Cerebral arterial gas embolism (CAGE) is a rare but serious cause for acute neurologic deficit that occurs most often in divers who breathe compressed gas at depth or iatrogenically from a variety of invasive medical procedures. We present a rare case of CAGE caused by inhaling helium from an unregulated, high-pressure gas cylinder. Following inhalation, the patient experienced loss of consciousness, neurologic deficits, pneumomediastinum, and pneumothorax requiring transfer and treatment at a hyperbaric facility with resulting resolution of neurologic symptoms. This case highlights the importance of rapid diagnosis and hyperbaric oxygen treatment (HBO), facilitated by close coordination among community emergency departments, pediatric tertiary care centers, hyperbaric facilities, and poison control. Cerebral arterial gas embolism (CAGE) occurs most often in divers who breathe compressed gas at depth or iatrogenically from a variety of invasive medical procedures –4. We prA previously healthy 12-year-old female presented to a community emergency department (ED) after a single inhalation of helium gas from a high-pressure gas cylinder purchased from a national retail store. Within a few seconds of inhaling helium, she began coughing and then quickly lost consciousness for less than one minute. Upon awakening, the patient was disoriented and complained of headache, chest pain, visual deficits, and weakness. She was unable to ambulate due to right-sided weakness.She was transported to a nearby ED where her vital signs, oxygen saturation, cardiac, and lung exam were normal except for sinus tachycardia. She was awake, alert, and oriented, with improved mentation since the inhalation event. Her right arm was objectively weak but able to move against gravity but she could not independently lift her right leg off the bed. Additionally, she had dysarthria and a right-sided facial droop.Chest X-ray revealed pneumomediastinum and subcutaneous emphysema of the neck . CT of tUpon arrival at the pediatric facility, the patient had persistent right arm and leg weakness with right facial droop, loss of right leg pinprick sensation, and dysarthria. She continued to be alert and oriented with GCS 15. Visual acuity was normal. After rapid assessment in the pediatric intensive care unit (PICU) to ensure stability and placement of arterial catheter for monitoring during HBO treatment, the patient was then transferred to the nearby hyperbaric center by ambulance accompanied by both a PICU physician and a PICU nurse. HBO began 6 hours, 45 minutes following helium inhalation. She received a total of 6 hours, 10 minutes of HBO according to US Navy Treatment Table 6, the standard protocol for arterial gas embolism (AGE). Complete resolution of neurologic deficits was noted after 90 minutes of HBO. The patient returned to the PICU for close neurologic monitoring. Her subsequent neurologic exam varied by provider between normal and minimal deficits; therefore, no additional HBO was given. An MRI and MRA of the brain and a transthoracic echocardiogram with bubble study were normal on hospital day 3, the day of discharge.We describe a case of pulmonary barotrauma and CAGE resulting in neurological deficits as a result of inhaling helium from an unregulated, high-pressure gas cylinder. The inhalation of helium under high pressure resulted in disruption of the alveolar interface with dissection of gas into the pulmonary venous circulation  as evideThe frequency of CAGE from high-pressure helium inhalation is unknown. There have been no reports in the medical literature since 2002, with three reports including one pediatric patient prior to that time , 12, 13.Diagnosis of CAGE is primarily clinical, including stroke-like symptoms, loss of consciousness, or death during or immediately after an at-risk activity or procedure. Because all previously reported cases of CAGE due to helium inhalation describe immediate loss of consciousness and pulmonary barotrauma , 12–15, Additional imaging studies are often obtained when CAGE is suspected but are not sensitive enough to exclude the diagnosis and should not materially delay HBO. The presence of gas within cerebral vasculature or brain parenchyma confirms the diagnosis, while gas within cardiac chambers or great vessels strongly supports the diagnosis . HoweverHBO is the primary treatment for CAGE  with shoDiagnosis and treatment of CAGE in children present several challenges. Lack of familiarity with helium inhalation causing CAGE may delay diagnosis. The vast majority of hyperbaric centers do not treat patients requiring emergent treatment, therefore lengthening travel time to facilities that do , 31. EmeWe have presented a case of CAGE due to inhalation of helium gas from an unregulated, high-pressure gas cylinder that resulted in pulmonary barotrauma. Immediate loss of consciousness or focal neurologic deficits after high-pressure gas inhalation should trigger consideration of CAGE. Rapid diagnosis and HBO treatment were facilitated by close coordination among community emergency departments, pediatric tertiary care centers, hyperbaric facilities, and poison control.


Stroke is one of the leading causes of death and disability worldwide. Reducing this disease burden through drug discovery and evaluation of stroke patient outcomes requires broader characterization of stroke pathophysiology, yet the underlying biologic and genetic factors contributing to outcomes are largely unknown. Remedying this critical knowledge gap requires deeper phenotyping, including large-scale integration of demographic, clinical, genomic, and imaging features. Such big data approaches will be facilitated by developing and running processing pipelines to extract stroke-related phenotypes at large scale. Millions of stroke patients undergo routine brain imaging each year, capturing a rich set of data on stroke-related injury and outcomes. The Stroke Neuroimaging Phenotype Repository (SNIPR) was developed as a multi-center centralized imaging repository of clinical computed tomography (CT) and magnetic resonance imaging (MRI) scans from stroke patients worldwide, based on the open source XNAT imaging informatics platform. The aims of this repository are to: (i) store, manage, process, and facilitate sharing of high-value stroke imaging data sets, (ii) implement containerized automated computational methods to extract image characteristics and disease-specific features from contributed images, (iii) facilitate integration of imaging, genomic, and clinical data to perform large-scale analysis of complications after stroke; and (iv) develop SNIPR as a collaborative platform aimed at both data scientists and clinical investigators. Currently, SNIPR hosts research projects encompassing ischemic and hemorrhagic stroke, with data from 2,246 subjects, and 6,149 imaging sessions from Washington University’s clinical image archive as well as contributions from collaborators in different countries, including Finland, Poland, and Spain. Moreover, we have extended the XNAT data model to include relevant clinical features, including subject demographics, stroke severity , stroke subtype (using TOAST classification), and outcome [modified Rankin Scale (mRS)]. Image processing pipelines are deployed on SNIPR using containerized modules, which facilitate replicability at a large scale. The first such pipeline identifies axial brain CT scans from DICOM header data and image data using a meta deep learning scan classifier, registers serial scans to an atlas, segments tissue compartments, and calculates CSF volume. The resulting volume can be used to quantify the progression of cerebral edema after ischemic stroke. SNIPR thus enables the development and validation of pipelines to automatically extract imaging phenotypes and couple them with clinical data with the overarching aim of enabling a broad understanding of stroke progression and outcomes. Stroke is the second leading cause of death throughout the world, and the leading cause of long-term disability . The manBrain imaging has a key role in providing further insights about complications after stroke. Indeed, most stroke patients have at least one brain imaging study performed during their acute hospitalization, primarily for diagnostic purposes on presentation. Follow-up scans are often obtained to evaluate the size of infarction and to exclude the development of hemorrhagic transformation. An endeavor is underway to describe the design and rationale for the genetic analysis of acute and chronic cerebrovascular neuroimaging phenotypes detected on clinical magnetic resonance imaging (MRI) in patients with acute ischemic stroke within the scope of the MRI-GENetics Interface Exploration (MRI-GENIE) study , 2020. AGiven the potential of imaging to advance our understanding of stroke and its complications, the GENISIS study endeavored to share all clinically performed brain imaging on enrolled subjects. The Stroke Neuroimaging Phenotype Repository (SNIPR) was created as a stroke-focused medical imaging repository that could serve as a platform for this and other stroke-related research. SNIPR is based on the open source XNAT imaging informatics platform, developed at Washington University in St. Louis (WUSTL) . SNIPR pStroke Neuroimaging Phenotype Repository was initiated in 2018 to manage the imaging data from the GENISIS multi-center acute stroke genetics study. SNIPR is a modified instantiation of XNAT, an extensible open source imaging informatics platform. The SNIPR image repository is built primarily to support images in DICOM format, the medical imaging industry standard . Users hAdditional data types such as imaging and clinical phenotypes can also be added by specifying new data type plugins which are based on XNAT’s standard XML Schema extension mechanism. Indeed, this capability to introduce additional data types empowers SNIPR to support managing multimodal data under a common research subject identifier. When research identifiers are determined by a project owner, then image studies can be directly pulled from PACS and anonymized during import, but clinical data can be downloaded from Electronic Data Collection (EDC) systems in spreadsheet format and uploaded into SNIPR. These anonymized data can be stored under a common research subject identifier in SNIPR so as to support subject level study for various stroke phenotyping at large scale. SNIPR manages subject-level clinical and derived radiological data  and sessAutomated imaging data analysis is implemented in SNIPR using XNAT’s Container Service. The Container Service uses a containerized computing architecture to encapsulate applications in portable structures that are readily distributed and deployed to large-scale compute clusters . The ConStroke Neuroimaging Phenotype Repository supports development and execution of automated and semi-automated processing