Real-time continuous monitoring of proteins in-vivo holds great potential for personalized medical applications. Unfortunately, a prominent knowledge gap exists in the fundamental biology regarding protein transfer and correlation between interstitial fluid and blood. Additionally, technological sensing will require affinity-based platforms that cannot be robustly protected in-vivo and will therefore be challenged in sensitivity, longevity, and fouling over multi-day to week timelines. Here we use electrochemical aptamer sensors as a model system to discuss further research necessary to achieve continuous protein sensing. Continuous monitoring or sensing of proteins.Biological challenges to continuous sensors for proteins.Biosensor sensitivity, longevity, and antifouling are areas for future research. It is, therefore, easy to envision that continuous monitoring of protein biomarkers could be revolutionary for the management of diseases such as heart disease, inflammatory bowel disease, diabetes, etc. Building on the successes seen in continuous glucose monitoring, the ideal protein biomarker is likely one which is present in interstitial fluid (ISF) at detectable concentrations, Furthermore, it is unclear how external stimuli from insertion of sensors into the body will affect protein transport between blood and ISF . Second, technologically, it is unknown if affinity-based sensing mechanisms such as redox-modified aptamers can accurately measure any type of protein in ISF over the multi-day to multi-week time scale needed for meaningful health status assessments. When deployed in biological fluids, if the sensors are to measure proteins, they cannot be robustly membrane protected like glucose sensors (or the protein targets would be filtered out). Additionally, the sensors will undergo multi-modal degradation based on interactions with small molecules and proteins. Therefore, it has yet to be determined if there is an effective method to protect sensing interfaces while still allowing affinity probes to specifically interact with protein biomarkers.However, the leap from small molecule monitoring to protein monitoring is quite large. There is a fundamental knowledge gap regarding both the biological and technological feasibility of continuous protein sensing. First, with respect to biology, it is yet to be confirmed with significant accuracy how large proteins  partition from blood to dermal ISF, leading to unknown correlations between protein levels in blood vs ISF. Even though aptamer sensors will be exclusively discussed, the problems they have are the same as any other affinity based platform which could be used . Therefore, the presented information can inform future research in all affinity-based platforms.The present perspective focuses on biological and technological concerns regarding continuous protein monitoring. The focus begins with the biology of protein monitoring and then moves into technological advances because without first establishing biological feasibility, continuous protein monitoring technology is not worth contemplation. Electrochemical aptamer sensors will be used as a model case study when discussing challenges to protein monitoring due to their previous in vivo demonstrations. saliva, sweat, and blood. While most of these biofluids can have niche instances of use for specific biomarkers, only blood and its next most similar biofluid ISF have a high potential for measuring proteins with clinical significance. ISF stands out further with potentially the most practical value because of its ease of access for continuous monitoring. ISF is readily available in the dermis and hypodermis of the skin where almost all analytes found in blood can also be found in ISF. Even though ISF presents an opportunity to continuously monitor proteins, several fundamental challenges ,ges Fig.  related The answers to these challenges will inform the technological design for continuous protein sensors and progress the field forward. Proteins smaller than albumin (∼66 kDa) can pass from blood into ISF unassisted through paracellular transport or assisted through transcellular transport. The size filtration of albumin from blood into ISF does cause albumin concentrations in ISF to be about 50% of blood concentrations. Therefore, proteins smaller than albumin see a similar or lesser decrease in relative concentrations in ISF. Proteins larger than the size of albumin do see a large concentration decrease in ISF due to their transcellular transport requirement. This concentration decrease could be below sensing threshold or highly variable and therefore poorly correlated with blood. Such concentration changes represent fundamentally important and yet unanswered questions.The first challenge concerns the ability of proteins to pass into ISF, how long the transport takes, and the dilution factor of the protein. Previous research has shown that most analytes (and therefore proteins) found in blood are also found in ISF; however, the size of proteins greatly