Prostate cancer (PCa) is a leading cause of cancer death for males in western countries. The current gold standard for PCa diagnosis - template needle biopsies - often does not convey a true representation of the molecular profile given sampling error and complex tumour heterogeneity. Presently available biomarker blood tests have limited accuracy. There is a growing demand for novel diagnostic approaches to reduce both the number of men with an abnormal PSA/ DRE who undergo invasive biopsy and the number of cores collected per biopsy. 'Liquid biopsy' is a minimally invasive biofluid-based approach that has the potential to provide information and improve the accuracy of diagnosis for patients' treatment selection, prognostic counselling and development of risk-adjusted follow-up protocols. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by tumour cells which may provide a real-time snapshot of the entire tumour in a non-invasive way. EVs can regulate physiological processes and mediate systemic dissemination of various types of cancers. Emerging evidence suggests that EVs have crucial roles in PCa development and metastasis. Most importantly, EVs are directly derived from their parent cells with their information. EVs contain components including proteins, mRNAs, DNA fragments, non-coding RNAs and lipids, and play a critical role in intercellular communication. Therefore, EVs hold promise for the discovery of liquid biopsy-based biomarkers for PCa diagnosis. Here, we review the current approaches for EV isolation and analysis, summarise the recent advances in EV protein biomarkers in PCa and focus on liquid biopsy-based EV biomarkers in PCa diagnosis for personalised medicine. Prostate cancer (PCa) is the most common solid-organ cancer in men and the second most common cause of cancer death in men in western countries. The American Cancer Society reported 164,690 new cases of PCa in 2018 in males and the lifetime risk of PCa is approximately 1 in 9 in the United States The current standard biomarker in blood testing, prostate specific antigen (PSA), has proven controversial as a screening tool and the harms may outweigh the benefits for asymptomatic men, because of its high false-positive rate absence of basal cells around glands to define prostate adenocarcinoma. Consequently, the significance of basal-cell differentiation in PCa diagnosis is excessively underestimated. These limitations of biopsy histology may be addressed by complementary liquid biopsy testing.In men with an abnormal PSA or DRE, the current gold standard to confirm or exclude PCa is a 12-30 core template needle biopsy via a transrectal route. This is associated with risks of anaesthesia, post-biopsy septicaemia, bleeding, urinary retention, pain, psychological distress and sexual dysfunction for patients Extracellular vesicles (EVs) are particles released from nearly all kinds of cells that are delimited by a lipid bilayer and cannot replicate. EVs were under-appreciated as “cell dust” for many years This article reviews current approaches for EV isolation and analysis including conventional and novel methods, summarises EV-based protein biomarkers used in PCa, and gives some typical examples on recent works on how EV biomarkers applied in liquid biopsy-based PCa diagnosis and monitoring, aiming towards the development of personalised medicine. Furthermore, we discuss the current difficulties in EV isolation, especially for plasma samples. Finally, we hope this review can shed some light on future directions in this field for PCa diagnosis.A liquid biopsy refers to using a biofluid sample such as blood, urine, cerebrospinal fluid and seminal plasma to detect and analyse biological markers to evaluate disease and determine PCa treatment options. This contrasts with needle biopsy in PCa, of which a small portion of tumour samples is removed from the prostate gland and analysed with histopathology. Normally, needle cores are performed using a core-biopsy gun for large pieces of tissue (up to 23mm/core) and 12, 18 or 24 samples per participant may be taken due to the heterogeneity of tumour, which may cause pain, bleeding, sexual dysfunction, frequency and urgency of urination, acute urinary retention or even life-threatening septicaemia in up to 2% of cases Figure 1). Compared to CTCs, EVs are significantly more abundant in plasma/serum (109-12/mL) Several types of biomarkers can be used in liquid biopsies: proteins such as PSA, circulating tumour cells (CTCs), cell-free DNA (cfDNA) and EVs which are new types of biomarkers studied recently  and specificity (purity). The populations of EVs are heterogeneous with variations in size distribution from typically 30 nm to 1000 nm as well as other physical properties including mass density and shape. Other than physical properties, differences in biochemical and physicochemical properties, such as solubilities, surface identity (proteins), charge, and hydrodynamic property are also being taken into consideration for isolating EVs and their subpopulations. Since EVs broadly exist in mammal biofluids, in which case each biological fluid presents specific biochemical and physical characteristics, it requires different isolation and purification processes. EVs and their subpopulations can be segregated to various extents using different methods or their combinations. Here, we discuss the common isolation methods and novel approaches of each method in PCa research in this section. The commonly used EV isolation methods in PCa are summarised in Table 2. The separation principle of this method is based on the sedimentation speed difference between EVs and other particles. UC has large sample capacity and low following cost. One stand out point is that UC can generate an EV pellet while most other methods maintain EVs within a solvent. This may be preferable for downstream analysis as it provides a richer concentration of EVs without being diluted by the buffer. Pellets of PCa EVs isolated by UC from the urine have been studied for miRNA profiling with good performance (using miR-19b versus miR-16 with 100%/93% and 95%/79% specificity/sensitivity) to distinguish PCa from healthy individuals Ultracentrifugation (UC) is presently the gold standard method for EV isolation. Current EV protein biomarker studies in PCa mainly focus on UC-based isolation methods, which can be seen from et al.Despite being the most commonly used approach for isolating EVs, UC has several limitations. EVs are nanoparticles with extremely close density to protein aggregates, apoptotic bodies and other non-EVs particles that may present in the EV fraction obtained by UC. Different subtypes of EVs such as those with different sizes may also sediment together during UC. Numerous recent studies have shown that the unexpected aggregation during the UC isolation process may lead to erroneous interpretation and interfere with EV surface antigen detection on subsequent analysis et al. et al.et al. Precipitation of EVs could be less time-consuming compared to UC by adding precipitation reagent and isolating EVs with lower centrifugal forces. The precipitation reagent can be hydrophilic polymers (e.g. polyethylene glycols), protamine, sodium acetate or event organic solvent which initiates hydrophobic interactions and thus precipitates EVs or proteins based on solubility change et al.et al. et al. demonstrated that small EVs (based on size ranging 30-100 nm) isolated from both healthy donors and PCa patients count to 95% and 90% of total EVs respectively using 100 nm UF membrane, and the isolated samples are positive to EV biomarkers CD63, CD9 and CD24 et al. Ultrafiltration (UF) is designed to separate EVs by applying membrane filters with appropriate pores size. Thus, particles with a specific size are concentrated on the membrane. Designed molecular weight cut-off (MWCO) of the membrane can be used to either remove or enrich particles with different molecular weight. For instance, Willms et al. el al.et al.UF is considered a safer and more time-efficient method by avoiding excessive centrifuge steps. However, applying UF may cause reduction in EVs quantity and EVs related proteins. Cheruvanky et al. et al. UF systems can either be dead-end flow or cross-flow in which the feed is passed through a bed or tangentially across the filter surface. Traditional dead-end flow can combine with SEC or other methods to purify the fraction with batch processes via the porous matrix according to their eluting time. Larger particles could rapidly pass through the column, whereas smaller particles come out in later fractions. Due to this property, SEC is available to separate EVs from blood plasma and urine samples et al.SEC, also called gel filtration, allows to separate molecules varying in their hydrodynamic radius by passing them through a column gel. During the process, different molecular weight substances can penetrate the pores with different efficiency in the stationary phase of chromatography. Accordingly, the specific Stokes' radius vesicles in the mobile phase would segregate via the binding between specific membrane markers  and the antibodies. This makes affinity binding outperform other methods in terms of selectivity and specificity as most non-membrane-based contaminants are removed. Affinity interactions can be also combined with other methods to make the isolation more efficient. For example, Mariantonia et al. et al.et al. et al.As specific proteins/lipids are exposed on the surface of the EVs, affinity interactions can be used to capture EVs by binding to their surface receptors. For instance, antibody binding to EV receptors is an attractive way to isolate specific types of EVs. This immunoaffinity method can be approached by covalently binding antibodies to a stationary phase, such as surface of beads, paper-based (cellulose) filters, membrane affinity column and microarray slides -9 to 10-18 litres) volumes of fluids compared to other isolation methods and is capable of handling viscous media in multiple channels The isolation methods aforementioned are mostly used for high-throughput but often require long processing time and bulky sample volumes which may bring extra stress to the patients. Recently, various methods have been developed for or applied on EV