication, maintaining cellular homeostasis and the transfer of functional biomolecules . These f1.1The first recorded use of the term EV was by Aaronson et al. in 1971, although evidence for the existence of EVs can be seen as early as the 1940s by Chargaff et al. , 11, 12.1.2EXOs are population of S‐EVs that originate from the endosomal system. EXO biogenesis begins with invagination of the parent cell plasma membrane, internalizing transmembrane proteins and forming early endosomes. The early endosome, as it invaginates, creates intraluminal vesicles (ILVs), this structure is subsequently referred to as a multivesicular body (MVB), which can fuse with the cells plasma membrane to release the ILVs into the extracellular space as EXOs . Various1.32+, leading to deactivation of flippases and activation floppases, two lipid transporter protein families, in addition to the activation of the enzyme family of scramblases resulting in a loss of membrane asymmetry . However, despite this, the authors noted that elution conditions might affect epitope binding efficacy and additional concentration may be required due to the isolate being diluted . Further validation is needed, but the method has shown promise for fast affinity isolation from complex biofluid [HPLC can be used to isolate EVs using affinity resins that antibody bind to, this has the benefits of an HPLC system including reproducibility and capability of running in series with other columns such as IEX to remove contaminants. Monoliths are single structure highly permeable support for liquid chromatography. Monoliths have the advantage of larger gaps between the matrix which facilities higher flow rates and may also help avoid damage of EVs as their laminar flow reduces shear forces introduced by turbulence . Monolitof S‐EVs , p. 61. 4.2.3Aptamers are nucleotide sequences such as DNA and RNA that can fold into a 3D structure capable of binding to a specific ligand . Aptamer4.2.4Microfluidics has begun to be utilized more in EV research, and while most of the interest lies in developing diagnostic devices, some approaches have been established to isolate EVs . MicroflEV isolation methods have also been proposed utilizing microfluidics, this includes affinity capture using conventional antibodies but can also be coupled with nanoscale topographies such as herringbone grooves, capable filtering particles of a particular size which can then be further selected based on affinity . Additio5EVs are membrane‐enclosed nanoparticles that have demonstrated roles in physiological processed such as cell‐to‐cell signalling, and pathophysiological roles such as neurodegeneration and cancer. Understanding the role of EV cargoes in their functions is important for furthering the field and providing insight into basic biology that can have implications for the development of therapeutics and diagnostics. Additionally, engineered EVs offer a method of therapeutic delivery by selectively loading therapeutics into EVs that can then be targeted to particular cell types by altering membrane proteins and lipid content . HoweverEV heterogeneity presents a significant challenge in EV isolation and characterization, particularly from biofluids. The overall complexity of biofluids not only from constituent proteins and cell debris but from heterogeneous nanoparticle populations makes a targeted isolation method a necessity to understand the signalling carried out be EV subpopulations. By isolating EVs based on composition, it is possible to isolate relatively pure populations that are more relevant for studying EV subpopulations and their functions . ProteomFurthermore, considerations to the effects of the isolation method on the isolated EVs need to be considered, such as aggregation, coating, damaging or altering EVs. For example, in affinity isolation surface proteins may lose their binding capabilities following elution of the antibody due to the harsh conditions of elution as well as damage EVs. Alternative affinity ligands such as aptamers may represent a way around this issue as they can be degraded under gentler conditions compared to antibody elution.Despite the heterogeneity of EVs, biophysical properties are still an important part of EV characterization. Particle size and count is an integral part of EV characterization. NTA is the most popular method of determining particle size and is based on the Brownian motion of particles . The resEV composition can be used in conjunction with the aforementioned characterization techniques. By targeting EV cargo it is possible to understand their function better, this can be with existing methods such as fluorescent NTA, flow cytometry or scanning electron microscopy. Morphology is another point of differentiation for EVs . TransmiTogether methods such as these can be utilized to understand EV subpopulations and their functionality. Current questions in the field, particularly for therapeutics, are based around finding and understanding the unique composition of the EV population responsible for the desired function. It is necessary as well to understand the number of EVs that are required to induce a response, in other words, potency, in addition to understanding the mechanisms involved. Determining the relevant EV subpopulation is critical, low uptake or cargo release may be due to differences in functional subpopulations. Particularly in vivo where it may be challenging to identify the origin of individual EVs and track populations without first knowing the exact EV composition.The heterogeneity of EVs introduces a level of complexity in explaining and understanding the functions of EVs. Size, while a useful isolation method is not able to resolve this heterogeneity. Affinity‐based methods targeting known protein differences in EVs might offer a useful alternative and tool for dissecting the functions of EVs. This will in turn contribute to the further development of EV‐based therapeutics and EV‐based biomarkers for diagnostics.The authors declare no conflict of interest.


COVID‐19 has pulled back the curtain on health system fragility to expose persistent and deepening inequities worldwide. The limited capacity of low‐ and lower‐middle income countries (LMICs) to respond to the pandemic and its impact on the health of populations – particularly the most vulnerable – presents a marked challenge. In this context, countries face the enormous task of rethinking the way essential services will be delivered. A critical and essential part of solving these challenges will be using information and communication technology and digital health to enhance direct communication with the public; scale proven and innovative service delivery models; and empower the frontlines. However, if the deployment, adaptation, or expansion of these innovations are not user‐centered for the most marginalized or do not learn from past lessons, it could be highly wasteful at best. At worst, such shortcomings could exacerbate pre‐existing weaknesses in the health care system such as exclusion of peripheral populations, disempowerment of health workers, and proliferation of unregulated private providers. We provide recommendations of which innovations should be prioritized and implementation principles to address the current challenges while responding to the need to fundamentally change service delivery for accelerated impact. While state of the pandemic remains urgent and overwhelming, novel and practical service delivery models can be implemented at‐scale and designed for the needs of populations who need them most. The novel coronavirus, COVID‐19, has pulled back the curtain on health system fragility exposing persistent and deep inequities. Across LMICs, approximately 8.6 million people succumb to causes amenable to routine health care provision annually  or Health Center by Phone. In less than a decade, CCPF has grown from providing RMNCHN services in a single district to scaling nationally and addressing all health topics with a quick pivot to include COVID‐19. Through the pandemic, the government has endorsed the national hotline as a reliable source for COVID‐19 information. Beyond Malawi, Viamo’s 3‐2‐1 interactive voice response (IVR) hotline has reached over 14 million listeners through upwards of 43 million message engagements across more than 25 countries to date worldwide – including Haiti, Nepal and Pakistan – through its COVID‐19 response Viamo, .The World Health Organization (WHO) has developed a communication platform through WhatsApp to disseminate trusted information directly to diverse populations globally in response to the pandemic. The WHO’s Health Alert service allows users to message ‘hi’ in one of seven languages, prompting a dialogue across a range of COVID‐19 topics – from symptoms to situation reports – enabled by chatbot technology. By partnering with a messaging service already embedded in the daily lives of citizens around the world, Health Alert can connect with as many as 2 billion people  is another way to reimagine the delivery and maintenance of essential services while unburdening health facilities. Globally, hundreds of thousands of CHWs already use smartphones and applications such as CommCare, Community Health Toolkit (Medic Mobile), and OpenSRP. In such settings, these applications can be rapidly updated with forms and automated protocols to assist with the pandemic response and perform tasks including triaging and referring suspected COVID‐19 cases, recording vital patient information, and building trust with community members through hygiene counseling and de‐stigmatization. By fortifying these community linkages, CHWs can play an integral role in reducing the need for patients to visit health facilities.Additionally, virtual peer‐to‐peer learning and tele‐mentoring platforms can facilitate the dissemination of new skills and information, particularly when the landscape of available guidance is changing in real‐time. One example is Project ECHO. This platform leverages videoconferencing to connect communities of practice virtually including, but not limited to, networks addressing COVID‐19 treatment and clinical operations. Over 800 ECHO programs exist across nearly 40 countries including India, Brazil and South Africa. As early as February 2020, Project ECHO co‐hosted specialized COVID‐19 virtual learning sessions connecting nearly 2,000 health providers in 20 (largely LMIC) countries across 400 sites  countries are favorably positioned to leapfrog traditional brick‐and‐mortar facilities that often lack efficient and sustainable service delivery and equitable health outcomes. With the uptake of new and improving technology diffusing across LMICs, the opportunity to reimagine service delivery through digital health innovation is real, not just theoretical.