determines their relative concentrations in ISF. This presents an opportunity for future research and sensor development because the best way to accurately measure lag times in ISF is through an indwelling sensor in the dermis or hypodermis, which should be more accurate in lag-time measurements than ISF extraction or microdialysis.The size exclusion from capillaries not only impacts ISF concentrations, but also the lag times between protein concentration spikes in blood and equilibration with ISF. Compared to relative analyte concentrations between blood and ISF, lag times for larger analytes have not been well studied. By being locally generated in ISF, the spike in concentration detected by an indwelling sensor could occur prior to the spike in blood, or vice versa if the infection or injury originates elsewhere in the body than the measurement site. Confounding factors related to proteins which are locally generated need to be considered during sensor development and in down-selecting potential clinical use cases.One challenge to correlating ISF protein concentrations to blood concentrations is that some proteins are produced locally in dermal ISF, but then circulated in the blood to other organs. Interleukin-6 (IL-6) represents an excellent example of local generation impacting blood correlations. IL-6 is synthesized in the local tissue environment in response to infection or tissue injury where it then enters the blood. Each layer contains unique cells and mechanical properties which will impact protein movement and transport throughout. The epidermis contains tightly packed cells connected through tight, gap, and adherens junctions to create an impermeable barrier to large solutes. The tight connections also contribute to a low volume of ISF available in this layer. The dermis is located directly beneath the epidermis and is a moderately vascularized matrix of collagen with a low cell density. Therefore, the vascularization and low cellular count of the dermis could make it an ideal location for continuous protein monitoring. The hypodermis contrasts the dermis with a highly dense cellular content of adipose cells and low ISF content; therefore, it is possible to speculate that the hypodermis could be inferior to the dermis. However, this all depends on the target to be measured, and if the measurement goal is not just blood correlation but rather tissue penetration of a protein. In the latter case, the hypodermis could be the preferred measurement location.The most accessible location for ISF sensing is in the skin which is composed of three layers: epidermis, dermis, and hypodermis. A similar situation occurs with brain natriuretic peptide (BNP) and adipose tissue where adipocytes have numerous receptors on their surface for BNP clearance. These results highlight the fact that protein transport and equilibration into ISF is a dynamic process with a multitude of factors which can impact protein concentrations in ISF and correlations between ISF and plasma.After arrival into ISF and dispersion throughout, many proteins are chemically altered or captured by receptors on cells for use and signaling. As a beautiful example, a study conducted by Yang and colleagues compared two similar proteins, inulin and insulin, and each protein’s transport from blood into ISF and lymph. Their results showed that although inulin reached an equilibrated concentration between ISF, plasma, and lymph, insulin did not reach the same equilibration. Instead, insulin concentrations were consistently lower in ISF and lymph as compared to plasma, showing insulin’s ability for uptake into cells. Additionally, many protein concentrations change throughout the day. For example, C-reactive protein has been shown to maintain stable concentrations in healthy individuals diurnally, whereas insulin varies based on time of day and circadian rhythm. Pharmaceutical drugs can have a broad array of effects on protein concentrations from impacting vascular perfusion and capillary exchange to modulating specific protein production. These are just a few factors which can ultimately have a large impact on a sensor’s ability to correlate relative concentrations in ISF with blood.Another challenge related to continuous protein monitoring concerns how factors external to fundamental capillary exchange can impact concentrations in ISF. How do factors such as skin temperature, inflammation, blood pressure, diurnal changes, and pharmaceutical drugs impact capillary exchange and therefore protein concentrations in ISF? Each factor may impact protein concentrations differently and needs to be considered both independently and collectively when interpreting protein correlations in ISF. Temperature, inflammation, and blood pressure are directly related with vasodilation/vasoconstriction and can therefore impact skin blood flow, and in the case of inflammation also modulate vascular permeability.We believe two factors will be the greatest obstacles to feasibility: variable dilution during capillary filtration and