pipelines. As an example, a containerized stroke edema pipeline was developed to automate image segmentation and measurement of CSF volume in serial CT scans in stroke patients . The pipAs shown in Web-based reports provide user-friendly access to the demographic, clinical, and imaging attributes captured in SNIPR. As listed in SNIPR was developed as a central repository to host and provide secure access to anonymized stroke imaging and associated clinical data, and to serve as a sandbox environment for developing computational pipelines to perform large-scale quantitative analysis. Data type plugins were added to XNAT to extend its support for stroke-related phenotype data type generated by these pipelines and extracted from the electronic medical record. To support scalable computing, advanced containerization tools, including Docker Swarm, were deployed along with a custom Docker scheduling plugin for XNAT. An automated processing pipeline to extract CSF volume in serial CT images was developed and subsequently deployed as a pipeline on SNIPR for use by other projects.While other repositories such as ENIGMA and MRI-GENIE are critically built with the focus on research MRI with limited longitudinal data, SNIPR offers longitudinal clinical CT (and MRI) which offers much larger numbers and broader representation of available imaging. Also, other repositories have focused on a variety of all neurological and psychiatric disorders or principally on stroke recovery, while SNIPR focuses on stroke-specific data, and it targets acute imaging and relevant acute phenotypes like edema and hemorrhagic transformation. Accordingly, SNIPR can equip researchers to launch processing pipelines at scale to study any stroke phenotype and its trajectory over time to study stroke and its dynamics effectively.Stroke Neuroimaging Phenotype Repository development is ongoing, with a particular focus on development of additional imaging biomarkers, including predictors of hemorrhagic transformation, measurements of collateral flow, and support of large-scale multicenter clinical studies to integrate different stroke phenotypic studies at massive scale and validate our new discoveries on various populations. The XNAT-based SNIPR database and IT infrastructure will be expanded further to support large-scale data contributions by external collaborators. Data structures will be developed to capture all stroke imaging modalities and multimodal phenotypes. Workflows will be implemented to upload and document data from all data contributors such as anonymization of image metadata, detection and removal of alphanumeric characters in images, and obscuring of identifying facial features. A suite of image quality control pipelines will be implemented to automatically assign quality metrics to uploaded images. A standardized imaging derived disease phenotype, including intracranial compartment, CSF, and infarct volumes, will also be generated for each patient exam and it will be integrated with associated clinical data. New algorithms will be deployed onto SNIPR using XNAT’s Docker container service to enable scalable reproducible processing and validation. A data dashboard will also be implemented as an information management tool that will visually track, integrate, analyze, and display demographic information and processing results for each subject and across all subjects in each project.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.HM drafted the initial manuscript and performed the pipeline development. RD collected the clinical data and revised the manuscript. YC, JG, and AH revised the manuscript. DM and J-ML supervised this project, reviewed the manuscript, and made critical revisions. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sarcopenia is characterised by the presence of diminished skeletal muscle mass and strength. It is relatively common in older adults as ageing is associated with anabolic resistance (a blunted muscle protein synthesis response to dietary protein consumption and resistance exercise). Therefore, interventions to counteract anabolic resistance may benefit sarcopenia prevention and are of utmost importance in the present ageing population. There is growing speculation that the gut microbiota may contribute to sarcopenia, as ageing is also associated with [1) dysbiosis, whereby the gut microbiota becomes less diverse, lacking in healthy butyrate-producing microorganisms and higher in pathogenic bacteria, and [2) loss of epithelial tight junction integrity in the lining of the gut, leading to increased gut permeability and higher metabolic endotoxemia. Animal data suggest that both elements may impact muscle physiology, but human data corroborating the causality of the association between gut microbiota and muscle mass and strength are lacking. Mechanisms wherein the gut microbiota may alter anabolic resistance include an attenuation of gut-derived low-grade inflammation and/or the increased digestibility of protein-containing foods and consequent higher aminoacidemia, both in favour of muscle protein synthesis. This review focuses on the putative links between the gut microbiota and skeletal muscle in the context of sarcopenia. We also address the issue of plant protein digestibility because plant proteins are increasingly important from an environmental sustainability perspective, yet they are less efficient at stimulating muscle protein synthesis than animal proteins. The expnd MyoD) . Muscle nd MyoD) , 41. Gutntenance , 42. Finntenance . In addintenance –45. Howentenance  and thatThe gut microbiota of a typical older person displays reduced species richness and higher interindividual variability, together with less beneficial butyrate-producing bacteria and tight junction integrity, and a greater prevalence of pathogenic gram-negative bacteria –49. The Claesson et al.  carried Probiotics can be defined as “live microorganisms that when administered in adequate amounts confer a health benefit to the host” . Recent Lactobacillus reuteri and Lactobacillus gasseri, but not Lactobacillus acidophilus) restored gut microbiota health from a baseline status of dysbiosis, reduced serum levels of pro-inflammatory cytokines, and increased muscle mass  and area under the curve (AUC) as proxy measurements for AA digestion and absorption. Cmax and AUC were higher after protein and probiotic co-ingestion in comparison with protein alone in both studies  and old  adults. Serum zonulin concentrations were significantly higher in older vs. younger adults, were positively associated with proinflammatory cytokine levels (TNF-α and IL-6), and were negatively associated with physical activity levels and skeletal muscle strength (n = 24) vs. old (60–75 years n = 24) individuals differs and is linked to markers of age-related systemic inflammation  that access systemic circulation. These harmful substances are known to trigger low-grade systemic inflammation, insulin resistance, and glucose intolerance in murine skeletal muscle , 116. ThEmerging evidence points towards a potential role for probiotic-mediated alleviation of the leaky gut. Probiotic supplementation improved markers of an intestinal barrier and inflammation in trained men under exercise stress in a randomised clinical trial , in rodeBarnesiella and Prevotellaceae and are both found in higher concentration in high- vs. low-functioning older adults, characterised based on the percentage of body lean mass and physical functioning  by improving the intestinal cell wall integrity, thus reducing metabolic endotoxemia and consequential inflammation linked to insulin resistance and anabolic resistance , and/or (2) by improving dietary protein digestion and absorption via gut-bacterial enzymatic activity, leading to enhanced amino acid availability for de novo protein synthesis. These points are relevant in the context of healthy ageing in the present ever-growing older population (de novo protein synthesis in humans, which may or may not be affected by the gut microbiota. Overall, improving the digestibility and absorption of alternative protein sources and their ability to stimulate muscle protein synthesis will ease the environmental and nutritional challenges of the future global population while also favouring functional ageing.Ageing is often associated with a reduction in muscle mass and function together with a reduction in abundance, resilience, and diversity of the gut microbiota , 61, 62.pulation , where epulation . Even thEM drafted the manuscript with advice from CM and HR. CM and HR provided critical revisions. All the authors approved the final version of the manuscript.EM is in receipt of a Ph.D. studentship co-funded by the Irish Research Council and Kerry. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Stroke Action Plan for Europe (2018–2030) calls for national-level secondary prevention plans that address lifestyle, in addition to prevention medications and surgical interventions. This scoping review examines national stroke care guideline and audit documents across WHO regions to identify non-pharmacological, non-surgical stroke secondary prevention recommendations and associated performance indicators. Using a snowballing methodology, 27 guideline documents met the inclusion criteria. Sixteen (59%) detailed, non-pharmacological, non-surgical stroke secondary prevention-addressing known, modifiable population attributable risk factors, of physical inactivity (N = 11), smoking (N = 11), unsafe alcohol consumption (N = 10), diet (N = 8), weight (N = 5), stress (N = 4) and depression (N = 2). Strategies recommended to address these risk factors were: assessment of stroke risk/risk factors (N = 4); provision of advice and information on reducing lifestyle related risk (N = 16); education and counselling for lifestyle behaviour change (N = 8) and onward referral for specialist management of risk (N = 4). Of the nine stroke audits/registries identified, only three (33%) included non-pharmacological, non-surgical quality indicators of documented provision of advice or information on the following: general lifestyle (N = 2); smoking cessation for current smokers (N = 2); reduction in alcohol consumption, where relevant (N = 1), exercise participation (N = 1) and diet (N = 1). Preventive quality indicators addressing the management of weight, stress or depression were absent. This review highlights current gaps in optimal stroke secondary prevention recommendations and their implementation. The burden of stroke is increasing, despite significant advances in acute stroke medical management  with disIdentified gaps in comprehensive stroke care include actionable messaging for secondary prevention . ImportaEmphasis on stroke prevention is now clearly reflected in the Stroke Action Plan for Europe (SAP-E) 2018–2030 . In secoGiven the pressing need to maximise stroke secondary prevention strategies