Here, we briefly review the human models that are currently being used in COVID‐19 research and drug development.Currently, therapeutics for COVID‐19 are limited. To overcome this, it is important that we use physiologically relevant models to reproduce the pathology of infection and evaluate the efficacy of antiviral drugs. Models of airway infection, including the use of a human infection challenge model or well‐defined, disease relevant  Abstract figure legend Current physiologically relevant models for studying COVID19. Image made using BioRender.com. The greatest risk factor for severe disease is age, with higher morbidity and mortality rates in the elderly population, despite younger people shedding similar levels of virus is an infectious disease caused by a newly emerged severe acute respiratory syndrome (SARS)‐coronavirus‐2 (SARS‐CoV‐2). COVID‐19 presents a diverse clinical spectrum, ranging from an asymptomatic carrier state to patients with life‐threatening multi‐organ failure and death . There is also evidence that some secretory cell types, notably the ‘secretory3’ cell type (an intermediate position between club or goblet cells and ciliated cells) may be an important viral target as they are ACE2+ and co‐express viral spike priming proteases transmembrane serine protease 2 (TMPRSS2) and/or furin . Severe cases also show greater enrichment for neutrophils, comprising >60% of cellular composition of upper airway samples   and Caco2 , whilst hepatic Huh7, renal HEK293T and neuronal U251 cells were also able to propagate the virus . Employing a range of functional analyses including RNA/protein crosslinking, splice reporter assays and surface sensing of translation (SUnSET) assays, NSPs were shown to disrupt mRNA processing. Mechanisms included suppression of global mRNA splicing (NSP16), binding to 18S ribosomal RNA causing global inhibition of mRNA translation (NSP1) and disruption of protein trafficking to the cell membrane (NSP8 and NSP9)  lack appropriate cell polarisation and many other distinguishing properties found in the lung, such as transport proteins, mucus and motile cilia. Fortunately, the relative accessibility to airway epithelium and air–liquid interface (ALI) culture techniques (described below) has allowed the development of a gold standard for in vitro model that resides at the top of the experimental hierarchy for preclinical airway epithelial model systems .In traditional airway epithelial cell culture systems, primary cells do not survive for more than a few passages and their characteristics can deteriorate rapidly with age. One solution of many  .Functional readouts from ALI‐specific assays have shown a transient decrease in epithelial barrier function and disruption of tight junctions, though infectious viral particles almost exclusively remain at the apical side of cultures Fig.  . To study this further, a modified ALI model has been developed that facilitates neutrophil transepithelial migration in response to airway infection or other stimuli  and infected, neutrophils or PMNs are added to the basolateral (medium) side of the epithelial cells so they can migrate in the physiological direction, from basolateral to apical surface. This model has already revealed important insights into the contribution of neutrophils to airway damage and viral clearance during RSV infection  across columnar epithelia is a key component of mucosal defence and inflammation, and it is becoming increasingly evident that COVID‐19 disease severity is associated with a dysregulation of the immune response, including increased neutrophil recruitment . The lung‐on‐a‐chip model has been employed successfully in some drug discovery and toxicity studies  co‐ordinate the uptake of nutrients from the media  Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. T.M. would also like to thank A.G Leventis Foundation for their support and help during her studies. C.M.S is currently a recipient of grants from Animal Free Research UK (AFR19‐20274), BBSRC (BB/V006738/1), GOSH Children's charity (COVID_CSmith_017) and the Wellcome Trust (212516/Z/18/Z).Funding for Open Access was approved for this article.Peer Review HistoryClick here for additional data file.Video abstract and summaryClick here for additional data file.


The coronavirus disease 2019 pandemic and the consequent global lockdown posed a particular challenge for youths with mental health problems. Crucial interference with their everyday lives likely increased psychological distress while accessibility of conventional mental health care was limited. Ongoing online trials offer a unique opportunity to analyse mental health status and help‐seeking behaviour of adolescents during the pandemic. The ProHEAD‐online trial aims at improving help‐seeking behaviour of children and adolescents with significant psychological impairment. From January to May 2020, 1,042 students had access to the ProHEAD‐online platform providing information on mental illness, monitoring, peer support and professional counselling. In the week from 11 March, when schools were closed in Germany, a drastic (more than 2 standard deviations) but time‐limited increase in utilization of the ProHEAD‐online services became apparent. This may indicate a worsened mental health status and an increased help seeking via digital services during the lockdown. Although this finding is purely observational, it speaks to the importance of evidence‐based online service in the field of mental health within the current crisis and beyond. Numerous commentary and viewpoint papers have recently discussed the expected direct and indirect economic, social and psychological effects of the coronavirus disease 2019 (COVID‐19) pandemic including the related global lockdown. Children and adolescents around the world were and still are affected by containment efforts such as school closings, stay‐at‐home orders and physical distancing which have led to abrupt and significant changes in their everyday lives and social relationships. While the exact impact of the current crisis and the various pandemic‐control decisions on the mental health and well‐being of young people is largely unknown, experts assume a significant increase in the burden of mental illness, and particular concerns have been raised about the worsening of pre‐existing mental health problems, discontinuity of care and an exacerbation of the already pervasive challenge of unmet treatment needs among young people , young people may face even more barriers to professional care than in prepandemic times. In response to the outbreak, and in line with the World Health Organization's timely strategic call to ‘reinforce or establish web‐based and other telemedicine platforms to provide direct clinical services and provide clinical decision support’ . Since the start of the ProHEAD consortium, 5,447 students from 77 schools at five study sites were screened for their mental health. Out of these, 1,143 students reported substantial mental health problems and were consequently eligible for participation in ProHEAD‐online. Participants were included in the trial independent of their current treatment status. While the automated monitoring of help‐seeking behaviours, treatment uptake and barriers in the intervention group was stopped as soon as participants reported that they took up treatment, they could still login to ProHEAD‐online and utilize the other modules. Between January and May 2020, we had 1,042 students  having access granted to the full ProHEAD‐online intervention (intervention group) or the limited version (control group). Among those, we observed a substantial, but clearly time‐limited increase in utilization of ProHEAD‐online (increase of more than 2 SD of the composite measure) starting in the week of 11th of March . We created a composite measure of ProHEAD‐online utilization including number of logins, clicks and chat participation aggregated by week  as well as in two other options: On the one hand, digital approaches allow us to significantly improve the continuity of care, for example in the context of stepped care approaches where online tools are used to provide pre‐ or post‐treatment support or in the context of the management of chronic mental illness where technology‐enhanced low‐threshold monitoring allows for the immediate detection and intervention of recurrent illness episodes. On the other hand, blended treatment approaches, that is the utilization of digital tools in parallel to face‐to‐face treatment, appear highly promising. Blended care either aims at improving treatment efficacy  or at improving treatment efficiency (when part of the treatment is delivered via technology and the costly time in the face‐to‐face setting is thus reduced).Future research and experiences from clinical practice will help to identify the best ways how to harness digital approaches in various care settings, service systems and during unforeseeable circumstances such as the COVID‐19 pandemic. In that sense, the present crisis actually offers promising opportunities for our field to work towards enhanced models of service delivery in order to mitigate the detrimental impact of mental illness on the lives of young people.Figure S1. Utilization of ProHEAD‐online over time . Dates represent the first day of the respective week.Click here for additional data file.