processing, aiming at reducing the sample volume and processing time and combining all isolation and analysis procedures into one lab-on-a-chip device. These methods are mainly based on microfluidic devices and chips. The microfluidic device is designed based on microscaled EVs isolation with manipulation of fluids through micro-channels. This approach processes with extremely small . They demonstrated that this approach has a superior yield, smaller input and enhanced EV concentration compared to UC, UC with density gradient, qEV SEC (Izon Science), and the exoEasy Maxi Kit (QIAGEN) from both serum and urine samples. The integrated nanoDLD arrays are composed of 1024 parallel pillar arrays on a single chip to fractionate smaller particles and larger particles with zigzag and bump modes respectively . Markers such as TSG101 and calnexin were used to identify EVs. They utilised RNA sequencing to profile small RNAs presented in EVs and reported the 50 most abundant PCa markers they found on RNA analysis . Their results showed an improved gene expression correlation compared to UC. Thus, this method is a simple, high throughput approach which has great potential for PCa diagnosis and the discovery of EV-based proteins and RNA markers.Smith et al. Figure ). The isolated EVs were lysed and measured by ELISA, and significant differences of optical density were observed between PCa and healthy donors among PSA, PSMA, epithelial cell adhesion molecule (EpCAM), heat shock protein 90 (HSP90) and epidermal growth factor receptor 1 (EGFR1) . Furthermore, this study also indicated that a combination of CD63, PSA, PSMA and HSP90 showed a higher sensitivity compared to HSP90 alone using support vector machine analysis. This platform has the potential to be used as a point-of-care system for early diagnosis of PCa as it is fast and fully automated with a higher yield and purity of EVs.Sunkara Overall, microfluidic device and chip-based methods have the potentials as low-cost, point-of-care devices for personalised cancer diagnosis. However, they are still in an early stage of development and have many disadvantages such as lower throughput, shear stress applied to the EVs, as well as mixing and clogging issues. And most importantly, the purity of the isolated EVs has not been properly or robustly validated for most of these methods. These issues should be addressed before microfluidic devices and microchips become practical in clinical settings.Quantitative and qualitative analysis of the EVs with specific cargoes is indispensable for different biofluids. Among the analytic methods, electron microscopy (EM) is used to characterize particle morphology; as an important method to study structural biology, cryo-EM can be used to observe natural state of EVs with a clear lipid bilayer. Cryo-EM can also differentiate EVs from dense particles such as lipoproteins and gives a direct understanding of sample purity et al. recently developed a thermophoretic aptasensor (TAS) Figure ). By using TAS, several surface proteins including CD63, PTK7, EpCAM, LZH8, HER2, PSA and CA25 were investigated. All markers demonstrated an elevated level from PCa patients compared to healthy controls, except PSA. This evidence further confirmed that the current PSA marker is not accurate for PCa diagnosis or as an indicator of biologic aggressiveness. As shown in Figure  a panel of the 7 protein markers can be used to distinguish PCa from BPH as well as recurrence from no recurrence. Furthermore, the results showed that the panel marker signature is superior to the PSA level detected from patients undergoing prostate biopsies . TAS can be used to handle a crude serum sample in 3 hours and costs around US$1 for detection of 7 EV protein markers. Thereby, TAS can be implemented into early cancer screening as a low-cost, non-invasive assay. These novel approaches may inspire further progress in future EV-based PCa diagnosis.Recently, some novel analytic methods combined with separation methods have been applied in the PCa diagnosis. For example, Liu Table 1). There is currently no perfect method for all EV applications, and which isolation method to use depends on the downstream application. Currently, combination of different separation methods (e.g. SEC combined with affinity interactions or precipitation combined with UF) become more promising for a higher recovery and specificity compared to single type of isolation methods. Some of the co-separation particles may not affect the research if the target focuses on all EVs or other particular components but not a specific EV subtype. However, isolating pure EV subtypes would satisfy the requirement of tremendously increased reproducibility, make less confusing and bring consistent outcomes in functions, thus, further discover the actual target of choice for diagnostic applications. Due to the heterogeneity of EVs In summary, each of the isolation methods has its own pros and cons  on the surface, which inhibits the function of CD8 T cells and facilitates tumour growth et al.et al.et al.et al.Modern “-omics” techniques including high-throughput proteomics (e.g. mass spectrometry) and genomics (e.g. next-generation sequencing) have been used to identify promising PCa biomarker candidates from cell lines, blood and urine. Proteomic analysis of EVs from PCa cell lines and cancer patients' samples in liquid biopsy showed that EVs are a rich source of intracellular proteins. Minciacchi et al.et al.et al. et al. et al. et al. et al. Apart from identifying novel EV diagnostic biomarkers using proteomics, it is also promising to investigate whether those reported biomarkers in blood or tissue have an outstanding performance on EVs, due to EV direct pathway from tumour and with less interference of same protein from different source as a multiparameter tool. As mentioned above, the diagnostic performance of serum PSA is modest in terms of specificity, however, PSA from EVs has shown great potential as a more reliable marker. Logozzi Glycan sugar groups, closely associated with PCa development and progression, are an interesting research area and holding promise in stratification of PCa patients through multi-omic platforms. For example, there are about 50 glycoforms of PSA that have been investigated and only some of them were found in aggressive PCa, especially glycoforms with α2,3-sialic acid Because of the enrichment of specific glycans to EV proteins, glycomics can be used to study EV surface glycans or glycoproteins such as prostatic acid phosphatase (PAP) and PSA to improve PCa diagnosis In summary, among EV protein biomarkers in PCa, several researchers have shown that quantitative assay of a combination of multiple EV-derived proteins might enhance the diagnostic efficacy compared to using a single serum-derived protein marker such as PSA. Because of the heterogeneity of EVs among different subtypes and different patients, a single marker may be lack of power to reflect the multifactorial essence of PCa, nor does it have the satisfactory discriminative precision for accurate risk-classification et al. As an abundant and easily extractable source of body fluid for liquid biopsy, blood is highly associated with EV study and further clinical applications. Injured, stressed or diseased cells such as tumour cells behave self-reaction and release molecules into the bloodstream. Since EVs are directly derived from their parental cells, exploring whether they can be used as a provider of potential cancer biomarkers is critical for cancer research. Clinical studies of EVs confirmed that EVs from blood of PCa patients are extremely abundant compared to BPH patients or healthy volunteers In addition, previous studies have shown that EV performance was sensitive to pre-analytical variables. During blood sample handling, artificial elevation of EVs matters as a result of platelet activation, which may increase platelet-EVs release, due to blood collection and transportation as well as unnecessary delays in plasma/serum separation PCa significantly affects men's health, and circulating EVs show promise as a key player in liquid biopsy. EVs play a significant role in transferring cellular signals to recipient cells under both physiological and pathological conditions. Investigating markers within EV cargos for clinical studies could be extremely valuable in discovering EVs as novel biomarkers for better diagnosis in PCa. In conclusion, this review summarises promising isolation methods that could promote the isolation and analysis of EVs in the clinical setting, including conventional methods and novel methods, and discusses the advantages and drawbacks of each. Cancer researchers should choose suitable isolation and analytic methods based on their own parameters such as original EV media, sample size, downstream analysis. We also summarised current EV-based protein biomarkers that have been identified for PCa from both cell lines and human samples such as blood and urine, which could substantially be used as a guide to PCa EVs biomarker investigation. Cancer-derived EV markers from various biofluids are valuable markers and can be taken a step further to correlate with EVs and PCa clinically especially in different stages for diagnostic purpose. Finally, we discussed several major hurdles that EV researchers may need to pay attention to before EVs can be applied in the clinic. Following studies can focus on these hurdles and avoid inappropriate handling that may affect EVs.For PCa diagnosis, it is crucial to choose proper isolation methods and analysis techniques as different methods yield different purity and subtypes of EVs. Different subtypes of EVs carry various information and may result in a conflict result for PCa diagnosis. Thus, there is a demand to instigate how EVs perform differently among different isolation and analysis methods. Such researches would largely improve both process and accuracy using EVs as diagnostic tool and promote the conversion of EV studies to the clinical, further benefit the clinical decision-making. Our review can serve as a basis for further investigation in PCa related EVs biomarker discovery. We believe that EVs hold a bright future in PCa assessment and can better-improve diagnosis and reduce cancer health disparities.