cellular uptake in the sensing environment. Determining unknown dilution factors will significantly add to the research required because correlations between blood and ISF will need to be determined individually. Additionally, protein uptake by cells impacts concentrations available for a continuous sensor to detect. Overall, we believe the greatest biological challenge will be one which variably reduces concentrations available for sensing.A final question, at least in the limited breadth of this perspective piece, concerning biological challenges for continuous protein monitoring in ISF is what if the summation of the described obstacles above cannot be overcome with technology? The next best biofluid containing proteins of interest would be whole blood, so could a sensor be placed in the bloodstream? Continuous sensing in blood solves the main problems of correlating concentrations and equilibration lag times but presents several new challenges concerning technological feasibility of a blood-based sensor. Sensor design would become a higher concern in blood to not disrupt blood flow, elicit an immune response, induce clotting, introduce infectious pathogens, or maintain stable positioning. Additionally, comfort and ease of use could be a concern in a commercialized product for patients. This being said, commercial catheters for patients who need regular dialysis exist. However, while this question currently does not have one answer, it should still be considered as a potential option if technology cannot accommodate the requirements for ISF sensing. current aptamer sensors suffer from sensitivity, longevity, and fouling concerns inhibiting their progression towards continuous protein sensing. Aptamer sensors will therefore be used as a model case study to discuss continuous in vivo protein monitoring, but each challenge persists across all affinity-based biosensors.Current sensing modalities are dominantly based on one of two fundamental mechanisms: affinity-based and reactivity-based interactions. Proteins will in almost all cases require affinity-based sensors because even if an enzyme could be developed that would react with a protein and create a detectable byproduct, the concentrations of proteins are typically so low and their diffusion rates to such a sensor would be so slow that limit of detection could become an insurmountable challenge. The most widely in-vivo demonstrated class of affinity biosensor is aptamer sensors, and more specifically, only redox-tagged aptamer sensors being shown to work in-vivo . Despite compelling initial demonstrations, For example, Somalogic has an aptamer for IL-6 with a binding affinity of 200 pM.Aptamers selected for proteins via the SELEX process can often have very low limits of detection due to increased binding affinity (Kd) between aptamer and protein. Simply, proteins are larger and more structurally and chemically complex than small molecules, allowing a greater degree of binding interactions with an aptamer. Commercially, companies such as Somalogic have further advanced binding affinity through chemically modified aptamers against >6000 proteins. sensitivity remains a bigger challenge for protein aptamer sensors. Unlike small molecule aptamers which show changes in redox tag current (sensitivity) over 100’s of percent throughout the detection range, aptamer sensors for proteins have been more limited with typical ranges of 10’s of percent. A portion of the challenge is that for small molecules many aptamers bind and form a linked stem geometry resulting in a strong redox current change between the electrode and redox-tagged aptamer. Proteins are often so large and have many aptamer-protein binding interactions that a powerful stem-loop or similar aptamer sensor architectures are typically not found in as-selected aptamers for proteins. Thus, further research is needed to optimize such aptamers for high sensitivity protein monitoring.Although binding affinities for proteins appear promising, Historically, aptamer sensor monolayers on gold electrodes have remained stable in simple buffers like PBS for 48 h under continuous voltammetric interrogation, but once deployed in vivo, sensors often degrade quickly over time spans of 6–12 h. Recent work has been performed to identify the driving mechanisms of degradation. Leung et al. reported that electrochemically driven desorption, and fouling of blood components are the primary sources of sensor degradation. Clark and Pellitero recently showed this degradation is a multi-modal process consisting of voltage induced desorption at low voltages (<−0.2 V), and competitive displacement from thiolated small molecules (i.e. L-cysteine) commonly found in biofluids. Scanning sensors at negative potentials is one major source of sensor degradation caused by: the reduction of oxygen into reactive oxygen species, thereby catalyzing the loss of monolayer elements from the sensor surface, the phosphate backbone of DNA being strained away from the electrode surface causing electrochemical