Angioma serpiginosum is a rare benign nevoid disorder affecting the small vessels of the upper dermis. We are presenting a case of this rare disease in an 11‐year‐old girl who presented with this condition on abdomen in zosteriform pattern which is rare presentation and is the first of its type. Angioma serpiginosum is a rare benign nevoid disorder affecting the small vessels of the upper dermis. We are presenting a case of this rare disease in an 11‐year‐old girl who presented with this condition on abdomen in zosteriform pattern which is rare presentation and is the first of its type. We are presenting a case of this rare disease in an 11‐year‐old girl with multiple progressive asymptomatic scattered and confluent punctate erythematous to violaceous macules over the left side of the abdomen in T8‐T10 dermatomal distribution with normal surrounding and intervening skin since birth. Histopathological examination (HPE) revealed few ectatic congested thin‐walled blood vessels in papillary dermis with few areas of downward growth of the rete ridges between these blood vessels. Based on the clinical and the HPE, a diagnosis of angioma serpiginosum was made. This rare vascular anomaly and abdomen involvement with zosteriform distribution are rarely reported in the literature.Angioma serpiginosum is a rare benign primary telangiectatic disorder affecting the capillaries of the upper dermis characterized by asymptomatic pinpoint macules that group together in linear, serpiginous, or gyrate patterns.2An 11‐year‐old girl presented with multiple asymptomatic erythematous lesions on the left abdomen which was first noticed by her mother as multiple red pinhead‐sized lesions during birth. The lesions progressively extended with new satellite lesions appearing around the previous ones with the increasing age. There was no family history of similar skin lesions. There were no visual or neurological symptoms.Cutaneous examination revealed multiple discrete and confluent punctate erythematous to violaceous macules on the left abdomen in T8 to T10 dermatomal distribution with normal surrounding and intervening skin  is a rare vascular disorder described first by Hutchinson in 1889 as a “serpiginous or infective nevus” and then named by Crocker in 1894.15The condition usually starts in childhood or birth with a female predominance.In our case, the patient presented with localized telangiectasia without skin atrophy. The differentials for the condition include Angioma serpiginosum, capillary malformation, unilateral nevoid telangiectasia, and purpuric conditions.None.KA: concept, manuscript preparation, and literature search. PP: manuscript editing, guidance, and final approval. RS: histopathological reporting. MB: manuscript preparation and literature search. SS: manuscript editing and correspondence Author.Consent obtained from the parents of the patient for publication and use of the images.


Adherence to direct oral anticoagulants (DOACs) remains a concern among non‐valvular atrial fibrillation (AF) patients. We aimed to assess patterns of adherence with DOACs and examine their association with ischemic stroke and systemic embolism (SE).This retrospective cohort study includes all adult members of Clalit Health Services, the largest healthcare provider in Israel, with newly diagnosed non‐valvular AF between January 2014 and March 2019, who initiated DOACs within 90 days of AF diagnosis and used DOACs exclusively. Adherence was assessed using the proportion of days covered (PDC) over the first year of treatment, and high adherence was defined as PDC ≥80%. Regression models were used to identify predictors of high adherence to DOACs and to examine the association between adherence and stroke or SE.2DS2‐VASc score ≥2. Risk of stroke and SE was lower among highly adherent DOACs users; adjusted HR 0.56 , compared to users with PDC <80%.Overall 15,255 patients were included in this study. The proportion of highly adherent (PDC ≥80%) DOACs users was around 75% and decreased slightly over the years. On multivariable analyses, the likelihood of high adherence to DOACs increased with age and across higher socioeconomic classes, and was more likely among females, Jews, statins users, and patients with CHAAdherence with DOACs is still sub‐optimal among non‐valvular AF patients, resulting in a higher risk of stroke and SE. High adherence to DOACs reduces the risk of stroke and systemic thromboembolism. With approximately 75% of patients highly adhering to DOACs, adherence is suboptimal. Older age, higher socioeconomic class, female sex, Jewish ethnicity, and CHA2DS2‐VASc score≥2 are predictors of high adherence. VKAs have several limitations such as high bleeding risk, slow onset of action, the need for frequent monitoring, and numerous drug and diet interactions. Direct oral anticoagulants (DOACs) have been recently introduced and have been gradually replacing VKAs owing to their distinct benefits in terms of efficacy, safety, convenience, more predictable effect and fewer drug and diet interactions.It has been shown that less than half of the patients adhere to OAC treatment over time.Adherence patterns are an integral part of clinical decision making and depend on various factors, either social or clinical. Understanding those patterns and factors is clinically relevant and may be helpful in better resources utilization and health planning.In this study, we use a population‐based real‐life data to assess patterns and trends of patients’ adherence with DOACs treatment. This study also aims to provide specific insight as for the implication of non‐adherence to DOACs on the risk of ischemic stroke and systemic embolism (SE).22.1This study is based on data from the computerized database of Clalit Health Services (CHS) which provides inclusive health care for more than half of the Israeli population. Health care coverage in Israel is mandatory according to the National Health Insurance Law (1995), and is provided by four groups akin to not‐for‐profit health maintenance organizations (HMO). All members of the different HMOs have a similar health insurance plan and similar access to health services, including low medications copayment. The electronic medical records (EMRs) of CHS include data from multiple sources: records of primary care physicians, community specialty clinics, hospitalizations, laboratories, and pharmacies. A registry of chronic diseases diagnoses is compiled from these data sources. Diagnoses are captured in the registry by diagnosis‐specific algorithms, employing International Classification of Diseases Ninth revision (ICD‐9) code reading, text reading, laboratory test results, and disease‐specific drug usage. A record is kept of the data sources and dates used to establish the diagnosis, with the earliest recorded date, from any source, considered to be the defining date of diagnosis. Several high‐quality population‐based studies have been conducted based on data retrieved from CHS database.2.2We used the computerized database of Clalit Health Services (CHS) to retrospectively identify all adults patients aged 50 years or older with newly diagnosed non‐valvular AF  between 1 January 2014 and 31 March 2019. For studying 1‐year adherence to DOACs treatment, we included patients who filled OAC prescription during the first 90 days after AF diagnosis, patients whose first OAC prescription was DOACs, patients who did not switched treatment from DOACs to VKAs during the first year of treatment and patients who survived for at least 12 months after first DOACs prescription date in order to keep homogeneity in the 1‐year adherence assessment. The date of first‐filled DOACs prescription constituted the index date. The flowchart in Figure The study was approved by the Review Board of the Lady Davis Medical Centre and conducted in accordance with the Declaration of Helsinki.2.3The use of OACs was determined based on CHS pharmacy records using ATC codes: B01AF01 (rivaroxaban), B01AF02 (apixaban), B01AE07 (dabigatran), and B01AA03 (warfarin). Data on medication use is considered to be complete because of the low copayment required in the CHS making it unlikely that prescription medications are purchased in non‐CHS pharmacies. Detailed prescription information on dates of prescriptions filled and number of treatment days supplied enabled us to estimate adherence.2 score ≥4 points. The indications for low copayment were updated in January 2014 to include non‐valvular AF patients with CHADS2 of ≥3, and later in January 2016 to include non‐valvular AF patients with CHADS2 of ≥2, and lastly in January 2019 to include non‐valvular AF patients with CHADS2 of ≥1.DOACs were included in the Israeli national healthcare insurance plan before 2014. At the beginning, DOACs were supplied at low copayment for patients with non‐valvular AF and CHADS2.4Adherence to DOACs was assessed using the proportion of days covered (PDC), as recommended by the Pharmacy Quality Alliance (PQA).We assessed the association between adherence and the composite outcome of ischemic stroke and SE, defined as a primary discharge diagnosis of ischemic stroke  and SE .2.52DS2‐VASc score, a widely used risk stratification score for stroke prediction in patients with AF.Demographic characteristics, and data on comorbidities were retrieved from the