Tumour angiogenesis and lymphangiogenesis are hallmarks of cancer and have been associated with tumour progression, tumour metastasis and poor patient prognosis. Many factors regulate angiogenesis and lymphangiogenesis in cancer including non-coding RNAs which are a category of RNAs that do not encode proteins and have important regulatory functions at transcriptional and post-transcriptional levels. Non-coding RNAs can be encapsulated in extracellular vesicles called exosomes which are secreted by tumour cells or other cells in the tumour microenvironment and can then be taken up by the endothelial cells of blood vessels and lymphatic vessels. The “delivery” of these non-coding RNAs to endothelial cells in tumours can facilitate tumour angiogenesis and lymphangiogenesis. Here we review recent findings about exosomal non-coding RNAs, specifically microRNAs and long non-coding RNAs, which regulate tumour angiogenesis and lymphangiogenesis in cancer. We then focus on the potential use of these molecules as cancer biomarkers and opportunities for exploiting ncRNAs for the treatment of cancer. The growth of new tumour blood vessels and lymphatic vessels is a key element of cancer progression having a close association with metastatic spread, poor patient prognosis and survival ,2. TumouPrevious work has shown that a range of growth factors, cell surface receptors and a multitude of signalling molecules drives the remodelling of the blood and lymphatic vasculatures in cancer ,4. RecenLong ncRNAs (lncRNAs) exhibit a range of different regulatory functions in distinct cellular compartments  Figure . They caAn important functional feature of miRNAs and lncRNAs is that they can exert effects on the regulation of complex biological responses: miRNAs by targeting mRNAs encoding multiple proteins involved in the same or a related molecular pathway, lncRNAs by controlling the remodelling of chromatin to modulate gene expression, or by targeting miRNAs. Moreover, a miRNA can act in concert with other miRNAs, lncRNAs or transcription factors to mediate gene silencing in a precise manner.It has recently been shown that ncRNAs can be encapsulated in extracellular vesicles called exosomes and thus take on a pivotal role in cell-to-cell communication which occurs in the tumour microenvironment and ultimately contributes to tumour progression, remodelling of blood vessels and lymphatics, and tumour spread ,25. In tde novo from endothelial progenitor cells or cancer stem cells in a process called vasculogenesis [Tumour cells require oxygen and nutrients to proliferate, and solid tumours therefore require blood vessels to facilitate their growth and spread . Tumour ogenesis . Tumour ogenesis . SeveralLymphatic vessels can undergo multiple changes in cancer which facilitate the spread of tumour cells to organs distant from the primary tumour, such as lymphangiogenesis and other forms of lymphatic remodelling . LymphanThe remodelling of the lymphatic vasculature in cancer is driven by a diverse range of growth factors that are produced by tumour cells and cells in the tumour microenvironment. These growth factors bind cognate receptors on LECs and activate several molecular pathways that ultimately drive lymphangiogenesis; they are described below. Some members of the vascular endothelial growth factor (VEGF) family of secreted proteins are inducers of angiogenesis and lymphangiogenesis in cancer. The human VEGF family is composed of VEGFA, VEGFB, VEGFC, VEGFD and placenta growth factor (PIGF). These growth factors bind and activate cognate receptors VEGFR1, VEGFR2 and VEGFR3 with different affinities ,39,40,41The VEGFC-VEGFR3 and VEGFD-VEGFR3 signalling pathways have been the most studied in lymphangiogenesis and lymphatic vessel remodelling in cancer and are considered major drivers of these processes . In a tuVEGFA is secreted by tumour cells and stroma and is the most studied inducer of tumour angiogenesis; its presence is correlated with tumour size, blood vessel density and metastasis . VEGFA iBesides the VEGFs, other growth factors and signalling pathways have been reported to stimulate lymphangiogenesis and angiogenesis. Angiopoietins 1 and 2 (ANGPT1 and ANGPT2) and their receptors (TIE1 and TIE2) on endothelial cells are required for both these processes . FibroblLymphangiogenesis and angiogenesis in tumours involve complex regulation by stimulatory molecules which modulate interacting signalling pathways to induce endothelial cell proliferation, sprouting, migration and eventually formation of new vessels from the pre-existing network. It is clear from the literature that the proteins which are best validated as lymphangiogenic factors in vivo, acting directly on LEC, are VEGFC and VEGFD. Likewise, VEGFA is the best validated angiogenic factor in cancer. These conclusions are based on many mouse genetic models, tumour xenograft models and clinicopathological studies ,4,57. Recently, miRNAs and lncRNAs have been found in exosomes in cancer . ExosomeExosomal miRNAs are capable of regulating lymphangiogenesis and angiogenesis within the primary tumour and at metastatic sites . Within Several research groups have also shown that tumour cells can secrete exosomal miRNAs which influence non-endothelial cell types in the tumour microenvironment such as fibroblasts and macrophages so they can, in turn, promote tumour angiogenesis and/or lymphangiogenesis. For instance, Fan and colleagues have shown that exosomal miR-210 derived from lung cancer cells induces the reprogramming of normal fibroblasts into cancer-associated fibroblasts (CAFs) by activating the JAK2/STAT3 pathway in these cells. Reprogrammed fibroblasts then upregulate the expression of several angiogenic factors, including VEGFA, which stimulate the remodelling of tumoural blood vessels . AnotherIn specific tumour settings, cells of the tumour microenvironment, including endothelial cells, can secrete exosomes that target tumour cells or other cell types and thereby indirectly regulate tumour angiogenesis. For instance, mesenchymal stem cells secrete exosomes containing miR-100 which inhibits angiogenesis by modulating the mTOR/HIF-1α/VEGFA signalling axis in breast cancer cells, thus reducing production of VEGFA by these cells . ExosomaRecently, lncRNAs have been shown to be involved in cell-to-cell communication in the context of tumour angiogenesis . lncRNAsFAM225A lncRNA has been shown to promote oesophageal squamous cell carcinoma (OSCC) progression and angiogenesis by sponging miR-206 in BECs. Specifically, OSCC cells secrete high levels of exosomal FAM225A which can then be endocytosed by BECs wherein this lncRNA inhibits miR-206 leading to upregulation of the expression of miR-206 targets . These iAnother example of how exosomal lncRNAs regulate tumour angiogenesis is that chondrosarcoma cell-derived exosomes carry lncRNA RAMP2-AS1 which is delivered to BECs. Here, RAMP2-AS1 acts as a competing endogenous RNA by sponging miR-2355-5p thereby upregulating the expression of one of its targets, the VEGFA receptor VEGFR2, leading to activation of angiogenic signalling .Some lncRNAs have been shown to directly upregulate the expression of VEGFA in endothelial cells thereby promoting angiogenesis. For example, it has been shown that exosomal lncRNA Hotair, produced by glioma cells, upregulates the expression of VEGFA in brain microvascular endothelial cells and thus stimulates their remodelling .https://mirxes.com/gastroclear/ (accessed on 9 February 2021)) [Tumour-derived exosomes are found in a variety of body fluids including blood, tears, urine, milk, saliva and ascites , and they 2021)) .We envisage a scenario in which cancer-derived exosomal ncRNAs that promote tumour angiogenesis and lymphangiogenesis (such as those discussed in previous sections of this article) are included in panels of ncRNA biomarkers for liquid biopsy to detect and stage cancers in the clinic. The purpose of monitoring ncRNAs that promote angiogenesis and lymphangiogenesis would be to provide information about the propensity of detected tumours to metastasize, via blood vessels or lymphatics, to distant sites in the body which is the most lethal aspect of cancer for most patients. We acknowledge there is considerable work that still needs to be done to clinically validate specific exosomal ncRNAs as biomarkers of cancer metastasis.In this article we have focussed on ncRNAs which promote angiogenesis, lymphangiogenesis and metastasis in cancer. In contrast, there are a range of ncRNAs which are known to be anti-angiogenic or anti-lymphangiogenic and could therefore form the basis of therapeutic strategies to restrict tumour growth and spread ,110,111.There are a range of non-cancer clinical settings in which it could be beneficial to promote angiogenesis and/or lymphangiogenesis such as wound healing, cardiovascular diseases and lymphoedema. ncRNAs which promote angiogenesis and lymphangiogenesis, such as those discussed in this article, could be exploited for these purposes by employing lipid-nanoparticle formulated, nucleoside-modified synthetic RNA. The potential of such a strategy has been exemplified by recent animal studies in which lymphangiogenesis was successfully stimulated in a therapeutic setting by delivering exosomal miRNAs, derived from adipose-derived stem cells (ADSCs), to LECs. ADSCs are multipotent cells located in adipose tissue that have been used in therapeutic settings  because While non-coding RNAs do not encode proteins they do play a critical role in regulating the levels of many cellular and extracellular proteins , and thuOur current insight into the regulation of tumour angiogenesis and lymphangiogenesis by non-coding RNAs can be exploited for development of new cancer biomarkers and therapeutics, as discussed here. However, further research is required for this to occur. In particular, specific non-coding RNAs which promote tumour angiogenesis and/or lymphangiogenesis will need to be clinically validated as useful biomarkers of metastasis before being incorporated in liquid biopsy strategies for detecting and staging cancers. The use of anti-angiogenic or anti-lymphangiogenic non-coding RNAs as therapeutics to restrict tumour growth and spread seems attractive, particularly in light of recent regulatory approvals for vaccines based on lipid-nanoparticle formulated, nucleoside-modified RNA. A key challenge for this approach will be to determine the best anti-angiogenic/anti-lymphangiogenic non-coding RNAs to use for restricting cancer metastasis, which will need to be carefully assessed in clinically relevant animal models of cancer.