and the importance of national guidelines and programmes, the purpose of this paper is to identify the recommendations and gaps for non-pharmacological, non-surgical stroke secondary prevention  in publiA scoping review of contemporary stroke guideline documents and published national stroke audits was undertaken. A scoping review was the methodology of choice as the purpose of this study was to identify and map the concept of non-pharmacological non-surgical stroke secondary prevention strategies in national guidelines and audits of stroke care across multiple jurisdictions. This proposed strategy allows examination of current best practice recommendations and their potential implications for policy makers, practitioners and consumers in stroke care who may lack resources to undertake the work themselves ,25. The The recommended Population, Concept, and Context (PCC) mnemonic  for scop(1) To identify and map the current recommendations for stroke secondary prevention in national stroke guideline documents to attributable, modifiable risk factors in ischaemic stroke, where pharmacological or surgical interventions are not the first line preventive treatment; (2) to identify where the current emphasis on non-pharmacological, non-surgical stroke secondary prevention is in clinical practice by examining the key performance indicators used in national clinical audits or registries of stroke care. Specifically, the scoping review asks the following: What lifestyle/behavioural  recommendations for stroke secondary prevention exist in national or multiple jurisdiction stroke guidelines?What key performance indicators are used for non-pharmacological, non-surgical stroke secondary prevention in practice in national clinical audits or registries of national stroke services?The most recent Clinical Stroke Guideline document developed by their national stroke organisation or governmental health department and available on the organisation’s website or in the published literature. Where a specific stroke secondary prevention guideline was published and available, it was to be identified in preference to a general stroke guideline document. Otherwise a general stroke management guideline was to be identified and the section addressing secondary prevention was to be reviewed.National audits of stroke care or, in the absence of a national audit, national stroke registry data that had defined quality criteria.In keeping with the guidance on scoping reviews  to comprFor any document identified, the most up-to-date version available on the organisation’s website or in the published literature was to be selected.Based on the geographical spread provided by this method, INSsPiRE members were then asked to identify further experts in stroke secondary prevention from their wider networks to cover additional jurisdictions that were not included by the initial search. Adequate representation from the continents of the Americas, Australasia, Asia, Europe and Africa were sought through this method.The targeted search was initiated in June 2020 and terminated in November 2020.To be included in this scoping review, guideline documents returned must have been at national or continental level and published by a national or international stroke organisation or governmental health department and relate directly to stroke care. Similarly, clinical audits of stroke care or stroke registry data with quality criteria were required to have been conducted at a national level and conducted by a nationally recognised body. Each guideline and audit document returned was reviewed independently against these inclusion criteria by two reviewers.The ‘descriptive analytical’ method within the narrative tradition was employed, applying a common analytical framework to all the primary reports included and collection of standardised information from each report was applied. Guideline documents and audits in languages other than English were read and translated by INSsPiRE network colleagues proficient in the given language. Author(s), year of publication, jurisdiction covered, whether the guideline was a general stroke care guideline or specific to stroke secondary preventionNon-pharmacological, non-surgical recommendation/s made listed under the heading of stroke secondary preventionThe approaches recommended for non-pharmacological, non-surgical stroke secondary preventionData were charted using a proforma in Microsoft Excel. We recorded information as follows from guideline documents:Author(s), year of publication and jurisdiction covered.Key performance indicators used for assessing current clinical practice in relation to non-pharmacological, non-surgical stroke secondary prevention strategies.Rating of achievement of the key performance indicators.For audits of stroke care, we extracted the following information:Data extracted to our proforma from the multiple documents identified were collated and presented in the following ways. Firstly, by summary statistics accompanied by a narrative synthesis of stroke guidelines addressing recommendations in the area of non-pharmacological, non-surgical stroke secondary prevention; secondly, by summarising the available clinical audits in stroke care and the key performance indicators used in non-pharmacological, non-surgical stroke secondary prevention. In total, thirty guideline documents were returned from the snowballing methodology employed. The guideline documents from Kenya, Uganda and Indonesia were excluded as they were more general healthcare guidelines that did not relate specifically to stroke. Guideline documents and clinical audits returned that were eligible for inclusion are summarised (N = 27) in Of the twenty-seven guideline documents identified, sixteen addressed non-pharmacological, non-surgical stroke secondary prevention recommendations; six of these were specific stroke secondary prevention guideline documents. The remaining ten were general stroke care guidelines. Most included a specific section detailing stroke secondary prevention recommendations that included non-pharmacological, non-surgical stroke secondary prevention. However, one of these referred to other guideline documents, relating to cardiovascular disease prevention in general, but not stroke specific and not secondary prevention.A total of nine national stroke care audits or registry-based surveys with quality indicators were identified, covering Canada, Australia/New Zealand, Denmark, Ireland, Netherlands, Norway, Scotland, Sweden, UK. Three reported quality indicators that addressed topic/s related to non-pharmacological, non-surgical stroke secondary prevention are presented in The scoping review presented in this paper was conducted as a staged body of work, adhering to a published framework ,65, and A key finding from this scoping review is that non-pharmacological, non-surgical interventions for stroke secondary prevention are not a current mainstay in guideline documents for stroke care and secondary prevention. Recommendations relating to risk reducing health behaviours were made in just over half of the guideline documents identified, indicating a lack of direction for clinicians on the important role of lifestyle-related risk factor reduction in stroke secondary prevention and the need to target and address all identified and modifiable PAR factors for stroke. Where lifestyle-related recommendations were present in the guideline documents identified, there was no consistency in the message delivered to clinicians across the documents. While the focus was primarily centred on smoking cessation, physical activity participation, healthy eating and safe alcohol consumption, as risk reducing strategies after stroke, important avenues for secondary prevention, including management of depression and psychological distress and weight management, were less frequently addressed and were never considered as secondary prevention performance indicators in the clinical audits conducted. Depression after stroke has been identified in a recent meta-analysis as an independent predictor of stroke recurrence in ischaemic stroke patients . SimilarSAP-E 2018–2030 addresses challenges facing stroke survivors and families, associated with life after stroke, including the assessment and management of modifiable risk factors in the prevention of recurrent stroke . IndividThe next key finding from this review relates to clinical audit. Overall, the promotion of guideline recommendations addressing modifiable health behaviours in stroke secondary prevention in routine practice, by means of clinical audit performance indicators, was lacking. Where present, performance indicators measured whether general lifestyle and/or smoking cessation advice was provided, rather than the behaviour change achieved. As a model, this tends to work less well in more chronic conditions, such as stroke, and is even less effective in the sphere of changing behaviour . Only onBoth the guideline documents and the audit performance indicators identified in this review reflect a focus internationally on the acute management of stroke. Post-acute stroke care has previously been identified as variable and fragmented , and witA number of limitations with respect to this scoping review are acknowledged. The quantity of international documents presented is not exhaustive and a decision was made to terminate the snowballing method for guideline identification in November 2020, when a sample deemed sufficiently representative of geographical areas was identified. We acknowledge that other stroke guideline documents exist and that some reported here have been updated since this scoping review was conducted . Indeed, both the AHA/ASA guidelines and the Australian living guidelines have been significantly revised since this scoping review was completed ,34. BothOur strategy to target risk reduction recommendations that were detailed under a secondary prevention heading in the identified documents means it is possible that issues, such as management of post-stroke depression, for example, may be addressed elsewhere in stroke guideline documents. While these recommendations in the management of depression may have the potential to impact stroke recurrence, we opted to only extract recommendations that were made specifically for targeted stroke secondary prevention and acknowledge that overlap between other recommendations may exist.This scoping review highlights, in the clinical guideline documents reviewed, a current lack of emphasis on non-pharmacological, non-surgical stroke secondary prevention recommendations addressing legitimate modifiable risk factors after stroke. This finding is further reflected in an absence of quality indicators relating to non-pharmacological, non-surgical stroke secondary prevention in national stroke care audits. These gaps identified provide a basis for future development in this area.