CHS‐computerized database for the calculation of CHA2.6p < .05 for the two‐tailed tests was considered statistically significant. Continuous variables are summarized with means and standard deviations (SD), and categorical variables are summarized with counts and proportions.Statistical analyses were performed in 2020–2021 using IBM SPSS Statistics 24.0 (IBM). For all analyses, Multivariable logistic regression using backward selection was used to identify predictors independently associated with increased adherence to anticoagulants (PDC ≥80%).vs. PDC <80%), as ordinal variable  with PDC <40% serving as reference category, and as continuous variable to estimate the HR for each 10% increase in PDC. Multivariable models that were used to assess the association between PDC and ischemic stroke or SE were adjusted for CHA2DS2‐VASc score.Kaplan–Meier curves were used to depict the distribution of time to composite of stroke or SE. Cox proportional hazard regression models were used to estimate the hazard ratios (HRs) and the 95% confidence interval (95% CI) for the association between PDC and stroke or SE. For this analysis the follow‐up started by the end of the 12 months required for PDC estimation and extended until the event of interest (stroke or SE), death or the end of follow‐up in 30 September 2020, whichever came first. PDCs were modeled as binary variable , during the first year of treatment, increased with age and with increasing socioeconomic classes, and was more likely among females compared to males, among Jews compared to Arabs, among patients treated with statins and among patients with CHAse Table .3.32DS2‐VASc score, was found to be 44% lower  among highly adherent DOACs users (PDC ≥80%) compared to DOACs users with PDC <80% (Table The risk of ischemic stroke and SE, after adjustments to CHA4This is one of the few large‐scale population‐based studies to assess adherence to DOACs. In this study, we found that approximately 75% of incident AF patients treated with DOACs were highly adherent to treatment, namely, were covered at least 80% of the days during the first year of treatment. The proportion of high adherence to DOACs in this study is consistent with the rates described in two recent large cohort studies conducted in France and Canada.2DS2‐VASc scores among their users over the years.Nevertheless, as much as these results seem encouraging compared to previous studies,2DS2‐VASc score ≥2 was demonstrated previously,Higher adherence rates among older ages, among higher socioeconomic classes, among women, among Jews and among statin users, as well as lower rates among smokers, stand in line with results from other studies.An important contribution of this study is providing an insight as for the implication of adherence level, estimated by PDC calculation, on adverse outcomes, using a real‐life data. In our study, high adherence to DOACs significantly reduced the risk of ischemic stroke and SE. While most studies demonstrated the protective effect of DOACs by comparing DOACs treated patients to warfarin‐treated patientsA notable strength of this study is being a population‐based study with a relatively large number of AF patients. The fact that healthcare services in Israel are public and medication copayment is low makes the documentation of medications prescribing and purchases, reliable. In addition, relying on these documentations rather than patients’ self‐reports avoids the potential recall bias.Nevertheless, our study is subject to some limitations. First, as an observational study, in which data is extracted from electronic health records, it is prone to misclassification bias or lacks data. Second, we relied on prescriptions and purchases data rather than actual taking the drugs by the patients. Thus, there might be an overestimation of adherence rates in cases in which patients stopped taking DOACs even though they had purchased the drugs. In addition, patients could have taken lower doses than purchased, hence being exposed to higher embolic risk. Finally, our study is prone to the healthy adherer effect.5Adherence with DOACs treatment is still far from optimal, resulting in a substantial higher risk of embolic complications. More efforts should be done in order to increase physicians’ and AF patients’ awareness of the importance of compliance to DOACs.The authors declare no conflicts of interest for this article.The study was approved by the Review Board of the Lady Davis Medical Centre and conducted in accordance with the Declaration of Helsinki.


Ferroptosis is a new form of regulated cell death, which is characterized by the iron‐dependent accumulation of lethal lipid peroxides and involved in many critical diseases. Recent reports revealed that cellular energy metabolism activities such as glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle are involved in the regulation of key ferroptosis markers such as reduced nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH), and reactive oxygen species (ROS), therefore imposing potential regulatory roles in ferroptosis. Remarkably, tumor cells can activate adaptive metabolic responses to inhibit ferroptosis for self‐preservation such as the upregulation of glycolysis and PPP. Due to the rapid proliferation of tumor cells and the intensified metabolic rate, tumor energy metabolism has become a target for disrupting the redox homeostasis and induce ferroptosis. Based on these emerging insights, regulatory impact of those‐tumor specific metabolic aberrations is systematically characterized, such as rewired glucose metabolism and metabolic compensation through glutamine utilization on ferroptosis and analyzed the underlying molecular mechanisms. Additionally, those ferroptosis‐based therapeutic strategies are also discussed by exploiting those metabolic vulnerabilities, which may open up new avenues for tumor treatment in a clinical context. Ferroptosis is regulated by a variety of metabolic pathways, but the involvement of energy metabolism is rarely investigated. This review discusses the functional roles of various tumor metabolic phenotypes on ferroptosis regulation in the tumor landscape, which may improve the understanding of the tumor ferroptosis network and provide implications for the development of novel ferroptosis‐based therapies. However, it has become increasingly evident that tumor cells could demonstrate intrinsic or acquired resistance to these apoptosis‐dependent antitumor modalities and substantially increase the risk of treatment failure and post‐treatment relapse. Therefore, it is of both clinical and practical significance to discover new non‐apoptotic regulated cell death pathways and explore their therapeutic usage. In 2003, Stockwell et al. employed high‐throughput screening method to interrogate the cytotoxicity of around 23 550 synthetic lethal small‐molecule agents against engineered tumorigenic cells and firstly observed that the two compounds of Erastin and RSL3 could selectively kill tumor cells expressing Small T oncoprotein and oncogenic RAS, of which the morphological features were distinctively different from those extensively studied cell death pathways such as apoptosis or necrosis. In a follow‐up study by the same group in 2012, the authors thoroughly investigated the morphological, biochemical, and genetic features of this emerging route of regulated cell death and evidently confirmed that it was different from any known form of cell death routes such as apoptosis, necrosis or autophagy. Due to its dependence on cellular iron but not other metal ions, this novel form of regulated cell death was eventually termed “ferroptosis”. The continuous progress in ferroptosis research has substantially expanded the arsenal of ferroptosis‐inducing agents for potential therapies, wherein some old drugs may also present pro‐ferroptosis activities. For instance, Sorafenib, originally used as a US Food&Drug Administration (FDA)‐approved inhibitor of multiple oncogenic kinases for tumor therapy, could induce ferroptosis by inhibiting the activity of SLC7A11 in a similar manner to erastin. Meanwhile, the FDA‐approved anti‐inflammatory agent Salazosulfapyridine could also induce ferroptosis by suppressing the System Xc–‐mediated exchange of cystine and glutamate. From a morphological perspective, ferroptotic cells usually demonstrate mitochondrial volume shrinkage, increasing mitochondrial membrane density and decreasing mitochondrial cristae, while no obvious changes were observed in the morphology of the cell nucleus and chromosomes. Meanwhile, ferroptotic cells are also associated with a series of characteristic biochemical features including the universal upregulation of cellular reactive oxygen species (ROS) and lipid ROS levels, accompanied with alterations in the expression levels of metabolism‐related genes such as glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1)  and NADPH supply to support vital biological events such as macromolecular biosynthesis, membrane‐protein integration, maintenance of cross‐membrane ion gradients and DNA replication. Nonetheless, many of these energy metabolic aberrations converge on ferroptosis and profoundly affect its initiation and execution. Typically, glucose is the primary energy source and biosynthetic substrate for most cell types. Under