The Pain Management Collaboratory (PMC) is a multi-site network of pragmatic clinical trials (PCTs) focused on nonpharmacological approaches to pain management, conducted in health care systems of the US Department of Defense (DoD) and Department of Veterans Affairs (VA) and co-funded by the National Institutes of Health (NIH). Concerns about potential research-site overlap prompted the PMC investigator community to consider strategies to avert this problem that could negatively affect recruitment and contaminate interventions and thus pose a threat to trial integrity.ClinicalTrials.gov. The second, remediation phase consisted of a series of moderated conference calls over a 1-month time period to develop plans to address overlap. Remediation efforts focused on exclusion criteria and recruitment strategies, and they involved collaboration with sponsors and stakeholder groups such as the Military Treatment Facility Engagement Committee (MTFEC). The MTFEC is comprised of collaborating DoD and university-affiliated PIs, clinicians, and educators devoted to facilitating successful pragmatic trials in DoD settings.We developed a two-step strategy to identify and remediate research-site overlap by obtaining detailed recruitment plans across all PMC PCTs that addressed eligibility criteria, recruitment methods, trial settings, and timeframes. The first, information-gathering phase consisted of a 2-month period for data collection from PIs, stakeholders, and Of 61 recruitment sites for the 11 PMC PCTs, 17 (28%) overlapped. Four PCTs had five overlapping Military Treatment Facilities (MTFs), and eight PCTs had 12 overlapping VA Medical Centers (VAMCs). We developed three general strategies to avoid research-site overlap: (i) modify exclusion criteria, (ii) coordinate recruitment efforts, and/or (iii) replace or avoid any overlapping sites. Potential overlap from competing studies outside of the PMC was apparent at 26 sites, but we were not able to confirm them as true conflicts.Proactive strategies can be used to resolve the issue of overlapping research sites in the PMC. These strategies, combined with open and impartial mediation approaches that include researchers, sponsors, and stakeholders, provide lessons learned from this large and complex pragmatic research effort. Pragmatic clinical trials (PCTs) evaluate the effectiveness of interventions in real-world scenarios involving a broad set of participants, rather than in the more tightly controlled conditions set by explanatory trials that aim to confirm a specific physiological or clinical hypothesis , 2. ReseGenerally speaking, clinical sites best suited for PCTs are those with practical experience conducting multiple studies with a large number of patients. However, it is difficult for researchers to draw accurate conclusions if study participants receive more than one intervention targeting the same health condition. Thus, careful evaluation of competing studies in multi-site PCTs is important to avoid research-site overlap as well as to ensure that investigators can achieve adequately powered sample sizes from which to draw valid conclusions. Research-site overlap is particularly problematic for investigations of the effectiveness of various nonpharmacological treatment modalities  for chronic conditions that share symptoms such as back pain because it can be difficult to ascertain that co-enrollment has occurred. Various factors contribute to the potential for research-site overlap, which poses a threat to trial integrity.The potential for research-site overlap was an early concern of the PMC. The phased timing of this effort—two PMC funding phases (2 years for planning and 4 years for implementation) and staggered PCT start dates—offered an important opportunity to avert potential co-enrollment of participants in multiple, concurrent trials (including non-PMC pain studies). In concert with PMC sponsors, the PMC Coordinating Center  oversaw this task of detecting, and resolving, overlap problems prior to final study design and participant recruitment/enrollment. Herein, we present effective strategies unique to PCTs that we used to address the problem of research-site overlap.ClinicalTrials.gov to identify potentially competing studies outside the PMC being conducted at target recruitment sites and shared this information with the PMC PIs. Recognizing the importance of a proactive approach for managing such a large group effort—and given that site-level information was often unavailable, outdated, or incomplete—the PMC3 developed a two-step strategy and implemented a second, remediation phase to resolve issues surrounding site selection.To identify potential overlap, after notification of funding, PMC PCT principal investigators (PIs) provided the PMC3 with proposed target sites, eligibility criteria, and recruitment strategies. The PMC3 also conducted a search of Because PMC PIs had used different nomenclature when referring to their recruitment sites, the PMC3 standardized each location using either the VA-designated Facility name and Station ID number or the Health System Name and Certification Number designated by the US Department of Health and Human Services (HHS) Centers for Medicare and Medicaid Services (CMS). This process identified 61 sites as targets for PMC trials, and this information was shared with PIs in an Excel-based pivot table that could be filtered by site location or PMC PI. Creating a searchable format was especially useful for those PIs who were still actively selecting sites.The PMC3 contacted PMC PIs to obtain detailed recruitment plans—including eligibility criteria, recruitment methods, trial settings, and timeframes—to further inform which PCTs might have overlap issues. The PMC3 observed that because studies could be recruiting from different clinical settings within the same health care system , these and other studies with distinct pain populations, unique recruitment settings, and/or differing timeframes would be unlikely to need any remediation plan.ClinicalTrials.gov to identify ongoing trials involving pain interventions (including opioid-tapering studies) that could interfere with PMC trial recruitment of similar patients in the same settings. This search identified 49 non-PMC pain trials with the potential for overlap at 26 PMC sites. Information describing these trials was incorporated into the Excel spreadsheet and included site name, condition being studied , type of intervention, trial start date, trial completion date, title of the study, and National Clinical Trial (NCT) number. Further curation of this information by PCT PIs ascertained the expected impact of overlap.Because interventional pain trials external to the PMC could also affect recruitment efforts, the PMC3 conducted a search of Whole Health Evaluation Program, which expands the availability of complementary and integrative health services [To ascertain real-world circumstances across recruitment sites, the PMC3 sought and obtained additional information from key stakeholders within PMC sponsoring agencies, to ensure that PMC interventions and data collection did not interfere with ongoing agency efforts. For example, VA representatives provided information about the rollout of its services  to meet A unique feature of the PMC is the formation and involvement of the Military Treatment Facility Engagement Committee (MTFEC) comprised of collaborating DoD and university-affiliated PIs, clinicians, and educators devoted to facilitating successful pragmatic trials in DoD settings. Using their expertise, knowledge, and access to key stakeholders, the MTFEC provided the PMC3 with up-to-date information on competing trials at specific MTF-recruitment sites. The MTFEC hosted regular monthly conference calls to facilitate exchange of key site-specific information among its members and provided this information to the PMC.ClinicalTrials.gov search, and (iv) a list of potential strategies to address conflict.While the overarching goal of remediation was to assist PIs in meeting recruitment goals and avoiding contamination in pain interventions, secondary goals included fostering a shared sense of community, reducing competition among PCTs, and maintaining collegiality within the PMC. A PMC facilitator  worked with PMC PIs to develop a plan to resolve issues surrounding site overlap, serving as an independent party to negotiate competing interests impartially and to maintain a collaborative spirit. In addition, the MTFEC moderated MTF-related issues. Together, the MTFEC and PMC3 operated as joint facilitators allowing the PMC PIs to identify, negotiate, and agree upon viable solutions. Given the involvement of 20 PMC PIs located across the country, the most efficient format for shared decision-making was small-group discussions hosted virtually. Prior to each discussion, the PMC3 provided the following information to PMC PIs: (i) summary of overlapping MTFs and VAMCs, (ii) inclusion/exclusion criteria and recruitment plans of proposed studies, (iii) summary of the Because individual PIs were most knowledgeable about their own pain populations, as well as how and from where potential participants would be recruited into their studies, the facilitator moderated discussions—identifying sites with overlap, describing concerns, and presenting general remediation strategies without dictating any specific course of action. Discussions began with PIs sharing information about their recruitment methods and design, including details not readily apparent from each protocol, given that many were still being developed. This activity allowed the PIs to determine the likelihood that overlap concerns were real, and if so, how to work together with the facilitator to determine an appropriate remedy. For example, in one instance, PIs recruiting participants from the same site determined that they could use different clinics  with minimal impact on outcomes. Such information was known only by those PIs, each of whom had a shared interest in ensuring rigorous study design and recruitment practices.ClinicalTrials.gov did not prove to be informative. Based on PIs’ experience during the site-selection process, the non-PMC pain trials identified by ClinicalTrials.gov neither provided new information  nor captured all relevant site-level activities . Collaboration with the MTFEC, in contrast, provided a viable path to identify active pain trials and other pain interventional initiatives at each of the participating MTFs, through direct engagement with clinical leaders  at those sites [Information about the 49 non-PMC pain trials obtained from se sites . The MTFThe PMC has 61 recruitment sites—seven are MTFs, 51 are VAMCs, and three are civilian facilities. Our analysis revealed that 17 PMC sites 28%) overlapped. Four PCTs had five overlapping MTFs . It is possible, however, that we did not capture all these instances, since some PIs used the information from the Excel pivot table to avoid selecting recruitment sites with competing PMC pain trials while still working through their site-selection process.As noted earlier, our attempts to derive strategies to identify and mitigate overlap of PMC trials with non-PMC trials appeared to be largely unsuccessful and possibly unnecessary—in most cases, PIs were aware of such trials external to the PMC. However, a thorough scan of potentially overlapping non-PMC pain trials exceeded the resources available within the PMC. Going forward, four of the 11 PCT PIs anticipated that they would collect information on non-PMC pain trials to use as a covariate in a sensitivity analysis to identify potential for contamination from site overlap. Previous reports have discussed using a factorial design approach to glean further statistical insights into addressing this problem .By design, PCTs recruit a diverse study population and usually have broad inclusion criteria, trading internal validity for greater generalizability, toward prompt implementation of effective interventions in clinical settings that mimic those of the trials themselves . There iWe believe that beyond specific strategies, the organizational structure and existing relationships within the PMC contributed to our success. The time and energy used to create the PMC allowed the PCT PIs to establish familiarity, build trust, and develop a shared sense of community. Other key determinants for successfully averting research-site overlap were gathering the right type of information for PIs to consider and discuss, as well as offering an impartial moderator with expertise in clinical-trial design to resolve potential conflicts. The PMC3 itself acted as a fair and