Extracellular vesicles (EVs) comprise a heterogeneous group of small membrane vesicles, including exosomes, which play a critical role in intracellular communication and regulation of numerous physiological processes in health and disease. Naturally released from virtually all cells, these vesicles contain an array of nucleic acids, lipids and proteins which they transfer to target cells within their local milieu and systemically. They have been proposed as a means of “cell-free, cell therapy” for cancer, immune disorders, and more recently cardiovascular disease. In addition, their unique properties of stability, biocompatibility, and low immunogenicity have prompted research into their potential as therapeutic delivery agents for drugs and small molecules. In this review, we aim to provide a comprehensive overview of the current understanding of extracellular vesicle biology as well as engineering strategies in play to improve their therapeutic potential. Numerous synthetic platforms, including polymeric nanoparticles and liposomes, have been investigated with only a small number successfully approved by the FDA . This gaExtracellular vesicles were first observed by electron microscopy in the 1980s, but regarded as no more than cellular garbage bags for expired protein until the early 2000s, when they were discovered to contain and transfer functional RNA to target cells . They arExosomes, a nanosized subset of EVs which originate during the formation of multivesicular bodies (MVB), are secreted constitutively by fundamentally all cells in physiological conditions. Their production can be stimulated, and the contents of their cargo regulated by stress or disease. They possess intrinsic biological activity through the expression of surface ligands and receptors and can carry therapeutic cargo, both of which are determined by the parent cell and environmental conditions from which they originate. For example, exosomes derived from mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs) have been shown to possess cardioprotective effects –9. FurthUpon discovery that exosomes could retain a sensitive cargo and move unabated from one location within the body to another, their potential as a therapeutic delivery vehicle garnered much attention. Exosomes encompass many of the features of an ideal delivery vehicle, including a long circulation time, low levels of clearance and degradation, and preservation of the therapeutic activity of its cargo . Phase Iexosomes, small (30–100 nm) vesicles of endocytic origin; (2) microvesicles, also known as shedding vesicles, ectosomes, or microparticles—medium sized (50–1000 nm) particles shed directly from the plasma membrane; and (3) apoptotic bodies, larger (50–5000 nm) blebs released by dying cells. There is a great deal of discrepancy in nomenclature and purification criteria in the literature with a definitive categorization yet to be achieved (Extracellular vesicles can be broadly classified into three main groups based on their mode of biogenesis: (1) achieved . As suchachieved . Interesachieved ,17.Exosomes are the most extensively researched sub-group of extracellular vesicles and are formed from the inward budding of endosomal membranes . When piThe majority of exosome formation at endosomes is dependent on the endosomal sorting complex for transport (ESCRT) machinery, although a subset of proteins  are sorted into ILVs independently of ESCRTs through raft-based microdomains –23. The Neutral spingomyelinase 2 (nSMase2) and the RAB family of small GTPase also play essential roles in exosome biogenesis through ESCRT-independent mechanisms . nSMase2In contrast with exosomes, microvesicles are plasma membrane-derived particles released into the extrcellular space by outward budding and fission of the plasma membrane . The bioAs opposed to the heavily regulated biogenesis and release of exosomes and microvesicles, apoptotic bodies are formed as a result of programed cell death . This clExtracellular vesicles contain lipids, nucleic acids and proteins, the content of which varies with their mode of formation and cellular origin. In a similar manner, while the membranes of each subset of EVs form as a lipid bilayer, the composition of proteins and lipids differs between EV populations secondary to their biogenesis.There are currently over 92,897 protein entries, 27,642 mRNA entries, 4934 miRNA entries, and 584 lipid entries in Vesiclepedia associated with EVs . The selThe selection of exosomal miRNA cargo also appears dependent on the parent cell and environmental factors. Some miRNA species, including miR-451a, are selectively enriched in EVs of multiple cell types . This soExosomes are also heavily enriched in lipids, as evidenced by their unique rigid lipid bilayer membrane . While bin vivo angiogenesis, likely contributing to increased plaque vulnerability  is one of the most widely used EV isolation techniques, separating particles based on their sedimentation coefficients . DiffereVarious commercial kits make use of volume-excluding polymers such as polyethylene glycol (PEG) and allow for rapid EV isolation from culture media or body fluids. Although easy to use and scale for larger isolations, these precipitation based methods lack isolation specificity, co-precipitating protein, polymeric materials, and other non-EV fractions . AdditioUltrafiltration is another popular EV isolation technique to concentrate EV fractions from supernatant based on their size and molecular weight. The force required to push EVs through the membrane however, can result in deformation of larger particles which are subsequently forced through the filter and erroneously incorporated into downstream applications and analysis . AdditioTechniques that pair ultrafiltration with ultracentrifugation into a sucrose cushion or polyethylene glycol (PEG) have both been used to purify EVs for clinical application ,64. AddiImmunoaffinity capture, or immunoaffinity chromatography, utilizes the interactions of EV surface proteins with specific receptors or ligands. The method can yield pure EV subpopulations, but is highly influenced by both the choice of affinity reagent and the ligand density on different EV types . This teRecent studies have directly compared different methods of isolation  on EV-RNA yield and purity. DGC was found to yield the highest purity, albeit 3–8× less protein and fewer particles than the commercial kits, highlighting the tradeoff of yield vs. purity that must be considered when addressing a specific research question . CurrentLess conventional than the aforementioned techniques and focused on the ability to process small volumes, microfluidic systems have also been developed for the isolation of EVs from bodily fluids . This onIt is crucial that extracellular vesicles are not only properly isolated, but also well characterized and quantified to enable accurate, interpretable, and comparable downstream analyses. While a number of EV quantification methods exist, there is currently a lack of consensus on the proper quantification . While oNanoparticle tracking analysis (NTA) is currently considered the best method available for exosome quantification. An analysis of Brownian motion via light scattering enables the quantification and size distribution of vesicles in a liquid suspension . By consWhile not a common tool for exosome quantification (secondary to exosome loss during dehydration and embedding), electron microscopy (EM) remains the gold standard for verifying the quality of EV preparations. Transmission and scanning electron microscopy are most frequently used in the analysis of EVs. SEM has been shown to be less-time consuming and able to achieve greater image resolution than TEM, enabling the optimal imaging EV native morphology .In addition to NTA and EM, exosome quantification is commonly reported in terms of total extracellular vesicle protein concentration. This is typically achieved with sample sonication followed by a total protein assay, with the total protein content serving as a surrogate measure of EV quantity. Western blot, ELISA, and/or flow cytometry is subsequently performed to identify and quantify specific EV proteins. The utility of this method of quantification is questionable when comparing exosomes from different cell sources or donors, as normalized protein content may not always equate to normalized bioactive load. Continuing to understand the mechanism/s of action of EV-based therapeutics remains essential for development of suitable potency assays and eventual clinical translation, as EV dosage may likely differ between samples quantified as “equivocal” by the methods discussed above ,81.in vivo fate and pharmacodynamic properties of extracellular vesicles including tissue distribution, blood levels, and urine clearance are paramount for future therapeutic applications. Most EV biodistribution studies are limited by their use of fluorescent and bioluminescent pre-labeled purified extracellular vesicles. It must be taken into consideration that each EV labeling technique has associated advantages and limitations and the method used should reflect the aims of the experiment , the biodistribution and clearance of systemically injected human HEK293T-derived EVs in mice was evaluated by analysis . EVs weranalysis .in vivo, however, studies suggest that EVs secreted by some cell types do exhibit target selection. For example, when comparing exosomes from C2C12 cells, bone marrow-derived DCs, and B16F10 melanoma cells (administered at the same dosage and consisting of the same EV size profile) DC-EVs had the highest distribution to the spleen   distributed to the liver and spleen without any of the observed side effects seen at the higher concentration  . Other sntration . It appentration .The majority of extracellular vesicles show limited cellular tropism, with a few exceptions ,103. ThiCell engineering techniques such as genetic modification, metabolic labeling, and exogenous delivery have been shown to change the surface expression and cargo of extracellular vesicles . Manipulin vitro and in vivo  with Lamp-2b to target exosomes to neurons and glia , which utilized a CD9-CIBN fusion protein and CRY2-conjugated cargo proteins  , lysosomand glia . Targete peptide . Purifieresponse . Finallyproteins . Taking proteins , cargo pet al. used the evolutionarily conserved late-domain (l-domain) pathway as a mechanism for loading exogenous proteins into exosomes  approach  and trans-acting proteins are considered to be the main driving forces of mRNA localization and post-transcriptional regulation ,117. Bolter mRNA . While tapproach . They foThe process of selectively loading miRNA appears to be more complicated, as RNA-binding proteins required for miRNA sorting have been shown to vary between cell types . Additioet al. demonstrated that curcumin, a polyphenol with anti-inflammatory and antineoplastic activity, is self-assembled into the lipid bilayer of exosomes via hydrophobic interactions, resulting in increased longevity of the drug  has been shown to result in enhanced bioactivity of EV-loaded agents when compared with sonication, possibly secondary to a higher degree of intact membrane proteins which results in more uniformity of their surface morphology and subsequent decreased clearance by macrophages .et al. used active EV engineering to modify EV membranes. Expression of modified arginine-rich cell-penetrating peptides resulted in activation of the macropinocytosis pathway, causing an increase in cellular EV uptake  to enhance their circulation time and prolong exposure to their target-specific receptor  can be achieved in breast and ovarian tumors when the chemotherapeutic agent is loaded into and delivered by EVs as compared to administered systemically. They also found a reduction in cardiotoxicity secondary to decreased crossing of DOX through the myocardial endothelium . The potential to deploy targeted EVs with specialized payloads has shown potential for circumventing the very limitations of current radiotherapy and chemotherapy. For example, Hadla othelium . In a siothelium .de novo miRNA cargo. O’Brien et al. found that loss of miR-134 in cells and their EVs was associated with increased cellular aggressiveness  is characterized by a resistance to insulin (type II) or the inability to produce insulin (type 1) and associated with end organ damage and life-threatening complications. More than one quarter of patients with diabetes will suffer from diabetic nephropathy which carries a significant increase in renal failure and mortality. Jiang at model . Followi effects . Compare effects .Cardiovascular disease (CVD) encompasses a group of disorders affecting the heart and vasculature, the most common of which is coronary artery disease. Current treatment, however, is limited in its regenerative potential. Preclinical cell therapy trials failed to demonstrate any significant engraftment or progenitor differentiation into new myocardium, but instead highlighted EVs as the key beneficial mediator of cell therapy. Work directed at understanding the cardiovascular bioactivity of various EVs has exploded in recent years with the goal of developing a novel means of cell-free cell therapy for heart disease.et al. first identified EVs in the conditioned media of MSCs and demonstrated their therapeutic valve in reducing infarct size in a myocardial ischemia/reperfusion (I/R) model  model . This suerfusion . GATA-4 erfusion . Exosomein vitro . Exosomein vitro .et al. engineered MSC-derived exosomes to carry increased levels of miR-133b in an effort to enhance brain remodeling after stroke  is responsible for their therapeutic potency . Furthermore, in engineered exosomes loaded with therapeutic cargo, loading efficiency should be expressed as both a percentage (cargo loaded into EV/cargo exposed to EV) as well as the number of therapeutic molecules/copies of loaded EV, to enable comparisons across different studies.Several hurdles still lie on the horizon which must be addressed prior to successful clinical translation. Fully realizing the therapeutic potential of EVs requires standardization of our methodology for isolation, quantification, and characterization. EVs produced from different cell types are markedly different; even EVs secreted from the same cell type can vary in shape, size, and cargo secondary to donor-to-donor variability and differences in cell culture conditions. To complicate matters further, certain populations of cells appears to generate exosomes with multiple subtypes ,146. Thi potency . While w potency ,81. Devein vivo properties of both exogenously and endogenously released EV from different cell types, isolated by different methods, and genetically engineered to express different surface receptors (Future studies on the potential correlation of EV size, dosage, and pharmacokinetic properties will provide additional insights into EV-mediated therapies. It would be useful to employ high sensitivity methods of exosome tracking to compare eceptors . AdditioEngineered EVs provide a relevant and exciting therapeutic tool for the treatment of a variety of diseases ranging from cancer to Parkinson’s to ischemic cardiomyopathy. Research in the field of EVs is currently advancing at an exponential rate. As additional engineering techniques are developed and applied to improve their ability as functional carriers, we believe the prospect of harnessing EVs as a clinically relevant therapy in the next decade will become a reality.