normal conditions, glucose was first converted to pyruvate via the glycolysis route then entered the tricarboxylic acid cycle (TCA) cycle and oxidative phosphorylation (OXPHOS) processes for complete metabolization. Alternatively, under hypoxic or malignant conditions, the glycolysis‐generated pyruvate was converted to lactate under the catalyzation of lactate dehydrogenase. Specifically, the internalized glucose molecules were first phosphorylated by hexokinase (HKs) to produce glucose 6‐phosphate, which would be further converted into converted to fructose‐6‐phosphate by glucose‐6‐phosphate isomerase to join the subsequent metabolic phases for energy production. Meanwhile, the fructose‐6‐phosphate may also be bypassed into the pentose phosphate pathway (PPP) by 6‐phosphate glucose dehydrogenase for complete oxidation, leading to the production of those necessary biosynthetic precursors and NADPH  was involved in the Erastin‐induced ferroptosis. VDAC is a class of porin ion channels located on the outer mitochondrial membrane, which is responsible for controlling the exchange of ions and metabolites between the cytosol and the mitochondria. Interestingly, erastin could inhibit the complexation of tubulin with VDAC and maintain VDAC in an opened state, consequently enhancing the mitochondrial respiration and increasing ROS production to promote ferroptosis. In contrast, downregulating the expression level of VDAC2/3 or treating cells with the ROS scavenger of N‐acetyl‐l‐cysteine (NAC) could reduce the mitochondrial ROS production, thus rescuing the cells from the erastin‐induced ferroptosis. The evidence above collectively demonstrates the indispensable regulatory role of mitochondrial energy production in the ferroptosis process.Mitochondrion is the primary site for oxidative respiration inside cells and also the major source of ROS.47 It  However, the metabolic rates of tumor cells are usually accelerated to support their rapid and uncontrolled proliferation, which would lead to enhanced cellular ROS stress and render them intrinsically susceptible to ferroptosis. Consequently, the glucose metabolism in malignant tumor cells is usually rewired to glycolysis to meet their increasing demands for energy and biosynthetic precursors as well as maintenance of redox homeostasis to prevent ferroptosis. For instance, it has been observed that tumor cells have high glycolytic rate to compensate its intrinsically low ATP‐generating efficiency. Meanwhile, the mitochondrial OXPHOS activity in these glycolytic tumor cells have been evidently inhibited to relieve the ROS stress. Nevertheless, the glycolysis‐induced suppression of OXPHOS is not a permanent state and could be reversed when the glycolysis becomes inhibited, accompanied with increasing susceptibility to mainstream chemotherapy as well as ferroptosis inducers. The underlying mechanism for the ferroptosis sensitization after glycolysis inhibition is that the metabolic rewiring to OXPHOS would elevate the cellular ROS levels to disrupt iron homeostasis and promote lipid peroxidation. Consistent with these insights, it has been observed that tumor cells undergoing erastin or RSL3‐induced ferroptosis showed substantially lower glycolytic activities, evidenced by the significant downregulation of three key glycolysis enzymes including HK II, platelet‐type phosphofructokinase and pyruvate kinase M2 (PKM2) in a recent report by Wang et al. The insights above are immediate evidence that ferroptosis‐inducing mechanism for both erastin and RSL3 is by reversing the cellular metabolic pathway from glycolysis to OXPHOS, further indicating that the coordination between glycolysis and OXPHOS have profound impact on the cellular ROS stress and could regulate ferroptosis in a dynamic manner.It is well established that ROS produced during TCA cycle and OXPHOS could also act as secondary messengers to regulate various cellular activities.63 How3 Typically, PPP branches after the initial step of glycolysis, during which the intermediate metabolite glucose 6‐phosphate (G6P) was bypassed into the oxidative and non‐oxidative branches of PPP to synthesize ribose 5‐phosphate (precursor of nucleotide and nucleotide coenzyme), erythrose 4‐phosphate (precursor of aromatic amino acids) and NADPH. Consistent with the central role of NADPH in the cellular metabolic network and redox homeostasis, it has been recently discovered that NADPH could regulate ferroptosis from multiple aspects. For instance, Wang et al. discovered that oxidoreductases on the endoplasmic reticulum such as NADPH‐cytochrome P450 reductase (POR) and NADH‐cytochrome b5 reductase (CYB5R1) were responsible for the catalyzation of lipid peroxidation during ferroptosis. Under normal conditions, POR and CYB5R1 would transfer the electrons of NADPH/NADH to the downstream proteins such as cytochromes P450. However, they may incidentally donate the electrons to the oxygen in the cellular environment under certain circumstances to generate H2O2, which would be further converted to hydroxyl radicals through the iron‐catalyzed Fenton reaction route. Due to the high reactivity of the hydroxyl radicals, they could cause the abstraction of hydrogen atoms from the methylene carbons in the polyunsaturated fatty acids (PUFAs) and readily initiate lipid peroxidation, leading to the disruption of the plasma membrane. NADPH could also act as electron carriers and donate electrons to enable the glutathione reductase‐mediated reduction from glutathione disulfide (GSSG) to glutathione (GSH). Meanwhile, NADPH could also support the SLC7A11‐mediated intake of cystine and further convert it into cysteine for GSH synthesis. GSH would subsequently participate in the glutathione peroxidase 4 (GPX4)‐mediated elimination of lipid ROS to inhibit ferroptosis. In addition to GSH regeneration and synthesis, NADPH also participates in the thioredoxin reductase (TR)‐mediated regeneration of thioredoxin (Trx) and form a functional system for the reduction of protein‐disulfides. Specifically, the reduced Trx could be oxidized by donating a proton from the sulfhydryl group to reduce other protein‐disulfides, and the oxidized Trx then reacts with NADPH under the catalyzation of TR and is converted back to the reduced form for recycling. This system is a critical biochemical component in redox homeostasis, antiviral defense, and ferroptosis regulation. For instance, the report by Evijola Llabani et al. showed that a small‐molecule compound ferroptocide could inhibit Trx in a targeted manner and initiate ferroptotic cell death. There are also reports that NADPH is involved in a non‐GPX4‐dependent ferroptosis route by cooperating with ferroptosis suppressor protein 1 (FSP1) to reduce Coenzyme Q10 (CoQ10) to CoQ10‐H2, which is a potent antioxidant capable of preventing the propagation of lipid peroxidation on the plasma membrane  via difolate reductase (DHFR) in addition to the de novo guanosine triphosphate cyclohydrolase I (GCH1)‐mediated synthesis route. Extending from the evidence above, it was discovered that inhibiting the NAD+ kinase (NADK) could reduce the cellular NADPH levels and enhance their susceptibility to ferroptosis induction by typical ferroptosis inducers such as erastin, RSL3, and FIN56, further validating the potential application of NADPH as a biomarker for assessing ferroptosis sensitivity. Recently, Leu et al. demonstrated that by replacing the proline (P47) at the codon 47 of the human p53 protein into serine (S47), the cells would show reduced glycolysis capacity while metabolism through PPP would be enhanced significantly, accompanied with increasing NADPH/NADP+ ratio. S47 cells showed distinctive metabolic profiled compared to P47 cells, characterized by lower ROS stress and expression of Activating Transcription Factor 4 (ATF4). However, enhancing the expression of ATF4 in S47 cells could reverse this trend and inhibit the PPP activities therein, leading to increasing sensitivity to ferroptosis‐inducing treatments. Overall, it could be concluded that the PPP‐generated NADPH could boost the antioxidant defense in tumor cells by supporting the biosynthesis of GSH, Trx, and CoQ10‐H2 to rescue them from ferroptosis, and targeting the abnormally elevated PPP in tumor cells could afford a robust strategy to disrupt the cellular redox homeostasis for ferroptosis‐based tumor therapy.Tumor cells not only exploit glucose as an energy source for the glycolysis‐mediated ATP production, but also show increasing activities of the PPP, through which glucose is consumed to obtain those biosynthetic precursors as well as to maintain the cellular redox homeostasis.69 Typ4 some of the tumor cells may have limited blood supply and be forced into a glucose‐deprived state, thus suppressing those glucose‐metabolism routes such as glycolysis and PPP as well as severely compromising cellular energy supply and biosynthesis. To ensure cell survival, tumor cells would activate the energy crisis censor adenosine monophosphate ‐activated protein kinase (AMPK) to enhance ATP production and prevent its decomposition. Specifically, the energy crisis due to glucose deprivation would elevate the AMP: ATP and adenosine diphosphate:ATP ratios and activate the phosphorylation of AMPK to phosphorylate the downstream effectors, thus promoting those