trusted third party to help PIs negotiate a set of remediation strategies balanced across all PMC trials. The final strategy for addressing research-site overlap was endorsed by the primary PMC stakeholders: the PMC Steering Committee composed of the sponsors, trial PIs, and the PMC3.The PMC3 learned several lessons from this exercise. These include the importance of prompt action; accurate and clear synthesis of information; and effective engagement of PIs, sponsors, and stakeholders. Above all, we learned the necessity of thinking ahead about potential pitfalls that could discount the validity of many person-years of work—and more pressingly, potentially delay delivery of effective treatments for people living with debilitating pain. The utility of PCTs is their ability to provide flexibility to adapt dynamically to results obtained in real-world conditions—thus requiring large and diverse participant populations to reflect what happens in real life. However, as for explanatory trials, the design and conduct of PCTs must adhere to the principles of scientific rigor to avoid participants either being double-counted or receiving more than one intervention, both of which would cloud data interpretation. The sponsors and PMC3 were quick to recognize the need for a well-articulated, collaborative process of shared decision-making to ensure that PIs are able to conduct their own research as well as preserve the integrity of the consortium’s shared vision.ClinicalTrials.gov search was resource-intensive, involving several people in the PMC3 to identify parameters, pull/clean data, and synthesize information in a timely manner. Ultimately, none of the overlapping sites identified through the ClinicalTrials.gov search provided meaningful information as this information was either already known to PIs or not relevant. In retrospect, however, such search information could have been helpful if it was introduced further upstream in the pre-planning stage (before beginning the site-selection process).Some activities in our information-gathering stage could have been streamlined or eliminated entirely. Gathering written recruitment plans about the PMC trials placed a burden on PIs and may have been unnecessary considering these details naturally arose in discussions on our many conference calls to develop a strategy to avert research-site overlap. Likewise, the Whole Health Evaluation Program that would affect recruitment at six VAMCs leading to the PMC decision to delay recruitment at these sites until May 2020 when the evaluation was expected to be complete. Because the PMC only conducts pain-relevant PCTs in military and VA health care systems, the strategies we have identified and presented herein may not be suitable to other collaboratories that conduct PCTs in disparate disease areas and conditions. Finally, we note that the process we used and have detailed herein may have been difficult to pursue for explanatory trials with less flexibility in study design and participant recruitment.Engaging PMC PIs, sponsors, and key stakeholders was critical for success, as each plays a vital but different role in the overall PMC effort. Individual PIs are the most knowledgeable about recruitment for their own PCTs, so they must be involved and invested in details of remediating site overlap. As important are people with comprehensive environmental knowledge and policy awareness, such as the MTFEC, who knew about programs and proposed initiatives that would affect recruitment more broadly for all PMC sites and who helped resolve MTF-specific issues. Input from sponsors and key stakeholders was also invaluable. For example, VA personnel identified the In reality, there is a continuum between pragmatic and explanatory approaches, and many individual trials are likely to possess some elements of both as appropriate to the research question at hand, the trial population, and other environmental issues . Our app


AIRE) gene that encodes a protein whose function has been uncovered by the generation and study of Aire-KO mice. These provided invaluable insights into the link between AIRE expression in medullary thymic epithelial cells (mTECs), and the broad spectrum of self-antigens that these cells express and present to the developing thymocytes. However, these murine models poorly recapitulate all phenotypic aspects of human APECED. Unlike Aire-KO mice, the recently generated Aire-KO rat model presents visual features, organ lymphocytic infiltrations and production of autoantibodies that resemble those observed in APECED patients, making the rat model a main research asset. In addition, ex vivo models of AIRE-dependent self-antigen expression in primary mTECs have been successfully set up. Thymus organoids based on pluripotent stem cell-derived TECs from APECED patients are also emerging, and constitute a promising tool to engineer AIRE-corrected mTECs and restore the generation of regulatory T cells. Eventually, these new models will undoubtedly lead to main advances in the identification and assessment of specific and efficient new therapeutic strategies aiming to restore immunological tolerance in APECED patients.Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare life-threatening autoimmune disease that attacks multiple organs and has its onset in childhood. It is an inherited condition caused by a variety of mutations in the autoimmune regulator ( Summary: In this Review, we discuss the in vivo and in vitro models available to study AIRE deficiency, and how they may contribute to restoring immunological tolerance in APECED patients. Preclinical research using experimental animal models of diseases is pivotal to advance the understanding of mechanisms involved in these diseases and to successfully translate bench research to the clinic. The selection of the model and its accuracy remain critical since the success rate of drugs reaching clinical development remains low. Worldwide, there are 7000 rare diseases recognized, affecting more than 350 million people, but only <10% of these diseases have an approved drug treatment . ClinicaAIRE) gene that is expressed in the thymus and whose protein product, AIRE, is essential for central immune tolerance  is one of these rare human autoimmune diseases . APECED olerance . AIRE isolerance .To better understand the human APECED pathology, AIRE-deficient mouse models have been generated since the identification of causative gene; they have been essential to study and get a better understanding of the APECED disease . Howeverex vivo models and organoids generated from embryonic or induced pluripotent stem cells, to better understand, challenge and assess immunotherapies.In this Review, we aim to describe the latest advances in the different APECED models, including a rat model generated by our lab and used to study the disease. Moreover, we discuss new approaches, such as AIRE mutation. Indeed, the clinical picture has evolved with the discovery of new manifestations and the documentation of prevalent symptoms from Whitaker's triad has declined  zinc finger of AIRE, which then suppress wild-type AIRE in a dominant-negative manner (Addison disease (AD), also known as primary adrenal insufficiency and hypocortisolism: an endocrine pathology affecting adrenal glands, resulting in a deficit in steroid hormone synthesis. Symptoms include weight loss, abdominal pain and weakness.• Alopecia: complete or partial loss of hair that can happen on the scalp, i.e. in patches  or the entire head , or over the whole body .• Aromatic L-amino acid decarboxylase : an enzyme that catalyzes several different decarboxylation reactions in the biosynthesis of various neurotransmitters and neuromodulators.• Chronic mucocutaneous candidiasis (CMC): chronic Candida spp. infection of the mucosa, nails and skin that persists, owing to an immune disorder linked to a T cell defect.• Chronic tetany: a condition characterized by spasms, cramps and overactive neurological reflexes as a result of low calcium blood levels that are often the consequence of hypoparathyroidism (see below).• Enamel hypoplasia: a developmental defect that weakens the surface of teeth, due to defective formation of the hard protective layer covering the outside of the tooth.• Epithelial cell adherence molecule (EpCAM): a common surface marker protein of epithelial cells.• Glutamate decarboxylase (GAD): an enzyme that catalyzes the decarboxylation of glutamate to GABA, the main inhibitory neurotransmitter.• Gonadic failure: a disorder in which testes or ovaries fail to produce either sex hormones or gametes, resulting in fertility issues.• Hypergonadotropic hypogonadism (HH), also known as primary or peripheral gonadal hypogonadism: the defective response of gonads to hormones, caused by problems with the pituitary gland or hypothalamus. It is the result of decreased testosterone or estradiol production, respectively, in males or females, inducing a delay in sexual development and diminished reproductive functions.• Hypoparathyroidism (HP): failure of the parathyroid gland to efficiently produce the parathyroid hormone, in fine leading to low blood calcium levels.• Immunological tolerance: capacity of the immune system to recognize the body's own components and not react to them, which is vital for the protection against autoimmune diseases. The tolerance process takes place in the thymus, whose main function is to control thymocyte development, and to discriminate between self- and non-self antigens.• Induced pluripotent stem cells (iPSCs): somatic cells reprogrammed back to an embryonic-like pluripotent state.• Keratoconjunctivis: simultaneous inflammation of the cornea and conjunctiva.• Medullary thymic epithelial cells (mTECs): a population of TECs located in the medulla of the thymus. mTECs express and present a large number of self-antigens to the developing T cell to ensure their education and prevent autoimmune reactions.• Protein-disulfide isomerase pancreas specific : a member of the protein disulfide isomerase (PDI) family, acting as a molecular chaperone that catalyzes the formation of disulfide bonds in secretory proteins.• Pernicious anemia: decrease of red blood cell due to B12 vitamin malabsorption in the intestines.• Regulatory T cells (Tregs): a subpopulation of T cells that help prevent autoimmune manifestations by regulating the activity of immune cells. They control the immune response to and self- and non-self antigens.• T cell receptor (TCR) repertoire: describes the T cell diversity within the immune system of an individual in a physiopathological context and it represents the repertoire of antigens encountered by the TCR.• Tissue-restricted antigens (TRAs): tissue constituents that are not ubiquitously expressed.• Type 1 diabetes (T1D): an autoimmune disorder that affects pancreatic Langerhans islet cells and results in very little to no insulin production.• Vitiligo: a progressive autoimmune disorder affecting the skin that manifests as patchy loss of pigmentation.