Giardiaduodenalis and HIV, peptic duodenitis, drug-induced enteropathy, common variable immunodeficiency, Crohn’s disease, Whipple’s disease, small intestinal bacterial overgrowth, eosinophilic gastroenteritis, tropical or collagenous sprue and autoimmune enteropathy. While mucosal adaptation due to malnutrition, leading to villous atrophy, has been reported in paediatric populations in Africa and South America, in hibernating animals and animal models of experimental starvation, there is very little literature on adult human subjects. We report a case of a 76-year-old gentleman, presenting with chronic metabolic acidosis and anorexia who was found to have villous atrophy on duodenal biopsy, in the absence of any evidence of coeliac disease or other non-coeliac enteropathy. The nutritional state improved with correction of the underlying metabolic abnormality, and serial endoscopic assessment showed improvement in the villous atrophy. We discuss the relationship between malnutrition and villous atrophy, and suggest a potential workup for non-coeliac villous atrophy.Coeliac disease is the most well-known cause of villous atrophy in duodenal biopsies. Other well recognised causes of villous atrophy include infection with  Giardia duodenalis and HIV, peptic duodenitis, drug-induced enteropathy, common variable immunodeficiency (CVID), Crohn’s disease, Whipple’s disease, small intestinal bacterial overgrowth (SIBO), eosinophilic gastroenteritis, tropical or collagenous sprue and autoimmune enteropathy. It can also simply be an artifactual finding, depending on the plane in which the biopsy has been taken and processed [In patients presenting with unformed stools or weight loss, a finding of villous blunting and mucosal atrophy on duodenal biopsy would point towards a diagnosis of coeliac disease in the vast majority of cases. Other possible causes of villous atrophy, albeit much less common, include a wide spectrum of conditions such as infection with rocessed ,2.Here we present the case of an adult patient, who was found to have villous atrophy on duodenal biopsy, but for whom coeliac and the other non-coeliac enteropathies listed above were conclusively excluded. The relationship between this patient’s malnutrition and his intestinal atrophy, which was shown to improve following the correction of his metabolic acidosis leading to restitution of his oral nutritional intake, makes for an interesting discussion of how villous atrophy can be worked up and managed. The longitudinal follow-up with serial biopsies over a period of several months, alongside incremental oral intake and the improvement in nutritional status, is unique, and represents a rarely reported aetiology for this common biopsy finding.Clostridium difficile toxin were all negative. CT-colonography showed a normal appearance except for some diverticulosis involving the sigmoid colon. Apart from the expected post-operative changes and an incisional hernia affecting the anterior abdominal wall, there was no suggestion of an extracolonic mass lesion. Upper gastrointestinal endoscopy was visibly normal but biopsies were taken from the proximal duodenum.Mr H, a 76-year-old retired school teacher, was referred by his general practitioner to the gastroenterology service in mid-2016 with iron deficiency anaemia and weight loss. His significant past medical history included urinary bladder cancer, which was definitively treated by radical cystoprostatectomy and ileal conduit surgery in mid-2015. He was referred to the cardiology services at the end of 2015 due to shortness of breath and had normal results on chest x-ray, electrocardiography and echocardiography and no explanation was found to account for his “breathlessness”. At the presentation to the gastroenterology clinic, he described feeling nauseous, lack of appetite, and a loss of more than 10% of his body weight over the preceding 6 months. He reported feeling dyspneic and lethargic, and passing unformed stools since his operation, but denied any bleeding. Blood tests showed iron deficiency anaemia, low folate level with macrocytosis, low phosphate and borderline low calcium levels and raised parathyroid hormone level. Testing for coeliac disease using anti-tissue transglutaminase IgA antibodies was negative with a normal IgA level. A review of historical test results showed peri-operative anaemia, partly corrected after iron supplementation, and a negative Bowel Cancer Screening Programme stool test result from 2 years ago. Stool cultures, tests for ova, parasites and cysts, and While awaiting the reporting of the duodenal biopsy and outpatient clinic follow-up, Mr H progressively became cachectic with increasing nausea and anorexia and started showing some cognitive changes. He was admitted to the hospital, as he could hardly tolerate any oral intake. There was tachypnoea, disorientation and apathy, and he was struggling to make any coherent conversation. On admission, he was found to be in profound metabolic acidosis with hyper-chloremia, and significantly reduced renal function. The additional blood results are shown in Table Ultrasound scan of the abdomen showed both kidneys to be of normal appearance, with no hydronephrosis or hydroureter, and a CT scan of the head revealed no overt abnormalities. A repeat CT scan of the chest, abdomen and pelvis, with contrast, showed normal lung fields, no filling defects in the pulmonary arteries, normal configuration of the ileal conduit with the parastomal hernia and a midline incisional hernia containing small and large bowel loops but no features of incarceration or strangulation. No enhancing mass lesion was noted.The patient was started on oral sodium bicarbonate replacement 500mg three times a day, to correct his metabolic acidosis. Within a few days the clinical picture rapidly changed; cognitive changes and anorexia improved, tachypnoea settled, the patient started tolerating oral intake and began to mobilise independently. Correction of electrolytes like phosphate, magnesium and potassium was done as needed with regular monitoring for re-feeding changes. The hyperchloremic metabolic acidosis was corrected with the continued use of sodium bicarbonate tablets. He was discharged, having significantly improved, after a hospital stay of three weeks.Tropheryma whipplei DNA PCR was negative on both blood and biopsy samples. As the cognitive changes resolved rapidly after correction of the metabolic acidosis, the cerebrospinal fluid study was not pursued. HIV testing was negative. The serum immunoglobulin electrophoresis showed normal levels of immunoglobulin IgG, IgM and IgA. A review of his medication history revealed none of the drugs associated with drug-induced villous atrophy, were involved at any point. While he had had previous small bowel surgery, there were no real blind loops and his serum folate was low so SIBO was not considered as the cause here. Whilst the antinuclear antibody test was positive, extractable nuclear antigen (ENA) antibodies and anti-double-stranded DNA antibodies were both negative. Anti-enterocyte IgG and anti-goblet-cell IgG antibodies were also negative.While he was an inpatient, the duodenal biopsy was reported as showing villous atrophy with moderate blunting of the villi. The lamina propria showed a moderately dense, mixed acute and chronic inflammatory cell infiltrate with some crypt hyperplasia. The overlying surface epithelium also showed infiltration by mixed inflammatory cells including scattered lymphocytes and neutrophil polymorphs. No dysplasia or malignancy was found. However, given the details of the case further serological and clinical correlation was advised. Repeat tests for anti-tissue transglutaminase IgA antibodies and anti-endomysial IgA antibodies were negative. HLA- DQ2 and DR8 genotypes were negative, thus coeliac disease was conclusively ruled out. Given the cognitive changes noted at admission, the possibility of Whipple’s disease was considered and Periodic acid-Schiff and Periodic acid-Schiff-diastase stains were carried out. However, no convincing Periodic acid-Schiff (PAS)-positive macrophages were identified and Following discharge from hospital and with continued clinical improvement from correction of his acidosis with sodium bicarbonate tablets, his nausea and anorexia resolved, his oral intake increased and he started to gain weight. At follow-up clinic review, given that we did not feel we had a full explanation of the villous atrophy, we arranged for repeat duodenal biopsies. The histology showed an improvement in the villous blunting, with some crypt elongation remaining but no increase in intraepithelial lymphocytes.When the patient was seen in the clinic later that year, he reported feeling the best he had felt since his original cystoprostatectomy, and the anaemia and weight loss had fully corrected. A further upper gastrointestinal endoscopy was arranged, two years since the first one, and duodenal biopsies showed nearly normal duodenal mucosa, with villous architecture within normal limits and certainly improved in comparison with his previous biopsies.The basic therapeutic intervention that had been instituted in this patient was the sodium bicarbonate tablets: by correcting the metabolic acidosis, his clinical condition dramatically improved and his appetite increased. We postulate that this patient’s villous atrophy was driven by a significant reduction in oral food intake and by reversing the starvation, the villous atrophy reverted as shown on longitudinal follow-up with serial duodenal biopsies. While this phenomenon has been demonstrated in animal experiments and in malnutrition in paediatric populations, we did not come across reported cases in adult populations where the recovery of the villous atrophy has been demonstrated with longitudinal follow-up.This case provides an example of the phenomenon of intestinal mucosal adaptation due to malnutrition, a less common but very likely under-reported cause of villous atrophy. It has been well established that prolonged periods of poor enteral intake can lead to intestinal failure via mucosal atrophy . Animal The adaptive re-sizing of the intestine is one of the well recognised physiological responses to tissue demands. Villous atrophy has also been reported in patients receiving total parenteral nutrition (TPN), even when their overall nutritional intake was adequate, but their enteral intake was significantly reduced or completely absent . HoweverThe changes of the atrophy of villi and increased intestinal permeability have been shown to start rapidly, as soon as four days after stopping enteral feeding, indicating a rapid response of intestinal stem cells to nutrient availability . In expeIn this gentleman’s case, the cause of hyperchloremic metabolic acidosis was thought to be his ileal conduit formation. This is a known complication of such urinary diversion surgery, affecting up to 42% of patients . The mecThis case provides a clinical example, with biopsy evidence, of the maladaptation of the intestinal mucosa to significantly reduced oral intake, as explained above. Furthermore, the series of repeat biopsies over a two-year period showed parallel improvement in the mucosal changes with ongoing nutritional recovery, once the metabolic acidosis which was causing the patient’s loss of appetite had been corrected.When investigating a patient who presents with chronic loose stools and weight loss, it is obviously important to rule out coeliac disease as a cause, especially in the presence of villous atrophy. Initial anti-tissue transglutaminase IgA antibodies testing is usually enough to establish this diagnosis in the vast majority of cases. If the serology is negative, it is important to review the pathology in light of the clinical picture and consider the possibility of non-coeliac enteropathies (NCE). Investigation of serum immunoglobulins to exclude IgA deficiency, checking for IgG anti-tTg antibodies, and HLA- DQ2/DQ8 testing will provide further guidance to exclude coeliac disease. In the absence of positive findings, it is reasonable to assume a diagnosis of NCE, especially if the patient has not seen an improvement on a gluten-free diet. We have demonstrated a series of investigations, exploring the causes of villous atrophy in this gentleman, trying to correlate the clinical features. Due to the paucity of clinical literature documenting such changes in adult populations, we did not initially consider the fact that the villous atrophy in this patient may actually have been the result of maladaptation of the small bowel mucosa to malnutrition, rather the cause of the malnutrition itself.