desorption of aptamer, and increased fouling from attracted proteins. Researchers recently reported no loss in DNA signal following continuous interrogation in the voltage window of −0.2 V to 0.2 V vs Ag|AgCl. While promising, this voltage range is outside the reduction potential of methylene blue, the benchmark redox tag. Recent work in the field led to the development of novel redox tags including, a tetrathiafulvalene based reporter (E°’ = 0.05 V vs Ag|AgCl), and an osmium-based redox reporter(E°’ = 0.18 V vs Ag|AgCl).Unlike most small molecule analyte targets, most proteins of interest change not in minutes but over hours or days; therefore, in practice, proteins require a sensor capable of operating days as opposed to hours to detect diagnostically relevant information. However, while sensors were able to stay intact, sensor responsivity suffered unless a protective membrane was added to prevent fouling, a sensor membrane so tight it would preclude protein monitoring, setting up the next topic of antifouling.While these results are promising, further work must be done to progress the field towards longer lasting gold electrode based sensors. A remaining issue is the inherent thermal stability of the monolayer of aptamers and protective molecules such as alkythiolates. Recent work from Watkins and Karajic showed that continuous voltametric scanning along with increasing alkyl chain length from the benchmark 6-mercapto-1-hexanol (MCH) to 8-mercapto-1-octanol (MCO) increases stability in biofluid conditions for over one week. A more challenging task is preventing fouling, which is a bigger challenge for protein monitoring because to monitor the protein, you must also allow common foulants such as albumin to reach the sensor surface  consideration of optical-based aptamer approaches which can alleviate the need to have the aptamer so immediately close to the substrate it is bound to and allow improved interaction with the protein .We hypothesize an ideal protein biosensor would contain a zwitterionic monolayer to aid in biocompatibility and reduce fouling at the sensor interface with a size exclusion membrane to selectively differentiate between proteins of interest, and non-specific foulant interactions. To combat the muted signal gain obtained during protein sensing two things may be helpful: (1) introducing chemically-modified DNAAffinity based sensors provide platforms capable of continuous protein monitoring to advance the field of personalized medicine. However, before technological advances can be made, fundamental questions in biology must be addressed which determine available concentrations and correlative value for detection. Once biological concerns are addressed, increasing sensor longevity and inhibiting fouling in biofluids without the use of membranes will be crucial for long-term sensing. Furthermore, maintaining high sensitivity of the recognition element in complex environments will be necessary to obtain accurate multi-day measurements. Nevertheless, continuous protein monitoring has the potential to revolutionize personalized medicine, changing the lives of millions suffering from chronic diseases all over the world.


As a leading cause of mortality and morbidity, stroke and its management have been studied extensively. Despite numerous pre-clinical studies identifying therapeutic targets, development of effective, specific pharmacotherapeutics remain limited. One significant limitation is a break in the translational pipeline – promising pre-clinical results have not always proven replicable in the clinic. Recent developments in virtual reality technology might help generate a better understanding of injury and recovery across the whole research pipeline in search of optimal stroke management. Here, we review the technologies that can be applied both clinically and pre-clinically to stroke research. We discuss how virtual reality technology is used to quantify clinical outcomes in other neurological conditions that have potential to be applied in stroke research. We also review current uses in stroke rehabilitation and suggest how immersive programmes would better facilitate the quantification of stroke injury severity and patient recovery comparable to pre-clinical study design. By generating continuous, standardised and quantifiable data from injury onset to rehabilitation, we propose that by paralleling pre-clinical outcomes, we can apply a better reverse-translational strategy and apply this understanding to animal studies. We hypothesise this combination of translational research strategies may improve the reliability of pre-clinical research outcomes and culminate in real-life translation of stroke management regimens and medications. Stroke is a pathological condition whereby brain damage occurs due to insufficient blood supply caused by either a blockage in the cerebrovasculature or a rupture and haemorrhage into the brain tissue. In 2019, stroke was the second leading cause of disability-adjusted life-years (DALYs) for both