• The first documented case of APECED syndrome was reported in 1929 by Thorpe and Handley, describing of a four-and-a-half-year-old girl suffering from chronic tetany . Most AIRE mutations that are not indels occur in the CARD domain; however, the most prevalent mutation, p.R257*, is located in the SAND domain  . WhereasAs discussed in more detail below, mouse APECED models in which the Aire-KO mice that do not express HEL in mTECs, few HEL-specific T cells were retrieved from wild-type (WT) mice, showing that AIRE-dependent HEL expression resulted in depletion of T cells able to recognize HEL peptides  using exon targeting to delete exons that encode functional domains of Aire ; mice of this model present with increased numbers of activated/memory T cells (CD44highCD62Llow) in peripheral lymphoid organs at the age of ∼2–3 months  provided molecular evidence that AIRE is potentially involved in shaping the TCR repertoire of Tregs  in vivo together with colitogenic cells, a phenotypic feature of APECED patients  genetic background that exhibited a more severe autoimmune phenotype , and nail overgrowth (nail dystrophy) – symptoms that are frequently seen in APECED patients  system that supports TEC survival and expansion has been developed, which draws on an in vitro model of skin development (Keratinocytes (skin cells) and TECs share many biological similarities . Indeed,Primary TECs show various biological similarities to keratinocytes in the skin  into EpCAMke cells , using ake cells . Here, t results . More re results . However results  (Otsuka  results , and so  results  preprint results  preprintAIRE gene or to correct AIRE function represent a promising approach to cure APECED. In support of this, similar approaches have been employed to treat other rare diseases, by using CRISPR-Cas9- or adeno-associated virus-based gene therapy to restore the correct expression of mutated genes (see also Aire-deficient mice (AIRE gene in patients could increase the risk of an APECED patient to develop cancer. One study, employing a more targeted approach, indicates how this problem might be addressed (in vitro in cell lines of thymic medullary or dendritic cell origin, as well as in bone marrow cells. In the cell lines, this approach showed reduced expression of TRAs. However, in bone marrow chimeras that had been generated using the transduced bone marrow cells, elevated expression of TRAs resulted in a delay of the symptomatic onset of EAE (Gene and cell therapy to correct a mutant see also . Howeverent mice , indicatddressed  by usingt of EAE . Transplt of EAE . In thist of EAE . To the Thanks to developments in tissue-engineering techniques, bioengineered artificial thymus organoids are also being developed with the aim of rejuvenating thymus function. Such organoids have been shown to successfully attract lymphocyte progenitors in nude mice, supporting the generation of a complex T cell repertoire and the induction of donor-specific tolerance . Howeverex vivo and in vitro experiments to the clinic is a real challenge. To diagnose patients at an early stage is of key importance as it allows therapies to take place before irreversible organ lesions have occurred. In addition, monoclonal antibodies represent a tremendously powerful tool that could be used to target specific effector T cells while preserving Tregs. Indeed, they might represent the next-generation therapies for APECED and would also help to avoid the deleterious, long-term side effects of the immunosuppressive drugs currently used to manage APECED patients (Given the broad spectrum of symptoms in APECED, translating findings derived from animal models and from patients . Indeed,patients . Treg cepatients . MoreoveFinally, although the absence of AIRE is a feature of APECED disease, significantly decreased levels of AIRE have also been observed in patients suffering from Omenn syndrome or Down syndrome, two disorders characterized by severe immunodeficiency and T-cell-mediated autoimmunity . This stAire point mutations that might be associated with specific disease phenotypes. The ex vivo 3D OTC and thymus-derived culture models also need to be adapted to the newly discovered broad spectrum of TEC sub-population (ex vivo, thereby providing a unique opportunity to restore a dysfunctional immune system through personalized cell therapy treatments. The comprehensive characterization of the complex molecular mechanisms that underlie the effect of AIRE on induction of TRAs, as well as the identification of additional molecular factors involved in the induction of central immune tolerance, will certainly be revealed by using newly developed single-cell transcriptomic and epigenetics approaches. Combined analyses of such new and existing data on TEC biology in human and rodent samples (The different models generated to study APECED and the development of systems for culturing primary TECs have considerably improved our understanding of the mechanisms that underlie immunological tolerance in the thymus. In addition, they have enabled the development of several pre-clinical therapeutic approaches for controlling autoimmunity in APECED . Howeverpulation  the spec samples  are key


Metapopulation persistence in fragmented landscapes is assured by dispersal of individuals between local populations. In this scenario the landscape topography, although usually neglected, may have an important role in shaping dispersal throughout the matrix separating habitat patches. Due to inter-sexual differences in optimal reproductive strategies, i.e., males maximizing the number of mating opportunities and females maximizing the offspring survival chances, topography-related constraints are expected to exert a different effect on male and female dispersal behaviour. We tested sex-biased topography effects on butterfly dispersal, with the following hypotheses: (1) females are constrained by topography in their movements and avoid hill crossing; (2) male dispersal is primarily driven by two-dimensional spatial structure of the habitat patches (i.e. their geometric locations and sizes) and little influenced by topography.Maculinea (= Phengaris) nausithous and M. teleius within a landscape characterised by an alternation of hills and valleys, we investigated sex-specific patterns in their inter-patch movement probabilities derived with a multi-state recapture model. In particular, we (1) analysed the fit of dispersal kernels based on Euclidean (= straight line) vs. topography-based  distances; (2) compared movement probabilities for the pairs of patches separated or not by topographic barriers; and (3) tested the differences in the downward and upward movement probabilities within the pairs of patches.Following intensive mark-recapture surveys of Euclidean distances between patches proved to be a substantially stronger predictor of inter-patch movement probabilities in males, while inter-patch distances measured along valleys performed much better for females, indicating that the latter tend to predominantly follow valleys when dispersing. In addition, there were significantly lower probabilities of movements across hills in females, but not in males.Both above results provide support for the hypothesis that topography restricts dispersal in females, but not in males. Since the two sexes contribute differently to metapopulation functioning, i.e., only female dispersal can result in successful (re)colonisations of vacant patches, the topography effects exerted on females should be considered with particular attention when landscape management and conservation actions are designed in order to maintain the functional connectivity of metapopulation systems. Dispersal is one of the key life-history traits which allow species to persist in fragmented landscapes. The proximate factors that drive an individual to actively move from its natal habitat patch vary but include local population conditions such as conspecific density, resource availability, stochastic environmental events, e.g., weather, habitat disturbance . UltimatAnalysing intra-specific variation in dispersal abilities is especially important in the case of endangered species occurring in highly fragmented landscapes. However, dispersal studies in the past often assumed dispersal abilities to be fixed species-specific traits, especially in the classic formulation of the metapopulation theory , 35, 91,Among other flying insects, butterflies have long been regarded as convenient models for dispersal studies due to their occurrence in discrete local populations that form metapopulation systems as well as to their ability to fly through an inhospitable matrix , 43. On In fact, three-dimensional topography should be considered as a major factor capable of influencing dispersal patterns through directing animal movements into particular pathways, as revealed by some recent studies into the movements of large mobile organisms such as birds or mammals e.g. , 10, 36), 3610, 3More importantly, its effects are likely to be different for male and female dispersal behaviour. The latter prediction derives from the physiological differences of males and females, which limit female dispersal due to their substantial resource investment in producing eggs . This dIn many butterfly species females are bigger and have greater wings than males . This chFurthermore, a strong evolutionary pressure is exerted on female dispersal behaviour, because female fitness is dependent on the ability to disperse safely and lay eggs in an appropriate habitat so as to assure the offspring survival. Females should normally move from a foodplant to nearby foodplant in order to lay eggs, but if foodplants are scarce, they should activate their ability to disperse in order to find another habitat patch . NeverthMales, instead, appear to be less subjected to similar limitations. The strategy to maximise the number of encounters with receptive females should presumably make them more prone to undertake more risk while dispersing. In terms of cost-benefit balance, the evolutionary advantage of leaving a natal patch in search of another one in the case of female scarcity or high male density in order to decrease male-male competition for mating , could fMaculinea (= Phengaris) nausithous and M. teleius, occurring sympatrically in a complex landscape of valleys and hills. Due to inter-sexual differences in optimal reproductive strategies added to morphological and physiological differences in the two sexes, topography is expected to exert a different effect on male and female dispersal behaviour. We tested the following hypotheses: (1) females are constrained by topography in their movements and avoid hill crossing; (2) male dispersal is primarily driven by two-dimensional spatial structure of the habitat patches  and little influenced by topography.Relaying on the above rationale, the purpose of the present study was to analyse how topography influences dispersal of males and females within metapopulations of the specialist butterflies M. nausithous and M. teleius are globally classified as Near Threatened and Vulnerable, respectively ) Maculinea [Analysing the probabilities of upward and downward movements within pairs of habitat patches, we found no particular tendency in males, while females were clearly more prone to move towards lower lying patches. This dissimilarity in the dispersal behaviour of the two sexes can possibly be once again attributed to the inter-sexual differences in morphology which, in turn are ultimately shaped by natural selection. The ‘oogenesis-flight syndrome’ postulate (cf. ) gives aaculinea . Greateraculinea . Therefoaculinea . Althougaculinea , 83), thMaculinea, thorax width was found to have no influence on the movement distances travelled within habitat patches [Conversely, butterfly males perform movements primarily in order to find more (or better) mating partners , and the patches , a more Maculinea  may resnea , 7865, 7Maculinea butterflies are able to efficiently cross the forest matrix surrounding their habitat patches, the present study, dealing with inter-sexual differences as well as the influence of topography on dispersal, makes it clear that only males are able to move relatively freely in all directions. In turn, females are likely to follow valleys, and hence their movements are affected by the topography of the landscape. It is noteworthy that, since only females are able to successfully colonise vacant habitat patches, the functional demographic connectivity of the entire system would ultimately depend on how females move through the landscape.Specifically, while the results of Nowicki et al.  suggesteTo summarize, the results of our study provide support for the hypotheses that topography affects dispersal in butterflies. More interestingly, in the present study, the same topography proved to differently shape dispersal in the two sexes of the focal species. It is evident that intersexual differences in dispersal have serious consequences for maintaining the functional connectivity of local populations, thus being crucial for the functioning of entire metapopulation systems. This is because only post-mating female dispersal can result in successful colonisations leading to the reestablishment of populations at the patches that have experienced local extinctions . ConsequComplementing our results with the actual dispersal routes followed by butterflies obtained through individual tracking (cf. ) might hAdditional file 1. Results - Sample sizes of butterflies captured throughmark-capture surveys in the Elbe river valley near Dečin, northern CzechRepublic.Additional file 2. Results - Testing of independence of movement probability estimatesderived for investigated Maculineabutterflies with the multi-state recapture model.Additional file 3. Results - Results of the negative exponential function (NEF) fitting performed toevaluate the effects of inter-patch Euclidean distances (ED) vs. topographic distances measured along the valley beds (VD) as well as of natal (= movementstart) patch areas (N) and targetpatch areas (T) on the movementprobabilities (ψ) of investigated Maculinea butterflies.Additional file 4. Results - Model selection based on the Akaike InformationCriterion corrected for small sample size.