ATP‐generating catabolic events while simultaneously inhibiting those ATP‐consuming anabolic events to maintain the ATP level. Recent studies indicate that AMPK would cause the phosphorylation of acetyl‐CoA carboxylase (ACC) to inhibit its activity and impose regulatory effect on tumor cell ferroptosis. ACC is a rate‐limiting enzyme in the biosynthesis process of fatty acids, and it has been observed in lipidomic analysis that inhibiting ACC in tumor cells would cause a substantial decrease in the cellular abundance of ferroptosis‐susceptible PUFAs, thus decreasing their susceptibility to ferroptosis inducing treatments . It is important to note that GLS has two subtypes that are GLS1 and GLS2, and only GLS2 is involved in the ferroptosis process. The underlying molecular mechanism is that only glutamate generated by GLS2‐mediated glutaminolysis may contribute to α‐ketoglutarate (α‐KG) formation and subsequently enhance the production of oxidizable lipids to induce ferroptosis. After entering the tumor mitochondria, the GLS2‐mediated glutamate could be converted to α‐KG under the catalyzation of aspartate aminotransferase (GOT) and glutamate dehydrogenase 1 (GLUD1) and thus maintain the TCA cycle function for metabolic compensation. It was also reported that tumor cells with intense TCA cycle activities were prone to glutamine addiction and demonstrating upregulated OXPHOS levels, thus elevating the cellular ROS stress and sensitizing tumor cells for ferroptosis  or an aspartate aminotransferase (GOT1) inhibitor (miR‐9) could both suppress the glutaminolysis in melanoma cells by disrupting the expression and function of their corresponding targets and negatively affect ferroptosis, validating their therapeutic potential of the ferroptosis regulation mechanisms discussed above.Glutamine is an important non‐essential amino acid in human body and widely involved in many metabolic events including the biosynthesis of proteins and GSH as well as energy production.102 La Specifically, System Xc‐ is a cystine/glutamate antiporter responsible for the regulation of cellular cysteine and glutamate levels, which is comprised of a light‐chain component (encoded by SLC7A11 gene) and a heavy‐chain component (encoded by SLC3A2 gene), in which the SLC7A11 subunit could take in extracellular cystine while simultaneously exporting intracellular glutamate at a 1:1 ratio. It should be noted that tumor cells are usually overexpressed with the SLC7A11 subunit to take in large amount of cystine and convert it into cysteine for the synthesis of GSH. This is an important process in maintaining the redox homeostasis, but it may also cause the rapid loss of cellular glutamate. To sustain the uptake of extracellular cystine, tumor cells often have upregulated glutaminolysis activities to replenish the cellular glutamate supply, which underlies the dependence of SLC7A11‐overexpressed tumor cells on glutamine metabolism. Consequently, it has been theorized that cystine deprivation could also suppress the glutamate export and the glutaminolysis‐generated glutamate would be decomposed through TCA cycle, leading to the production of high level of ROS to promote ferroptosis. Based on this rationale, inhibiting key targets in tumor glutamine metabolism could offer new opportunities for ferroptosis‐based tumor therapy.Recent insights also reveal that the glutamine metabolism in tumor cells is strongly affected by the cellular transport/metabolism of cystine.111 Sp6 The primary driving force of ferroptosis is the cellular ROS stress generated by the energy metabolic activities. Excessive cellular ROS would react with the PUFA components in cell membrane under the catalysis of iron species to initiate and propagate lipid peroxidation, eventually leading to cell death. Remarkably, the ROS levels in tumor cells are usually elevated compared to normal cells due to the intense metabolic activities, which renders them intrinsically susceptible to ferroptosis. These studies above collectively demonstrate the central role of energy metabolism in coordinating the tumor response to ferroptosis while also highlighting the potential application of ferroptosis as a therapeutic modality for tumor treatment.Despite the tremendous progress in antitumor research in recent years, tumor therapy remains one of the great clinical challenges. Conventional treatment modalities such as chemotherapy and radiotherapy would inevitably induce adverse side effects against normal cells and tissues, and their efficacy would decline rapidly due to the emergence of intrinsic and acquired treatment resistance in tumor cells. Consequently, It is urgent to develop new treatment strategies based on alternative tumor inhibition mechanisms. Ferroptosis is a recently discovered route of regulated cell death, which is distinctively from those previously discovered form of regulated cell death such as apoptosis, pyroptosis, and necrosis in terms of morphological, biochemical, and genetic features and shows particular promise for therapeutic purposes.13 The From a general perspective, tumor cells usually demonstrate upregulated glycolysis and PPP activities, which could not only inhibit the mitochondrial respiration to reduce ROS generation but also replenish the NADPH supply, thus contributing to the maintenance of the redox homeostasis to ensure cell survival. Meanwhile, several characteristic genetic mutations in tumor cells such as RAS, PI3K, and P53 could substantially boost the energy metabolism in tumor cells and affect the initiation and progression of ferroptosis therein. It could be thus concluded that disrupting the tumor energy metabolic pathways would not only alter the ferroptosis sensitivity of mutated tumor cells but also breading down their antioxidative defense to promote ferroptosis. Typically, Dannielle DeWaal et al. reported that downregulating the glycolysis in tumor cells could force the cellular glucose to enter the TCA cycles for OXPHOS, thus elevating the ferroptosis susceptibility of tumor cells under cystine deprivation. Meanwhile, interrupting the PPP route in tumor cells could downregulate the NADPH levels to inhibit the expression of downstream bioreductors such as GSH, CoQ10, and Trx, which was also beneficial for enhancing ferroptosis in the tumor landscape. Additionally, the intense glycolysis of tumor cells would also reshape the tumor microenvironment, leading to the upregulation of lactate levels in the tumor extracellular compartment. Interestingly, lactate could not only be exploited as an energy source by tumor cells, but also function as a signaling molecule to activate the hydroxycarboxylic acid receptor 1 (HCAR1) on tumor cell membrane to promote the production of anti‐ferroptotic monounsaturated fatty acid (MUFA). Based on this rationale, blocking the tumor lactate signaling pathways could be a viable strategy for ferroptosis sensitization. According to the report by Zhao et al., the authors confirmed that the lactate species in the tumor microenvironment could upregulate the production of MUFAs in tumor cells via the HCAR1/MCT1‐SREPB1‐SCD1 axis, which was capable of suppressing the lipid peroxidation in hepatocellular carcinoma (HCC) cells and ensuring the survival of HCC cells under ferroptosis induction with erastin or RSL3. In contrast, treating HCC cells with the combination of an MCT1 inhibitor (AZD3965) and RSL3 could efficiently initiate lipid peroxidation and induce ferroptosis both in vitro and in vivo. This study provides direct evidence on the anti‐ferroptotic roles of HCAR1 and MCT1 in tumor cells, highlighting their potential applications as molecular targets for ferroptosis‐based HCC therapy.As already discussed above, the cellular energy metabolism is directly connected to the ferroptosis process as it could regulate the antioxidative defense by mediating the synthesis of biomacromolecules and bioreductors such as NADPH.119 Fr However, James A. Olzmann et al. reported that certain types of tumor cells were still insensitive to ferroptosis induction after GPX4 inhibition or knockdown, indicating that ferroptosis is actually regulated by a complex network and more regulators still remain to be discovered. For instance, tumor cells were capable of removing the lipid ROS via FSP1–CoQ10–NAD(P)H pathway in a non‐GPX4‐dependent manner, which may act in parallel with the GSH‐dependent antioxidative defense to prevent ferroptosis. In another study by Jiang et al., the authors discovered that immortalized MCF10A cells showed upregulated Prominin2 expression under pro‐ferroptotic stimuli, which is a pentaspanin protein capable of promoting the formation and secretion of ferritin‐containing multivesicular bodies and exosomes. Specifically, inhibiting GPX4 in breast cancer cells would activate the prominin2‐MVB‐exosome‐ferritin pathway to reduce the cellular iron abundance and prevent ferroptotic cell death. Recently, Xuejun Jiang et al. reported that PI3K‐AKT‐mTOR pathway mutation, a common mutation that frequently occurs in a wide variety of cancers, could enhance the ferroptosis resistance of cancer cells by upregulating the synthesis of anti‐ferroptotic fatty acids through the SREBP1‐SCD1 axis. The authors demonstrated that the combinational treatment with mTORC1 inhibition and ferroptosis induction could lead to near‐complete