the 50–74 and 75+ years of age groups , such as alteplase , a protoDespite the discovery of many potential therapeutic targets, such as N-methyl-D-aspartate (NMDA) receptor antagonists , interleImproving the translatability of drugs requires an improvement to the drug discovery pipeline both in pre-clinical studies and interpretation of clinical outcomes. Many attempts to refine the pre-clinical pipeline have already been made and reviewed extensively . Here, wVR technology facilitates the simulation of an alternative environment either through immersive or non-immersive means, allowing the user to perceive and interact with a reality that may include features or objects absent from their physical environment. Clinical application of this technology has previously shown success in the treatment and quantification of parameters in neurological diseases. Here, we look at the possibility of using VR for the quantification of post-stroke assessment of human patients to measure stroke severity and the therapeutic efficacy of drugs. We hypothesise that these data can be used in a reverse translation approach to refine pre-clinical studies and improve the effort to develop novel, clinically relevant stroke therapeutics.The authors searched for current literature based on a set of search terms: ‘translational medicine’, ‘reverse-translational approaches’, ‘virtual reality’, ‘stroke modelling’ and ‘stroke rehabilitation’ in the context of neuroscience research. Searches were conducted up until October 2022 on PubMed MEDLINE. Case reports and opinion pieces were excluded. When referring to clinical guidelines, the authors utilised the National Institute of Clinical Excellence (NICE) guidelines , 2019.Failures in the translational pipeline have been reviewed extensively elsewhere, however for stroke in particular, current clinical practice does not parallel with the pre-clinical strategy of measuring stroke severity and outcome. Changes to practice in both the laboratory and the clinic can be made to increase the likelihood that data collected from both models and patients are better comparable. To measure stroke severity pre-clinically, both ischaemic and haemorrhagic stroke models tend to measure cerebral histological changes such as infarct volume and peri-haematomal area respectively, and conduct motor tests to measure spatial awareness, memory and fine motor skills. However, motor tests are not always consistently carried out between studies, and some do not have parallels between mice and rats. Contrastingly, discrete classification in clinical testing such as the Glasgow Coma Scale (GCS), National Institute of Health Stroke Scale/Score (NIHSS) and modified Rankin Scale (mRS) is less sensitive to changes during stroke recovery. In addition, such tests are mainly behaviour-focussed assessments that measure multiple aspects of a post-stroke deficit such as language, gross and fine motor response, and ability to carry out Activities of Daily Living (ADLs), and so are unable to be measured equivalently in rodent models. The difference between pre-clinical assays and clinical tests means that comparing outputs across species is complex and not always indicative of a drug’s therapeutic power, contributing to the stall in drug discovery.The need for novel therapies for stroke is compounded by the increasing incidence of risk factors in the general population, such as ageing and hypertension . HoweverIn the following sections, we will discuss how implementing VR into quantifying patient outcomes to reverse translate to pre-clinical studies may help bridge the translational gap, with the potential to increase the proportion of successful stroke therapies entering development.Common rodent models of ischaemic stroke and haemorrhagic stroke reflect the human condition to varying extents and tend not to mimic clinical populations where patients exhibit comorbidities  includinClinically, assessments of post-stroke neurological abilities focus on physical or cognitive deficit. Some of the more commonly used clinical tests, such as the GCS, NIHSS and the mRS, output a single number based on the assessment of both physical and cognitive performance post-stroke. These assessments have low sensitivity and rely on patient interpretation of their own performance and abilities. When compared to pre-clinical assays, there are few direct parallels. One example of such a translatable test is the Tübingen–Boston Rat Coma Scale (RCS) developed by We hypothesise that by increasing the sensitivity in clinical assessments, and by interrogating outcomes similar to those in pre-clinical assays, we can generate more comparable data across the pipeline. One such strategy to develop more translatable measures after stroke may be to employ VR technology to quantify and standardise measurements of patient performance  to draw Specifically in stroke research, thus far the use of VR has been predominantly rehabilitative rather than assessment-focussed, with a particular focus on upper limb rehabilitation. A