The Randomized Embedded Multifactorial Adaptive Platform for COVID-19 (REMAP-COVID) trial is a global adaptive platform trial of hospitalized patients with COVID-19. We describe implementation at the first US site, the UPMC health system, and offer recommendations for implementation at other sites.To implement REMAP-COVID, we focused on six major areas: engaging leadership, trial embedment, remote consent and enrollment, regulatory compliance, modification of traditional trial management procedures, and alignment with other COVID-19 studies.We recommend aligning institutional and trial goals and sharing a vision of REMAP-COVID implementation as groundwork for learning health system development. Embedment of trial procedures into routine care processes, existing institutional structures, and the electronic health record promotes efficiency and integration of clinical care and clinical research. Remote consent and enrollment can be facilitated by engaging bedside providers and leveraging institutional videoconferencing tools. Coordination with the central institutional review board will expedite the approval process. Protocol adherence, adverse event monitoring, and data collection and export can be facilitated by building electronic health record processes, though implementation can start using traditional clinical trial tools. Lastly, establishment of a centralized institutional process optimizes coordination of COVID-19 studies.Implementation of the REMAP-COVID trial within a large US healthcare system is feasible and facilitated by multidisciplinary collaboration. This investment establishes important groundwork for future learning health system endeavors.NCT02735707. Registered on 13 April 2016. A Randomized Embedded Multifactorial Adaptive Platform (REMAP) trial combines features of adaptive platform and pragmatic point-of-care trials to simultaneously evaluate multiple treatment strategies and maximize trial conduct efficiency , 2.REMAP-CAP is a global adaptive platform trial of patients with severe community-acquired pneumonia (CAP) admitted to the intensive care unit (ICU) that was launched in 2016 . The triIn light of challenges conducting research relevant to pandemic infection uncovered by the 2009 H1N1 influenza experience, the REMAP-CAP trial was initially drafted with a pre-specified Pandemic Appendix to be activated in the event of an emergent pandemic. In February 2020, as the Coronavirus Disease 2019 (COVID-19) pandemic spread, the Pandemic Appendix to REMAP-CAP was activated and the trial labeled REMAP-COVID . REMAP-CTo implement REMAP-COVID, we focused on six major areas Table : engaginhttps://clinicaltrials.gov/ct2/show/NCT03861767. https://clinicaltrials.gov/ct2/show/NCT03861767) [The UPMC system is comprised of multiple community hospitals, regional tertiary referral centers, and one quaternary referral institution, all predominantly located in western and central Pennsylvania. For several years, the system has worked to develop a learning health system and previously launched a REMAP trial  exemptions, rather than an investigational pharmacy. To speed study launch, we began the trial only with interventions with IND exemptions. We educated pharmacists at the system level for each investigational treatment and emphasized medication order verification differences for COVID-19 versus non-COVID-19 indications.Third, UPMC deployed a telemedicine program to facilitate local patient care and minimize COVID-19 transmission and provided tablet computers to help hospitalized patients communicate. We used these resources to aid remote connection with patients.Embedment into the EHR facilitates low operational complexity at the bedside, despite complex internal trial machinery. We launched the trial at the 20 UPMC adult acute care hospitals that use Cerner . To facilitate both clinical care and clinical research for COVID-19, we created a “one-stop shop” for operational efficiency—a COVID-19 tab in the provider facing interface of the patient’s electronic record. Physicians and advanced practice providers can use the tab to access treatment protocols and order sets, as well as complete a COVID-19 intake form  if they would be interested in hearing about potential additional therapies for COVID-19. An affirmative response represents assent to be approached about research, and intake form completion generates an automated email to research staff, which triggers the informed consent and enrollment process (described in the next section).After obtaining informed consent, randomization and in-trial notification processes are embedded within the EHR. Research staff complete a web-based application Enrollment Form, linked within Cerner, and hosted on a local server behind the health system firewall. The application references a response adaptive randomization table to generate treatment arm assignments for each domain , which are then written by the application to a custom-built table within Cerner. Each unique trial recipe triggers a custom series of pop-up Cerner Discern alerts, which appear the next time after enrollment a provider enters a patient order. The alerts contain guidance for clinicians regarding eligibility criteria and protocol procedures. Where appropriate, the alerts pre-populate investigational treatment orders corresponding to the assigned trial recipe, which clinicians are requested to sign unless previously undetected exclusion criteria are present, or an investigational treatment is not thought to be in the patient’s best interest Fig. . To furtWe developed a centralized remote consent and enrollment process Fig.  applicaband physician investigator via secure video conference; and (4) signature capture via a software platform  that allows the patient to electronically sign their name using a smart device. We provide the software platform by texting or emailing a link to the patient’s smart device, investigators use the same platform to sign, and the final signed consent form is electronically sent to the patient. Bedside clinicians are engaged to assist with the process on an ad hoc basis, including providing patients who do not have a personal smart device a hospital phone or tablet, and assisting in the videoconferencing process if needed. Clinicians follow UPMC infection control procedures when assisting. On rare occasions, we have used paper consent, in which case a provider or investigator in personal protective equipment provides the paper forms and sends pictures of the signed forms to the research team.Both the state of Pennsylvania and the University of Pittsburgh Institutional Review Board (IRB) require physician consent for clinical trials , with thA team of coordinators and multidisciplinary physician investigators trained in the protocol, informed consent, and Cerner currently screen and enroll 7 days a week, approximately 12 h a day. Once the informed consent signature is obtained, research staff then complete the web-based Enrollment Form, which triggers the randomization and alerts as described above.www.clinicaltrials.gov is updated with each newly activated domain (NCT02735707).UPMC and the University of Pittsburgh serve as both a REMAP-COVID site and the US Regional Coordinating Center. As most UPMC hospitals are under the purview of the University of Pittsburgh IRB, we obtained local approval to launch the trial. In parallel, we worked with the US sponsor, the Global Coalition for Adaptive Research (GCAR) to centralize protocol review at the Western Institutional Review Board  for implementation across the USA. We presented each IRB the core protocol and appendices for each domain making the approval process modular and adaptable. Repurposed FDA-approved drugs are frequently granted an exemption from the Investigational New Drug (IND) application process, whereas experimental agents require IND approval. In partnership with GCAR, we submit documentation to the FDA for approval and adverse event monitoring where necessary. Trial registration at We regularly update trial management procedures as the adaptive design necessitates frequent updates to our internal work environment , workfloWe use a combination of automated EHR detection alerts, traditional coordinator oversight via manual monitoring of enrolled patients, and input from clinical staff to monitor adverse events and protocol adherence. A REMAP goal is to rely on automated EHR detection alerts where possible as the trial progresses, and to optimize alerts by comparing them to manually adjudicated events. Medical monitoring is managed in a two-tiered system with a local investigator providing oversight of IND exempt interventions and a central medical monitor contracted by the sponsor for oversight of IND domains requiring FDA oversight. As the coordinating center for the US region of REMAP-CAP, essential documents are collected and filed with the UPMC program management team.Trial-relevant data are continuously and automatically extracted from the Cerner database and curated into a dataset suitable for export to the international data coordinating center at Monash University in Australia. Data are stored in a MySQL Server database, processed using a scheduled combination of SQL statements and Python scripts, and formatted and exported for trial reporting. An unblinded investigator reviews initial exports in detail to ensure data accuracy. EHR data are reviewed for completeness and validity at the time of entry into the EHR, the time of extraction into a trial database, and following transformation into trial-relevant elements. Validation and process refinement are ongoing, to account for impurities in EHR data as well as frontline context of care and documentation practices that have changed due to the pandemic. A key challenge has been the need to rework both workflow and analytic models to keep pace with the adaptive nature of the trial and the evolution of the pandemic. To support adaptive randomization, relevant patient characteristics and outcomes are iteratively transferred to the international data coordinating center as a Health Insurance Portability and Accountability Act limited dataset using Globus  secure file transfer services. Post-hospital discharge outcomes are collected by a dedicated follow-up team via phone calls and query of national mortality databases. Secondary outcomes during hospitalization are captured from the EHR.The COVID-19 pandemic sparked intense interest within academia and industry. To minimize competition for protocols, temper inconvenience to patients, regulate biospecimen volumes, and promote the “Learning While Doing” philosophy, UPMC and the University of Pittsburgh centralized all COVID-19 clinical trial recruitment through the REMAP infrastructure. As previously reported , the REMThe pandemic challenged all REMAP-CAP sites to respond with innovation and efficiency. As a new site, we had limited preparation time before trial launch. We went live April 9, 2020, with two domains comprising three investigational treatment arms . We enrolled our first patient on April 12, added an immunoglobulin  domain on May 8, discontinued the initial two domains in response to emerging international data in mid-June –16, addeWe offer the following implementation recommendations, outlined in Table We found an inclusive and transparent approach effective, while in retrospect we should have created and disseminated a preliminary organogram at the outset to rapidly communicate the complex trial structure. Although we had advantages of institutional desire and scale, in practice, implementing a learning health system (and REMAP-COVID) is achieved at the bedside and therefore local challenges with daily workflow and staffing require individualized attention. Similar conditions likely exist in other large health systems. Alignment of institutional and trial goals is imperative. Thus, simultaneously engaging leadership while keeping daily bedside realities in the forefront is essential. The optimal ways to engage leadership will vary by institution, as will unification needs, barriers, and solutions. Institution-level incentives for individual and group participation may increase bedside engagement and will also vary by institution. Participation in REMAP-COVID can help institutions lay the groundwork for future development as learning health systems. Smaller institutions may lack scale, but can likely move faster, and in the authors’ experience can match or exceed trial enrollment performance compared to larger institutions.Embedment into routine care processes