There is a need for timely, accurate diagnosis, and personalised management in lung diseases. Exhaled breath reflects inflammatory and metabolic processes in the human body, especially in the lungs. The analysis of exhaled breath using electronic nose (eNose) technology has gained increasing attention in the past years. This technique has great potential to be used in clinical practice as a real-time non-invasive diagnostic tool, and for monitoring disease course and therapeutic effects. To date, multiple eNoses have been developed and evaluated in clinical studies across a wide spectrum of lung diseases, mainly for diagnostic purposes. Heterogeneity in study design, analysis techniques, and differences between eNose devices currently hamper generalization and comparison of study results. Moreover, many pilot studies have been performed, while validation and implementation studies are scarce. These studies are needed before implementation in clinical practice can be realised. This review summarises the technical aspects of available eNose devices and the available evidence for clinical application of eNose technology in different lung diseases. Furthermore, recommendations for future research to pave the way for clinical implementation of eNose technology are provided.The online version contains supplementary material available at 10.1186/s12931-021-01835-4. The field of pulmonary medicine has rapidly evolved over the last decades, with increasing knowledge about pathophysiology and aetiology leading to better targeted treatment strategies. Nevertheless, many chronic lung diseases have non-specific, often overlapping symptoms, which delays the diagnostic process and timely start of adequate treatment. Moreover, even specific disease entities can be very heterogeneous with varying phenotypes, and thus disease courses and optimal treatment strategies vary per patient. Accurate, non-invasive, real-time diagnostic tools and biomarkers to predict disease course and response to therapy are currently lacking in most lung diseases, but are indispensable to achieve a personalised approach for individual patients.An emerging tool that has the potential to meet this need is an electronic nose (eNose). This device ‘smells’ exhaled breath for clinical diagnostics, a concept probably as old as the field of medicine itself. Exhaled breath contains thousands of molecules, also known as volatile organic compounds (VOCs). These VOCs can be divided into compounds derived from the environment (exogenous VOCs) and compounds that are the result of biological processes in the body (endogenous VOCs). Endogenous VOCs can be associated with normal physiology, but also with pathophysiological inflammatory or metabolic activity , 2. IdenThe aim of this paper is to review the current clinical evidence on eNose technology in lung disease, regarding diagnosis, monitoring of disease course and therapy evaluation. In addition, technical aspects and available eNose devices are discussed.In the time of Hippocrates, it was already acknowledged that exhaled breath can provide information about health conditions . For insExhaled breath analysis by use of eNose technology is recently gaining increasing attention. An eNose is defined as “an instrument which comprises of an array of electronic-chemical sensors with partial specificity and an appropriate pattern recognition system, capable of recognising simple or complex odours” . SensorsDetection and recognition of odours by an eNose is similar to the functioning of the mammalian olfactory system Fig. . First, To analyse eNose breathprints, pattern recognition by machine learning is most commonly used. A machine learning model uses algorithms which automatically improve due to experience with previously presented data. These models are in general established using a five step process: data collection, data preparation, model building, model evaluation, and model improvement. Machine learning is categorised into unsupervised, supervised, and reinforcement learning . In supeSeveral machine learning models have been proposed as appropriate algorithms for modelling complex nonlinear relationships in medical research data, such as breathprints. These models include, amongst others, artificial neural networks , ensemble neural networks , and support vector machines  , 15. A cVarious eNose devices have been developed and studied in different lung diseases. Table The stage of development towards a clinically implemented tool differs substantially per device and disease. Before clinical implementation, each specific eNose has to be tested as a proof of concept and consecutively in substantial cohorts for each specific disease. Subsequently, data validation and clinical implementation needs to be assessed in real-life cohorts. To give more insights in the stage of development for each eNose per lung disease, we divided studies in five different stages: (1) proof of concept study; 2) cohort size of diseased participants less than fifty; (3) cohort size of diseased participants equal or more than fifty; (4) study cohort with an external validation cohort; (5) evaluation of clinical implementation. An overview of the progress per eNose and disease is visualised in Fig.  cohort sOn 21 October 2020, a systematic literature search was performed in the databases Embase, Medline (Ovid), and Cochrane Central. Search terms and selection criteria are described in the Additional file Asthma is a chronic lung disease characterised by reversible airflow obstruction with airway inflammation and hyperresponsiveness. Common symptoms, such as cough, chest tightness, shortness of breath and wheezing, are variable in severity and often non-specific . VariousThe existence of multiple asthma pheno- and endotypes with different underlying pathophysiological mechanisms is increasingly acknowledged . In receA currently ongoing study is evaluating whether eNose technology can be used to predict response to monoclonal antibody therapy (NCT03988790).In general, the diagnosis of asthma in children is challenging. Lung function tests are often difficult to perform and do not always provide a diagnosis. Interestingly, a study in 45 children demonstrated that eNose measurements were fairly well repeatable, both in healthy and asthmatic participants .Moreover, two studies showed that eNose technology distinguishes children with asthma from healthy controls , 25, 34.Although COPD is one of the major causes of death worldwide, epidemiological studies indicate that it remains largely underdiagnosed . COPD isSeveral studies have evaluated the ability of eNose technology to diagnose COPD. Exhaled breath analysis discriminated between COPD and (smoking) healthy controls with an accuracy of 66–100% , 37–41. eNose technology can theoretically be useful in early detection of inflammation and acute exacerbation of COPD (AECOPD), as inflammatory processes influence breathprints. This hypothesis was confirmed in a cross-sectional study evaluating the association of breathprints with different inflammation markers in sputum; eNose breathprints highly correlated with inflammatory activity . In patiRegarding prognostic value of eNose technology, one study demonstrated that eNose data correlated better to change in 6-min walking distance over one year, than the current GOLD classification . A few sCystic fibrosis (CF) is associated with bronchiectasis, recurrent infectious exacerbations, and progressive deterioration of lung function due to exacerbations .A few studies using different eNoses showed that patients with CF could accurately be distinguished from healthy controls and asthma patients based on their breathprint , 52, 53.Interstitial lung disease (ILD) is a heterogeneous group of relatively uncommon diseases causing fibrotic and/or inflammatory changes in interstitial lung tissue. Disease course and treatment strategies widely vary for different ILDs, and even within individual ILDs disease course often varies. Diagnosis is based on integration of clinical data with imaging and if needed pathology data. Diagnosis is often complex and diagnostic delays are common , 56. eNoA large single centre cohort, including various ILDs, found that breathprints of ILD patients could be distinguished from healthy controls with 100% accuracy. Results were confirmed in a validation cohort . A few oWorldwide, lung cancer is the leading cause of cancer deaths and has the highest incidence of all cancer types. More than 80% of patients suffering from lung cancer are former or current tobacco smokers . Early dWhether an eNose can differentiate lung cancer patients from healthy controls, patients with benign lung nodules or (former) smokers, has been investigated in different cohorts. All studies in (non-) small cell lung cancer ((N)SCLC) showed significant results, albeit with a wide range in reported sensitivity (71–99%) and specificity (13–100%) –80. SmokPrediction of response to therapy is investigated for anti-programmed death (PD)-1 receptor therapy in NSCLC patients. Breathprints were collected before start of pembrolizumab or nivolumab therapy. Exhaled breath data could predict which patients would respond to therapy with an AUC of 0.89, confirmed in a validation cohort. By setting a cut-off value to obtain 100% specificity, the investigators were able to detect 24% of non-responders to anti-PD-1 therapy. In this regard, eNose seems to be more accurate than the currently used biomarker PD-L1 . AnotherSchmekel et al. investigated the ability of eNose to predict prognosis in patients with end stage lung cancer. They collected breathprints before start and several times after start of palliative chemotherapy and applied different prediction models. Patients with less than one year survival and more than one year survival could be separated based on breathprint . The autAt the moment, the gold standard for diagnosing obstructive sleep apnoea (OSA) is (poly)somnography which is a costly and time-consuming test. eNose technology has been investigated as an alternative modality to diagnose this condition and assess treatment effect.It was shown that breathprints from OSA patients and healthy controls can be distinguished reliably –91. HoweOther researchers investigated OSA, OSA-COPD overlap syndrome and COPD. OSA could be distinguished from the overlap syndrome, but eNose technology could not discriminate well between the overlap syndrome and COPD. Also here it is not clear whether true OSA can be detected or other factors, such as COPD, are picked up , 92. WheThe effects of continuous positive airway pressure (CPAP) treatment in patients with OSA has also been studied. The breathprint of OSA patients changed significantly already after one night of CPAP treatment . SignifiPathogenic micro-organisms, such as viruses, bacteria or fungi, can cause severe pulmonary infections. Identification of specific micro-organisms with sputum cultures can take up to several days, and is only possible if a specimen with sufficient quality is obtained. Specificity and sensitivity also depend on the causative micro-organism, experience of laboratory observer, and prior treatment . Therefo, breathprints of bronchiectasis patients