Cochrane review analysing the use of VR in the post-stroke rehabilitation of the upper limb found that it was not a more effective treatment option compared to standard care, and that the studies included were classed as low/moderate quality of evidence based on the GRADE system . AlthougThe BTS–Nirvana VR device is an example of a minimally intrusive sensor used for rehabilitative purposes which utilises optoelectronic infrared sensors  to monitIn addition to motor rehabilitation, Regarding the treatment of wider neurological conditions, VR was found to be more effective than standard care in treating depression, anxiety, pain and fatigue . One exaSimilarly, VR has been used in physical therapy of paediatric patients suffering from burns to reduce subjective pain associated with burn rehabilitation therapy . This VRBased on the methodology and results of the rehabilitation methods described above, it can be proposed that similar mechanisms can be used to quantify memory, unilateral spatial neglect (USN), coordination and motor function to use as analogues to pre-clinical behavioural tests that focus on assessing corresponding deficits occurring after a strokeA study with simple applications was conducted by Similar programmes have been developed for neurological assessment. For example, Choi et al. developed a 2D-screen mobile game-based VR tool to aid with rehabilitation of the upper limb , which cRecently, a case–control pilot study was conducted by Zúñiga et al. (2021) assessing post-stroke motor function of the trunk and upper limb using a 3D avatar of the body to monitor movement without the attachment of markers to the subject, which showed that the VR quantification was more sensitive than clinical scales and found a correlation between the mRS and degree of body sway. Another example of VR application providing useful quantification of functional outcomes is the work of Crucially, adaptations to pre-existing VR programmes must closely follow pre-clinical behavioural tests without losing ecological validity or the ‘game’ aspect that promotes gratification, motivation and engagement. An example of this may include developing a simulated task specifically tailored to spatial memory and locomotor function resembling the Morris Water Maze (MWM), or memory and anxiety, such as the NOR with the programme being set in a real-life situation, such as a simulation of an ADL, similar to Reh@City . There aA key limitation of using VR clinically relates to the scalability which may prove to be hindered by access to reliable supportive technology including monitors, a reliable source of electricity and the cost of equipment. An estimate of cost for one VR MWM with full equipment is 5290 USD . SimilarAnother aspect to consider regarding the reverse-translatability of outputs is the standardisation of sensitivity between different softwares and equipment, as this could alter the assimilated results reaching pre-clinical trials. This is a problem currently present in forward translation where the loss of standardised pre-clinical tests affects the translation of drugs from animal to human subjects. Therefore, in order for reverse translation to objectively target this shortcoming and bridge the translational gap, this must be addressed before the use of VR outputs in stroke research.The pre-clinical focus remains on finding biological targets rather than assessing the feasibility of targeting them using therapeutics causing a lower clinically relevant output despite increased research funding . An examHere, we have discussed the hypothesis that adaptations to the translational pipeline may increase sensitivity in translation between animal and clinical data and ultimately advantage the development of stroke therapeutics that has remained stagnant for so long. VR has shown promise as a method for reverse translation in stroke research, particularly for USN and upper limb function for which VR-based scores have correlated with clinical assessment scores. With modification of programmes already in use and availability of clinical data sets from rehabilitation, VR may allow for the reproducible quantification of multiple post-stroke assessments ranging from motor to cognitive function to parallel the pre-clinical investigation. To validate the comparability of clinical data generated using VR in this manner, quality randomised control trials with large sample sizes using VR to assess multiple post-stroke deficits are required. The results of this would then produce evidence on the strength of the correlation between patients and pre-clinical models, commenting on the translational integrity of this approach. Ideally, this approach would be repeated for testing the validity in multiple cognitive domains, such as motor function, memory and spatial awareness. This strategy has the potential to provide a more robust comparison between pre-clinical models and patients with equivalent quantitative data opposed to current patient stroke scales assessing individual cognition.