is essential as an implementation philosophy and design. We found partnering with existing institutional information dissemination, pharmacy, and telemedicine structures effective, and recommend this approach for efficient embedment, and as a means of engaging local leadership and front-line personnel.While adding REMAP messages into existing information channels is relatively straightforward, constant and consistent re-education of frontline personnel is also required, particularly as investigational treatments are added or dropped. For drugs that require IND approval and are not available in clinical pharmacies, engagement of an investigational pharmacy is required, which may not be available in all hospitals. We have embedded the trial into the investigational pharmacies at the UPMC flagship academic hospitals and are expanding access to community hospitals using a combination of flagship hospital investigational pharmacy outreach and community hospital pharmacies.. We are also considering expanding intake form completion ability to pharmacists.Many health systems have increased their telemedicine capabilities in response to the pandemic. This shift to telemedicine will likely be sustained, and opportunities to embed trials within existing telemedicine infrastructure should be sought. Our initial intent was to deploy telemedicine critical care nurses and physicians for informed consent and enrollment discussions. However, we abandoned this plan due to the complexity of ensuring all were trained in research ethics and protocol nuances, and uncertainty as to whether trial duties could coincide with telemedicine duties both operationally and ethically under current clinical research regulations. Nonetheless, it is our long-term goal to further embed trial operations into routine care processes and rely less on research personnel.Embedment into the EHR is an optional feature, and traditional trial processes are utilized effectively in most regions of the world enrolling in REMAP-COVID. If feasible though, EHR embedment, while requiring significant upfront investment, renders trial operations more efficient than traditional processes and reduces both clinician and research staff burden, and positions institutions for future embedded research. In addition, EHR embedment allows enrollment at hospitals geographically distant from a centralized research team. Therefore, we recommend leveraging a system’s existing EHR to implement the trial if possible.Customization of EHR-based workflow to support trial infrastructure, as has been accomplished within Cerner and Epic at UPMC, can conceivably be adapted to a range of other non-Cerner EHR systems. Most large health systems likely have the requisite information technology resources and expertise, while smaller systems may need to rely on traditional trial processes until standardized EHR solutions can be established. To this end, interactive web response system (IWRS) and electronic data capture (EDC) platforms have been established for REMAP-COVID to support trial deployment and software training resources are available from the coordinating center. Seamless to sites, data are collated, verified, and cleaned at the data coordinating center for analysis by the REMAP-CAP statistical analysis committee. In addition, data mapping across traditional IWRS/EDC systems and EHR-based approaches are handled at the coordinating center level.We found remote consent and enrollment most effective when combined with engaged bedside care providers. Thus, as with implementation of any hospital-based project, stakeholder engagement cannot be overemphasized. Regulatory requirements for consent vary by state and institution. Videoconferences should be secure and electronic signature software should be compliant with part 11 of Title 21 of the Code of Federal Regulations . If teleWe have streamlined the regulatory compliance and oversight processes to simplify onboarding of new sites in the US region in partnership with GCAR and the Western IRB. We recommend interested sites engage their local IRB to partner with the Western IRB for centralized ethics approval, and our group for protocol compliance, contract, and other key areas to facilitate rapid onboarding and activation of participant enrollment.Frequent adaptive trial updates and the lack of traditional milestones such as target enrollment numbers can be challenging for classically trained frequentist investigators and trial personnel. As recommended by other platform trials groups, we have found that identifying, pausing, and briefly celebrating natural points of progress and contributions from local champions aid morale . VirtualFinally, implementation of the REMAP-COVID core protocol has enabled coordinated and collaborative clinical research focused on COVID-19 at UPMC. Other institutions have similarly centralized COVID-19 research , 19. EstWe envision using the REMAP-COVID trial infrastructure to develop a learning healthcare system approach beyond COVID-19. The infrastructure can serve to implement best practices, facilitate quality improvement, and expedite clinical research. Participation of community hospitals is essential, expanding enrollment and generalizability beyond quaternary academic centers, as well as facilitating enrollment of vulnerable populations, who often disproportionately seek care locally rather than at large academic centers. Enrollment of patients by bedside clinicians as is done in the UK for the RECOVERY trial  is a lonIn conclusion, implementation of the REMAP-COVID trial within a large US healthcare system is feasible and facilitated by multidisciplinary collaboration. This investment establishes important groundwork for future learning health system endeavors.


Coronary artery spasm (CAS) defined by a severe reversible diffuse or focal vasoconstriction is the most common diagnosis among INOCA (ischemia with no obstructive coronary artery disease) patients irrespective to racial, genetic, and geographic variations. However, the prevalence of CAS tends to decrease in correlation with the increasing use of medicines such as calcium channel blockers, angiotensin converting enzyme inhibitor, and statins, the controlling management of atherosclerotic risk factors, and the decreased habitude to perform a functional reactivity test in highly active cardiac catheterization centers. A wide spectrum of clinical manifestations from silent disease to sudden cardiac death was attributed to this complex entity with unclear pathophysiology. Multiple mechanisms such as the autonomic nervous system, endothelial dysfunction, chronic inflammation, oxidative stress, and smooth muscle hypercontractility are involved. Regardless of the limited benefits proffered by the newly emerged cardiac imaging modalities, the provocative test remains the cornerstone diagnostic tool for CAS. It allows to reproduce CAS and to evaluate reactivity to nitrates. Different invasive and noninvasive therapeutic approaches are approved for the management of CAS. Long-acting nondihydropyridine calcium channel blockers are recommended for first line therapy. Invasive strategies such as PCI (percutaneous coronary intervention) and CABG (coronary artery bypass graft) have shown benefits in CAS with significant atherosclerotic lesions. Combination therapies are proposed for refractory cases. Coronary artery spasm (CAS), which is a reversible vasoconstriction driven by a spontaneous vascular smooth muscle hypercontractility and vascular wall hypertonicity narrowing the lumen of normal or atherosclerotic coronary arteries compromising the myocardial blood flow, is recognized recently under the chapter of myocardial infarction with nonobstructive coronary arteries (MINOCA) , 2. SeveThe concept of CAS was first postulated by Prinzmetal et al. by describing a nonexertional angina occurring at rest or during regular daily activities , which cRecently, the coronary artery spasm hypothesis was confirmed and demonstrated in several experimental studies, especially after the introduction of either the provocative test that induces vasospasm , 11 or cCAS prevalence varies widely among races and between countries, but it remains the main cause of ischemic heart disease with nonobstructive coronary lesions . It was The length of the CAS episode is important in terms of the large variance of clinical manifestations from the asymptomatic event to the different aspects of ACS  and sudden cardiac death –25. NoneKounis syndrome is an unfamiliar form of CAS appearing in a specific situation characterized by the simultaneous occurrence of ACS and hypersensitivity reaction. It is known as “allergic angina” or “allergic myocardial infarction” . CAS conThe main risk factors for CAS are age, smoking, hypertension, LDL-cholesterol, diabetes mellitus, and Hs-CRP –36. MoreA long-term mental stress , a lightThe exact pathophysiology of CAS is not clearly understood. However, CAS phenomenon is multifactorial implicating the autonomic nervous system, endothelial dysfunction, inflammation, oxidative stress, smooth muscle hyperreactivity, atherosclerosis, thrombosis, and genetic predisposition .The complex relation between CAS and the autonomic nervous system is illustrated via the contribution of its two components: the sympathetic and the parasympathetic nervous system . The rolThe nitric oxide (NO) produced by a normal functional endothelium enhances vasodilatation via suppressing the release of vasoconstrictors agents such as angiotensin II and endothelium I , 62. TheThe relationship between inflammation and CAS was first described by Lewis et al. in a case report of variant angina and pericarditis . An incrOxygen free radicals stimulate vasoconstriction and endothelial damage via degrading nitric oxide released by endothelial cells . The incATP channels [Phosphorylation and dephosphorylation of the myosin light chain (MLC) regulate coronary smooth muscle reactivity , 76. In channels , 80.CAS and atherosclerosis are two distinct clinical entities that share common risk factors such as male gender, smoking, elevated Hs-CRP, dyslipidemia, and diabetes mellitus , 81. BotGenetic polymorphisms or mutations coding for angiotensin converting enzyme , paraoxoThe contributing role of microvascular dysfunction in the pathophysiology of INOCA is a current trend in cardiology. Female gender, low BMI (body mass index), decreased adenosine triphosphate-induced coronary flow reserve (ATP-CFR), borderline diastolic to systolic velocity ratio (DSVR), and minor ischemic ECG findings at rest are the identified predictors for microvascular CAS . MicrovaThe most reliable method for diagnosing CAS is the provocation test adjunctive to coronary angiography  with sevCurrent modalities of coronary imaging detect CAS-specific findings. For example, intravascular ultrasound study (IVUS) provides clues to differentiate vasospastic angina from nonvasospastic angina. It identifies a small lesion site plaque volume and burden and a diffuse coronary intimal thickening reflecting intimal hyperplasia in vasospastic angina  as well Currently, cardiac magnetic resonance imaging (CMRI) is the gold standard, as it is safe and noninvasive for assessing MINOCA patients , but itsSublingual nitrate remains the main treatment to relieve acute attacks of CAS . SmokingSeveral invasive approaches to drug refractory CAS were proposed. The feasibility and success rates of the coronary artery bypass graft are considered according to the underlying characteristics of CAS, as a focal, nondiffused atherosclerotic site of CAS is an essential determinant . On the Finally, the indication for sympathectomy or implantable cardiac defibrillator was limited to life-threatening situations defined by cardiac arrest survivors and episodes of ventricular arrhythmia –143. DesThe first 3 months following the CAS attack is the critical period with highest risk for cardiovascular events . SmokingCAS is a complex multifactorial disease that can lead to serious complications. A wide clinical spectrum is attributed, and sudden cardiac death may well be the uncovering event. As a result, an early recognition and appropriate medical approach is primordial. The prevalence of CAS tends do decrease with wide spread use of ACE inhibitors and statins and diminished performance of spasm provocation tests which are considered time consuming in highly active cardiac centers.Although CMRI plays a key role in the differential diagnosis of MINOCA, further studies are needed to emphasize CMRI's utility in CAS diagnosis.