tumor regression in xenograft mouse models with PI3K‐mutated breast cancer or PTEN‐defective prostate cancer. As shown by the reports above, ferroptosis could be a powerful tool for tumor therapy in a clinical context.It is well established that the ferroptosis process could be affected by several key factors including GSH synthesis, GPX4 expression, activity of ferroptosis‐related enzymes and cellular iron abundance, and preliminary research exploiting these vulnerabilities has already generated positive therapeutic responses on xenograft tumor models in vivo.126–15γ to inhibit the expression of SLC7A11 in tumor cells and impair the cystine uptake, thus inducing tumor cell ferroptosis. Extending from the interaction between ferroptosis and immunotherapy, the authors designed an engineered cystine‐degrading enzyme called cyst(e)inase, which could synergize with PD‐L1 immunotherapy for enhanced tumor inhibition efficacy. Meanwhile, some studies have shown that ferroptosis was a contributing factor to the radiotherapy‐mediated tumor inhibition, and treating tumor cells with ferroptosis inducers can effectively promote their sensitivity to radiotherapy via compromising the ionizing radiation‐induced SLC7A11/GPX4 induction and disrupting the ROS‐scavenging system. Ferroptosis inducers may also cooperate with other ROS‐reliant chemotherapeutic drugs such as cisplatin by simultaneously elevating the nutrient and oxidative stresses. These findings strongly support the therapeutic synergism between ferroptosis and other mainstream antitumor modalities including chemotherapy, radiotherapy, and immunotherapy, potentiating novel combinational therapies with improved clinical transability.The distinct biochemical characteristics of ferroptosis also offer emerging opportunities for the development of new combinational therapies, which may afford improved antitumor efficacy compared to mono‐therapies due to the beneficial therapeutic interactions. Typically, it was demonstrated that CD8+ T cells activated by immunotherapy could release IFNroptosis.128 Ex Typically, GoX could convert glucose into non‐metabolizable gluconic acid and H2O2, of which the latter could act as an endogenous oxidant to overcome the low H2O2 abundance in tumor cells under bioenergetic stress to enhance lipid peroxidation, thus potentially amplifying the ferroptotic damage. Nevertheless, it was also acknowledged that the GoX usually demonstrates indiscriminate cytotoxicity and is very susceptible to denaturation upon entering the physiological environment, thus necessitating the development of new delivery technologies to perverse its bioactivity and enhance the tumor‐targeted uptake. Remarkably, recent reports collectively demonstrated that the clinical translation of GoX‐assisted ferroptosis therapy could be substantially accelerated by the integration of nanotechnology. From an overall perspective, nanoparticulate therapeutic systems usually have superior tumor‐targeting effect than free drugs as well as multiplexing capabilities for other non‐therapeutic applications such as imaging or diagnosis. For instance, Yang et al. synthesized cancer cell membrane‐coated metal‐organic framework for the facile incorporation of GoX, Fe3+ ions, and doxorubicin for tumor‐targeted triplet ferroptosis/chemotherapy/immunotherapy. The released GoX could catalyze the oxidation of glucose to produce abundant H2O2, while the Fe3+ would react with the intracellular GSH and reduced to Fe2+, which would not only break down the antioxidative defense in tumor cells but also confer greater reactivity of the Fenton system for ferroptosis initiation. Similarly, GoX has also been used in conjunction with other metal ions with Fenton catalytic activity such as manganese and copper. Typically, Fu et al. developed an intelligent nanocatalytic theranostic system based on polyethylene‐glycol‐modified GoX (PEG‐GoX) to selectively enhance the production of hydroxyl radicals in tumor cells via Fenton‐like reaction routes. The authors employed PEG‐GoX as a biomolecular template to obtain doxorubicin‐loaded copper‐incorporated calcium phosphate nanoparticles. The nanoparticles could be rapidly hydrolyzed in tumor cells and release the Cu2+ ions, PEG‐GoX, and doxorubicin. The Cu2+ ions could react with the glutathione in the tumor intracellular compartment and be reduced to Cu+, leading to the restoration of its Fenton‐like catalytic reactivity. Meanwhile, PEG‐GoX could rapidly deprive the tumor cells of glucose and enhance the generation of H2O2, which could readily supply the Cu+‐mediated production of hydroxyl radicals. The combination of GoX and Fenton catalytic systems enhanced the hydroxyl radical production efficiency without inducing apparent toxic side effects in vitro and in vivo, further demonstrating the excellent clinical translatability of this strategy for ferroptosis‐based tumor therapy.By exploiting the markedly upregulated glucose uptake in hyperglycolytic tumor cells, glucose oxidase (GoX), a natural‐occurring enzyme that has been extensively explored for biocatalytic therapy, demonstrates significant relevance for ferroptosis‐based tumor treatment.132 Ty Dichloroacetate (DCA), a PDK inhibitor that has already been approved for the treatment of metabolic diseases such as lactic acidosis and diabetes, could readily inhibit the activity of PDK4 in tumor cells and enhance their susceptibility to the ferroptosis induction by imidazole ketone erastin. Nevertheless, considering the universal presence of PDK4 in both normal cells and tumor cells as well as the unsatisfactory pharmacokinetic features of DCA, new nanoformulation strategies are still urgently needed for enhancing the tumor‐targeted delivery of DCA. Alternatively, Guo et al. exploited the elevated PPP activities in tumor cells to develop an electron‐accepting micelle for tumor‐targeted ferroptosis therapy, which comprised of a PEG backbone modified with nitroimidazole‐conjugated polypeptide through a sheddable azobenzene linker. As the hypoxic tumor microenvironment favors the expression of nitroreductase and NADPH quinone dehydrogenase 1 (NQO1), the azobenzene linkers could be readily cleaved by reacting with the NQO1 and NADPH to remove the PEG moieties and enhance the uptake of nitroimidazole, which could afterward react with nitroreductase and NADPH to further deplete the intracellular NADPH, leading to the impairment of both the GSH and Trx redox cycles for ferroptosis induction. Considering the examples above, It is anticipated that the integration with nanotechnology could profoundly improve the efficacy, safety, and controllability of energy metabolism regulation in tumor cells for ferroptosis treatment.Alternative to the usage of GoX, some synthetic energy metabolism inhibitors have also demonstrated promotional effects on ferroptosis‐based treatment. Typically, Song et al. demonstrated in a recent report that pyruvate dehydrogenase kinase 4 (PDK4) was a top gene that regulated ferroptosis resistance in multiple cancerous cell lines, which could directly inhibit the activity of pyruvate dehydrogenase (PDH) and repress the PDH‐mediated pyruvate oxidation, thus preventing the glucose‐derived pyruvate from entering the TCA cycle and reducing the production of ferroptosis‐associated PUFAs to enhance the ferroptosis resistance.141 Di7The rewiring of energy metabolism in tumor cells is one of their most important metabolic features, which is necessary to provide the energy for sustaining the tumor growth and proliferation. Targeting vital processes in the tumor energy metabolism may offer the opportunity to regulate tumor susceptibility to ferroptosis and potentiate novel and effective ferroptosis‐based tumor therapies. Nevertheless, despite these therapeutic opportunities, the clinical translation of ferroptosis‐based tumor therapies is impeded by a series of scientific and practical issues. For instance, metabolic inhibitors that could specifically and precisely inhibit those tumor‐unique energy metabolic activities have yet to be developed, which should have optimal efficacy and safety profiles under clinical conditions. Meanwhile, the actual effectiveness of tumor ferroptosis sensitization through energy metabolic interference remains to be elucidated. It is also necessary to identify and summarize characteristic features of tumor indications that have positive response to ferroptosis therapies after energy metabolic interference, as well as its potential impact on the tumor immunogenicity. Nevertheless, It is also important to note that the clinical translation of these energy‐metabolism‐targeted ferroptosis‐inducing strategies is still impeded by some practical obstacles. For instance, most of the potential therapeutic targets in the energy metabolism pathways are universally present in both normal cells and cancer cells. Moreover, the pharmacokinetics of existing regulatory agents for tumor energy metabolism is still not satisfactory. These issues highlight the necessity of developing new nanotherapeutic strategies to enhance the antitumor efficacy of these energy metabolism regulators while lowering their potential adverse side effects, which may open up new avenues for improving ferroptosis‐based therapies in a clinical context.The authors declare no conflict of interest.