Over the past 15 years, there has been great interest in the potential to repurpose the diabetes drug, metformin, as a cancer treatment. However, despite considerable efforts being made to investigate its efficacy in a number of large randomised clinical trials in different tumour types, results have been disappointing to date. This perspective article summarises how interest initially developed in the oncological potential of metformin and the diverse clinical programme of work to date including our contribution to establishing the intra-tumoral pharmacodynamic effects of metformin in the clinic. We also discuss the lessons that can be learnt from this experience and whether a further clinical investigation of metformin in cancer is warranted. A drug is described as being ‘repurposed’ when it exhibits clinical benefit for the treatment of cancer patients, despite being initially developed for an unrelated indication. As drug development costs rise for new entities, there is a growing attraction in looking to off-patent medicines that have established safety and pharmacokinetic profiles potentially reducing the time to entry into the oncology clinic. Indeed, a recent estimate suggested that the median cost to develop a single cancer drug is $648 million , althougMetformin is the most widely prescribed medicine for type 2 diabetes worldwide and on the World Health Organisation’s list of essential medicines. A series of epidemiological studies which suggested that metformin may reduce cancer incidence in diabetic populations sparked great interest in its potential as a cancer treatment. However, there remains some debate as to its pharmacodynamic effects in tumours and recent randomised trials have not clearly demonstrated clinical benefit for any cancer indication to date. In this perspective article, we present a summary of the history of preclinical and clinical studies that informed the repurposing of metformin, discuss how this programme of work could have been better focused and coordinated and lastly describe oncological indications where there remains a strong rationale for investigation.As has typically been the case drug repurposing programmes, the interest in metformin was serendipitous. Two decades ago, researchers trying to understand the metabolic effects of metformin in diabetic patients discovered that it inhibits Complex 1 of the mitochondrial respiratory transport chain, the consequence of which was the activation of the cellular regulator of energy homoeostasis, AMP-activated protein kinase (AMPK) , 4. AMPKSaccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In the same study the administration of metformin to mice inhibited the growth of control xenografts but not those expressing NDI1 [Despite great effort and multiple published preclinical studies, the actual mechanism of action of metformin in tumour cells remains a topic of debate. As described above it is clear, at least when cells are treated with high doses, that metformin inhibits Complex-1 activity and cellular respiration. This was demonstrated in a series of elegant experiments in which metformin-resistant ing NDI1 . In modeing NDI1 . Howevering NDI1 –10. Metfing NDI1 .However, an alternative hypothesis focussed on metformin’s effects on systemic ‘host’ metabolism has been proposed. Within the liver, activation of AMPK has been shown to reduce gluconeogenic gene expression in hepatocytes , increasIn the clinic, metformin’s anti-cancer effects were initially evaluated in several small pharmacodynamic clinical trials. These typically used ‘window of opportunity’ designs often prior to surgery and compared assays using the diagnostic and surgical tumour sample with a course of metformin in between to assess the drug’s effects on cancer biology. The endpoints and findings of these studies are described in Table Results have now been presented from a number of randomised trials in different settings which in most cases have not demonstrated clinical benefit for metformin as a cancer treatment. These studies have assessed the combination of metformin with chemotherapy, endocrine and other targeted therapy in a number of different tumour types as summarised in Table When the MA.32 study commenced recruitment in 2010 there was very limited information regarding the pharmacodynamic effects of metformin in breast cancer at therapeutic doses from the clinic. This impacted on the ability to develop rationale clinical trial designs that took into account markers of selection, resistance, treatment combination and dosing. As has been frequently been demonstrated in drug development without appropriate patient selection clinical benefit may be masked.Initially, there was little effort to establish the tolerability and possible advantage of higher dose levels of metformin in the context of treatment for cancer where a greater risk/side effect