significantly differed between those colonised with Pseudomonas Aeruginosa and those colonised with other pathogenic micro-organisms or non-colonised [Until now, two in vitro studies aimed to differentiate micro-organisms by analysing breathprints of their headspace air , 100. Moolonised . For detolonised , 102. ThVentilator-associated pneumonia (VAP) is a common nosocomial infection in ventilated patients and has an incidence and mortality around 9% , 103. InIn pulmonary tuberculosis (TB) patients, detection and screening with eNose technology has been studied in different countries and compared to different control groups –112. As Due to the Corona Virus Disease (COVID-19) pandemic, much research effort is being put in the evaluation of eNose technology as a fast and non-invasive tool for the detection of COVID-19 . To date, one study tested the accuracy of eNose technology for COVID-19 screening prior to surgery in non-symptomatic patients and found a negative predictive value up to 0.96. Reverse transcription-polymerase chain reaction on a pharyngeal swab and antibody testing were used to confirm presence or absence of COVID-19 .A number of eNose studies have been performed in other lung diseases. In acute respiratory distress syndrome (ARDS), eNose technology could discriminate between mechanically ventilated patients with and without ARDS, with moderate accuracy in a training and validation cohort .One small proof-of-principle study has been performed in patients with suspected pulmonary embolism, defined as a high clinical probability according to the Well’s score or elevated D-dimer. Breathprints of non-comorbid patients with and without pulmonary embolism could be distinguished with an accuracy of 85%. However, in patients with comorbidities known to influence VOCs  the accuracy dropped .Finally, eNose technology could be useful for follow-up and monitoring lung transplant recipients. One study found a significant association between breathprint and plasma tacrolimus levels, suggesting that eNoses might be used for non-invasive therapeutic drug monitoring .A clinical trial in lung transplant recipients is currently conducted (NL9251) looking at discrimination of stable lung transplant recipients, acute cellular rejection, and chronic lung allograft rejection.In the past decades, multiple eNoses have been developed and tested in numerous clinical studies for a wide spectrum of lung diseases. So far, the vast majority of studies evaluated the ability of eNose technology to distinguish lung diseases from healthy controls, and to discriminate between different diagnoses. A small number of studies have been performed for prognostic or therapeutic purposes, and only a handful of studies have focused on clustering patients by breathprint and identifying phenotypes. Results in lung diseases are overall very promising, but several issues should be addressed before eNoses can be implemented in daily clinical practice.One of the issues is the use of various eNose devices with different qualifications, types of sensors and breath sample collection methods as summarised in Table Given important differences between the various devices, it is difficult to compare data of the different eNose devices. Hence, each eNose needs to be validated for every clinical application. This implies that knowledge about characteristics of eNose devices is essential before initiating eNose research, as the type of device cannot easily be changed during the trajectory of developing a clinical tool. Additionally, the influence of endogenous  and exogenous factors  on breathprints needs to be further elucidated.Furthermore, studies differ significantly with regards to study design . As illustrated in Fig. Moreover, most studies do not explain the rationale for choosing a certain machine learning model for analysing eNose data. This prevents insights in and discussion regarding the optimal analysis techniques and algorithms. Machine learning models are complex to execute and interpret, and if not used in the right way are prone for overfitting. To avoid inadequate modelling, data scientists should always be involved in these complex analyses and models should be validated independently to exclude overfitting. To allow for comparison of different modelling techniques, we recommend an extensive world-wide shared database per eNose with FAIR  and open source data, including patient characteristics and other pre-test probabilities. This database would ensure optimal training, validation, and application of models.Finally, a factor that hampers eNose implementation is the need for a strong gold standard to establish a diagnosis or to evaluate therapeutic effect. High quality data input is required for optimal validity when developing a new technique. Some of the diseases mentioned in this review lack a gold standard, and even if a gold standard does exist, there is always a range of uncertainty. There is a potential for unsupervised machine learning models in this regard, as such analyses could help to identify previously unrecognised phenotype clusters. Discovering such new clusters can help to generate hypotheses about the existence of unravelled disease subtypes or overlap between diagnoses, and might eventually guide new diagnostic standards.In conclusion, eNose technology in the field of lung diseases is promising and at the doorstep of the pulmonologist’s office. To facilitate clinical implementation, we recommend conducting prospective multicentre trials including validation in external cohorts with a study design and analysis method relevant for the research aim, and sharing databases on open source platforms. If supported by sufficient evidence, research can subsequently be extended to clinical implementation studies, and finally, use in daily practice.We believe that eNose technology has the potential to facilitate personalised medicine in lung diseases through establishing early, accurate diagnosis and monitoring disease course and therapeutic effects.Additional file 1. Sensor technology explained.Additional file 2. Search strategy.


Communication in noise is a complex process requiring efficient neural encoding throughout the entire auditory pathway as well as contributions from higher-order cognitive processes  to extract speech cues for perception. Thus, identifying effective clinical interventions for individuals with speech-in-noise deficits relies on the disentanglement of bottom-up (sensory) and top-down (cognitive) factors to appropriately determine the area of deficit; yet, how attention may interact with early encoding of sensory inputs remains unclear. For decades, attentional theorists have attempted to address this question with cleverly designed behavioral studies, but the neural processes and interactions underlying attention’s role in speech perception remain unresolved. While anatomical and electrophysiological studies have investigated the neurological structures contributing to attentional processes and revealed relevant brain–behavior relationships, recent electrophysiological techniques  may provide novel insight regarding the relationship between early sensory processing and top-down attentional influences. In this article, we review relevant theories that guide our present understanding of attentional processes, discuss current electrophysiological evidence of attentional involvement in auditory processing across subcortical and cortical levels, and propose areas for future study that will inform the development of more targeted and effective clinical interventions for individuals with speech-in-noise deficits. Attentional selection of relevant inputs is a phenomenon that has been extensively studied in the auditory, somatosensory, and visual sensory modalities. In the auditory domain, attentional selection applies in everyday communicative interactions as the listener must attend to signals of interest while ignoring, or suppressing, competing signals present in the soundscape. Difficulties extracting important speech information from background noise have been linked to a variety of communication disorders in individuals of all ages for several decades. In children, these difficulties may impair a child’s ability to access the phonological structure of language ,2 and coBoth bottom-up perceptual and top-down cognitive processes contribute to a listener’s ability to extract a salient auditory signal from a competing stream of background information. Uni- and bi-directional components of the auditory brain that contribute to adequate processing of speech in background noise mature at different rates across development and are subject to a variety of acquired factors across adulthood and aging. Moore  noted thBoth Moore  and HumeAs speech-in-noise (SIN) deficits are present even in normal hearing listeners ,7, theseAt the same time, global cognitive processes and functions influence how auditory information is processed and perceived by a listener. Factors such as processing speed ,26, workAttention is a cognitive process that bridges bottom-up sensory input with identification and perception and contributes to speech understanding in challenging listening conditions. Attention serves to alert and orient us to when and where stimuli of interest occur within our environment . It alsoSuch studies would also elucidate at which stages of processing attentional modulations occur. Electrophysiologic studies provide unique insight into the underlying mechanisms of auditory processing across all levels of the auditory neuroaxis. For years, scalp-recorded electrophysiologic measures have been used as a non-invasive method to infer the underlying neural mechanisms contributing to behavior. Electrophysiologic recordings reflect the temporal and spatial summation of neural activity contributing to the processing of sensory inputs and cognitive processes with remarkable temporal resolution . These rOver the course of time, theorists have proposed numerous hypotheses regarding how and when attention plays a role in sensory processing with these attentional effects ultimately affecting perception and behavioral responses. As most of these theories were based on behavioral studies, the application of theoretical suppositions to electrophysiologic studies enables the establishment of brain–behavior relationships and the mapping of selective attention to neuroanatomical structures. This article aims to bridge these literatures by (1) establishing a framework of selective attention by reviewing relevant proposed theories, (2) evaluating electrophysiologic evidence of selective attention throughout the auditory neuroaxis focusing on the controversial attentional effects on early processing within the brainstem, and (3) identifying areas for future research to clarify the potential role of attention in early auditory processing and the clinical implications of such studies.Selective attention is necessary given the limited amount of information that humans can process at one time. If multiple inputs are presented simultaneously, individuals are only able to consciously detect a few items. Thus, attentional selection reflects an individual’s prioritization of relevant information to