Pediatric Surgical Oncology is a challenging sub-speciality field requiring strong discipline, resilience and a “never quit” attitude. Pediatric cancer patients are frail, vulnerable and have life limiting illness were the skill(s) of the operating surgeon may determine “cure” and survival or untimely death from inadequate tumor resection with disease progression and / or “never event” misadventure. It could be stated therefore that a career in surgical oncology may not be for the “faint hearted” surgeon. Training in a high volume center(s) accredited in the delivery of high quality care and clinical excellence driven by inspirational leaders in surgical oncology with motivated multidisciplinary teams is key. International surgery oncology fellowship(s) add significant credits to a residents' clinical training crucially also yielding opportunities for gaining research skills. On the journey toward subspeciality accreditation the aspiring surgeon oncologist must first demonstrate skill set(s) proficiency in general pediatric surgery residency training to advance to the next phase(s). This article offers a “snap shot” overview synopsis of pediatric surgical oncology training in its broadest perspective(s) for those seeking further information and career guidance. You Only Train Once?” …..“Getting started or wishing to train as a pediatric surgical oncologist is often influenced by exposure to leaders and role models during early surgical residency. My own personal journey was greatly influenced by working in a university department adult surgical oncology service led by an inspiring Professor of Surgery Niall O'Higgins at University College Dublin, Ireland—During these formative years along with completing a residency in Pediatric Surgery I had trained in General Surgery, Vascular Surgery, Thoracic Surgery, Plastic Surgery and Trauma with many great surgeons - I firmly decided I was going to be a university pediatric surgeon with a subspeciality oncology practice. It may be said here at this point that an academic pediatric surgeon can enjoy the benefit(s) of a rewarding career where clinical job diaries with “surgical research time” are contracted and honored accordingly allowing a perfect match for surgical oncology.Young surgeon(s) must be wholly mindful of the landscape in their own nations if they wish to train in pediatric surgical oncology. Where are the busy “high volume” surgery oncology centers where you can get and be assured of surgical “hands on” training? Be cognisant that pediatric cancer whilst the second leading cause of death in childhood in many developed world nations remains a rare disease and index cases notably solid tumor malignancies such as Wilms tumor (80 new cases/annum in the UK—CCLG) and neuroblastoma (100 new cases/annum in the UK—CCLG) are greatly surpassed by hematological cancers and CNS tumors. You will not want to work therefore in a surgery oncology center with a low index case load volume of solid tumors split amongst several residents and the consultant staff.A surgical skills set acquired by spending attachements in vascular, plastic and thoracic surgery departments are invaluable (including urology) prior to a pediatric surgery residency programme before advancing to become a surgical oncologist. Technical expertise acquired in the “basic principles of surgery” here cannot be overemphasized. Surgical oncology demands the operator to have capabilities in a wide range of techniques e.g., vascular access, vessel dissection, isolation and control, vascular suture repair, reconstruction, organ sparing procedures and precision tissue handling—Get busy then with your search when applying for these highly competitive surgery posts. At this stage if truly vocationally committed to pediatric surgical oncology also consider international fellowship training (see later). Ask yourself also what research is going on in these leading surgical centers? Will it be profitable for you? Is it exciting and worthwhile to explore? Crucially what is the track record of the surgical department(s) and their faculty staff? Do they inspire? What do staff (past & present) say about them? Are the faculty national and international leaders in their field? Do your homework. Choose (if you can) what best “fits for you.”Having completed pediatric surgery training hopefully in a busy hospital programme or consortium hospital network partnership with exposure to surgical oncology do then consider further opportunities with advanced progression to seeking out a surgical oncology fellowship. Currently the best programmes available for international medical graduates are located in North America. Memorial Sloan Kettering Cancer Center, New York, USA and St Jude's Hospital Memphis Tennessee for example publicize fellowship positions regularly in the major pediatric surgical journals including with other professional organizations such as the International Society Of Pediatric Surgical Oncology (IPSO). It is greatly hoped in the near future further fellowships can be actively developed in other world nations through IPSO network partnerships. Different nations  may offer varied new opportunities. So regularly check it out! ….In the UK currently there are too many pediatric surgery programmes  providing cancer services for children. As a result individual surgical centers manage and treat few index solid tumor malignancies to provide wholly sufficient training in pediatric surgical oncology with residents competing with consultant surgeons who now frequently share operating in teams to maintain skill competencies. Similar scenarios may exist in other world countries requiring a critical reappraisal with the considered future development of pediatric cancer surgery “super centers”. As an example in the Netherlands  pediatric cancer surgery (2014 –) is now restricted to a single major center located at the Princess Maxima Center in Utrech, NL. A health care model such as this concept is considered “forward thinking” in vision to create a national center of excellence teamed and “tooled up” with a highly skilled infrastructure to sustain credibility.Perhaps the same thoughts and ideas may be uttered for other world countries with their pediatric cancer health care systems. We must shift the imbalance. Without pediatric surgical oncologists and cancer specialists working together and sharing a leadership role in health care many of our hospitals seeking to deliver world class services will suffer. Surgical training programmes working toward national ranking(s) in residency and oncology fellowship applications should also “think smart” to be competitive. The drive toward “evidence based surgical oncology” health service provision, training and innovation(s) in cancer care have never been greater than in our current times. The best hospitals in the world will reassuringly always value well-trained inspirational pediatric surgical oncology leaders. I wish you all—“Good Luck”. . Get started now on your journey!.A Career In Surgical Oncology Is Life Long Learning”…“PDL served as Chair UK CCLG Surgeons Group (2015–2017). PDL is currently President Elect - International Society Of Pediatric Surgical Oncology (IPSO).The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The author confirms being the sole contributor of this work and has approved it for publication.The author declares 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.