account for limitations of the processing system ,49,50. TEarly researchers of auditory attention likened sensory processing to communication transmission systems of the time. Early models suggested a limited amount of information could be transmitted along a given channel at one time from the sender to the receiver, yet theorists disagreed about when selective filtering of inputs occurred. Some theorists believed that selective filtering happened early in processing to prevent overloading the channel with excessive inputs ,52 whileProposing an early filter model, Broadbent  hypothesBased on the findings of Cherry and Moray, Treisman  expandedDeutsch and Deutsch  disagreeTaking a slightly different approach, other theorists focus on the limited capacity of the system rather than the filtering mechanism. Like many early theories of attention, limited capacity theories share similarities to previously proposed theories yet add unique perspective by attempting to address more complex scenarios, particularly by incorporating interactions between perceptual and higher-order cognitive processes. Whereas filter theories propose serial processing mechanisms for attention, capacity theories describe how bottom-up and top-down mechanisms interact to influence attentional selection and behavior.Kahneman  proposedMore recently, Pichora-Fuller et al.  adapted In an alternative limited capacity approach, Lavie  attempteOverall, behavioral studies suggest that perceptual limitations are more influential in attentional selection than response competition ,63 whichUltimately, the selection process in voluntary attention requires a determination of what inputs are most relevant and will become the object of attentional focus . This suFrontal, temporal, and parietal areas, together with heteromodal sensory association regions, all contribute to a distributed network of cortical connections that subserve attention . AttentiEfforts in electrophysiology help refine the time course of selective attention in speech processing. Although attentional theories provide an analytical framework and anatomical studies identify key neurological structures involved in attentional processes, the question remains as to where within the auditory system attention begins to influence the encoding and subsequent processing of sensory inputs. Most studies have focused on the role of auditory cortex in selective attention ,87,88,89Studies evaluating attentional effects on cortical activation reveal attentional modulation of auditory cortical responses . Late auNaatanen  suggestsOther non-obligatory, event-related potentials have also been used to evaluate aspects of attentional processing including stimulus salience and novelty. These non-obligatory responses are elicited using special paradigms that include frequent, standard and rare, deviant stimuli that differ in acoustic characteristics . The mismatch negativity (MMN) is a response occurring around 150–250 ms following a deviant stimulus . It is aIn sum, unattended auditory inputs demonstrate less robust responses in early cortical measures than attended signals, and neural representations of ignored signals are notably reduced or absent in later, higher-order cortical responses ,97. SimiOn the other hand, some studies focusing on attentional modulation of brainstem responses suggest that corticofugal projections alter early neural encoding within the ascending auditory pathway and that the relay nuclei within the brainstem may serve as the early filtering mechanism ,37,73,99Attentional effects have been noted for both transient and sustained responses . As defined by the American Speech-Language-Hearing Association, APD is “a deficit in neural processing of auditory stimuli that is not due to higher order language, cognitive, or related factors” . IndividAssessments that identify unique weaknesses in specific auditory processing skills have greater potential to lead toward targeted interventions. In some areas of auditory clinical practice, individualized and client-centered treatments, together with appropriate counseling strategies, have resulted in greater adherence to recommended interventions, improved functional status, and enhanced patient satisfaction ,131. Cli


The dorsolateral prefrontal cortex (dlPFC) generates the mental representations that are the foundation of abstract thought, and provides top-down regulation of emotion through projections to the medial PFC and cingulate cortices. Physiological recordings from dlPFC Delay cells have shown that the generation of mental representations during working memory relies on NMDAR neurotransmission, with surprisingly little contribution from AMPAR. Systemic administration of low “antidepressant” doses of the NMDAR antagonist, ketamine, erodes these representations and reduces dlPFC Delay cell firing. In contrast to the dlPFC, V1 neuronal firing to visual stimuli depends on AMPAR, with much less contribution from NMDAR. Similarly, neurons in the dlPFC that respond to sensory events  rely on AMPAR, and systemic ketamine increases their firing. Insults to NMDAR transmission, and the impaired ability for dlPFC to generate mental representations, may contribute to cognitive deficits in schizophrenia, e.g., from genetic insults that weaken NMDAR transmission, or from blockade of NMDAR by kynurenic acid. Elevated levels of kynurenic acid in dlPFC may also contribute to cognitive deficits in other disorders with pronounced neuroinflammation , or peripheral infections where kynurenine can enter brain . Much less is known about NMDAR actions in the primate cingulate cortices. However, NMDAR neurotransmission appears to process the affective and visceral responses to pain and other aversive experiences mediated by the cingulate cortices, which may contribute to sustained alterations in mood state. We hypothesize that the very rapid, antidepressant effects of intranasal ketamine may involve the disruption of NMDAR-generated aversive mood states by the anterior and subgenual cingulate cortices, providing a “foot in the door” to allow the subsequent return of top-down regulation by higher PFC areas. Thus, the detrimental vs. therapeutic effects of NMDAR blockade may be circuit dependent. The recent discovery that the NMDA receptor (NMDAR) antagonist, ketamine, can produce rapid, antidepressant actions has stirred interest in the possible mechanisms underlying these therapeutic effects, and why blockade of NMDAR can produce such a swift change in mood. The current review discusses how NMDAR-calcium mechanisms are needed for sustained neural representations, e.g., such as the persistent representation of visual space in working memory by circuits in the dorsolateral prefrontal cortex (dlPFC), and suggests that parallel mechanisms in the cingulate circuits mediating mood and emotion may be overactivated in depression, and aided by NMDAR blockade , 2.GRIN2B gene expands across primate cortical evolution (NMDAR are heterotetramers composed of GluN1 and GluN2 (A-D) or GluN3 (A-B) subunits- usually with two GluN1 and two GluN2 subunits . Althougvolution , and acrvolution . The folThe paper will briefly review PFC circuits in primates and their regulation of the cingulate cortices, and then discuss the critical role of NMDAR for generating mental representations in dlPFC, the expansion in NMDAR-GluN2B transmission across the cortical hierarchy and across cortical evolution, and the role of NMDAR-GluN2B in the cingulate cortices mediating affective pain responses and depression. It will briefly discuss how stress exposure impairs higher PFC regulation, and will close with an exploration of the idea that ketamine's rapid antidepressant actions may involve blocking mental representations of aversive mood state in the cingulate cortices.The PFC greatly expands and differentiates over brain evolution, allowing representations of information in the absence of sensory stimulation. The primate PFC is topographically organized across multiple dimensions, e.g., with “simpler” representative functions found more caudally and more complex  more rostrally in the frontal pole , 13. TheThe more newly evolved, rostral and lateral areas of PFC provide top-down regulation of the more primitive medial and caudal areas. For example, the dlPFC can regulates emotion via direct projections to BA24 , 21, andAn important note about species differences: rodents do not have rostral PFC areas  or a dlPFC, and even the medial and orbital PFC areas they do have are much less developed and differentiated than those in primates . Indeed,The primate dlPFC has the remarkable ability to generate and sustain mental representations without sensory stimulation, the foundation of abstract thought . dlPFC “not extra-synaptic, consistent with their direct mediation of neurotransmission (Immunoelectron microscopy (immunoEM) showed that NMDAR-GluN2B are expressed exclusively within the post-synaptic density (PSD) in layer III dlPFC spines, and are smission . The abismission  and in Dsmission .2+) block within the NMDAR pore, permitting NMDAR actions  and subgenual cingulate (BA25) cortices mediate the emotional responses to pain . Hiiewed in , and mayiewed in . In contiewed in –83, thusiewed in –86 in adiewed in , 77, 78.iewed in . The ciniewed in .This state of weakened higher PFC circuits and stronger BA24/BA25/amygdala control of brain responding is codified by chronic stress, which induces spine loss and dendritic retraction in PFC neurons which correlate with impaired working memory and attention regulation –90. MuchThe loss of rostral PFC and dlPFC activity in concert with increased cingulate and amygdala activation would shift mental state from an outward, cognitively-engaged frame of mind to one focused inwardly on aversive experience. This is common in depression, where there is often loss of perspective, reduced empathy for others, anhedonia, and an urgent need for relief of mental anguish . SymptomWe have hypothesized that ketamine interrupts the self-perpetuating cycle of primitive circuit activity that is sustained by BA25 overactivity, allowing higher PFC circuits the opportunity to restore more normal functioning [Intranasal ketamine or esketamine administration may produce ultra-rapid antidepressant effects by delivering the drug directly to the anterior and subgenual cingulate cortices, which reside directly caudal to the nasal epithelium . Ultra-rin vivo (prior to spine re-emergence (Support for this hypothesis comes from a remarkable recent rodent study, where dendritic spine changes in medial PFC could be monitored in vivo . Prolongin vivo . Howevermergence , suggestIn closing, we are learning that NMDAR transmission is especially important for persistent neuronal firing. It is possible that the sustained neuronal activity underlying mood state, and particularly an aversive mental state, similarly relies on NMDAR transmission, and thus is relieved by NMDAR blockade from ketamine.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.AA and Yale University receive royalties from Shire/Takeda from the USA sales of Intuniv. They do not receive royalties from generic or nonUSA sales. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.