profile might be acceptable. Efforts have now been made to evaluate different dose levels for metformin and its biguanide cousin phenformin in various therapeutic combinations –31. HoweAn example of a well-structured programme of work that could be taken as template to repurpose an anti-mitochondrial agent for cancer therapy is the ongoing evaluation of the anti-parasitic drug atorvaquone as a radio-sensitiser. A decade ago, a group of investigators carried out a high throughput screen for drugs that reduced oxygen consumption and hence, potentially tumour hypoxia. Atovaquone is an anti-malarial agent and ubiquinone analogue that inhibits mitochondrial complex III and was identified as a ‘top hit’ in this screen. In vivo, atovaquone reduced tumour hypoxia and sensitised xenograft models to radiotherapy . To deteThe contrast here with the approach to metformin is clear. The work was led by a team of collaborators who worked together throughout each stage of the drug development project to ensure that each step was informed by the prior. At an early point during clinical evaluation detailed pharmacodynamic assessment of the drug was carried out at several dose levels. We believe this stepwise approach to pharmacodynamic characterisation prior to Phase II/III efficacy trials optimises the chances of success in a drug repurposing programme.Given the results of the randomised efficacy trials enthusiasm to develop further clinical studies of metformin as a treatment for established cancers is waning. However, preclinical and clinical pharmacodynamic data obtained since the design of early clinical efficacy studies has now informed new avenues of investigation.Markers are now being established that may define response to Complex-1 inhibitors such as mutations in the SWI-SNF complex . MitochoA number of animal and human studies have shown that metformin can alter the metabolism of gut microbiota , 40. TraMetformin, by inhibiting oxidative respiration and hence oxygen consumption has been shown to reduce hypoxia in tumour models  and moreMetformin and its role in cancer prevention is an area that has been underexplored in prospective studies. Indeed, the epidemiological data provide a strong rationale for testing this hypothesis in selected groups of patients, for example, obese or insulin-resistant individuals and now early clinical trial data is emerging in support. One clinical study investigated metformin’s potential utility in preventing tamoxifen-induced endometrial hyperplasia showing reduced endometrial thickness on transvaginal ultrasound for metformin-treated patients compared to the placebo control group . MetformTP53 tumour suppressor gene [TP53, metformin increases their cancer-free survival [mTP53 carriers. On this basis, randomised clinical trials are now moving forward to evaluate whether metformin can reduce cancer incidence in this high-risk population.However, prevention studies designed to identify differences in cancer incidence are notoriously difficult to execute given the numbers of patients needed to properly power such a trial and the length of time it takes to complete adequate follow-up. However, opportunity lies in investigating the potential of metformin as cancer preventative for patients with cancer predisposition syndromes which will allow for smaller studies and shorter follow-up. For example, Li-Fraumeni syndrome (LFS) is a rare inherited cancer predisposition syndrome with a lifetime risk of cancer close to 100% by age 60 years in women and 73% in men. LFS is caused by germline pathogenic variants in the survival , 55. ThiIn summary, outcomes from late-phase efficacy studies testing metformin as a repurposed cancer therapeutic have been disappointing. In a rush to establish its potential utility, such trials were designed prior to due diligence with regard to patient selection, mechanism of action and appropriate combination. New avenues of investigation in selected populations including the assessment of combination with immunotherapy, and potential as a cancer preventative agent still warrant well-designed clinical investigation.


Care for the critically ill patients is often considered synonymous with a hospital having an intensive care unit. However, a focus on Essential Emergency and Critical Care (EECC) may obviate the need for much intensive care. Severe COVID-19 presented a specific critical care challenge while also being an exemplar of critical illness in general. Our multidisciplinary team conducted research in Kenya and Tanzania on hospitals’ ability to provide EECC as the COVID-19 pandemic unfolded. Important basic inputs were often lacking, especially sufficient numbers of skilled health workers. However, we learnt that higher scores on resource readiness scales were often misleading, as resources were often insufficient or not functional in all the clinical areas they are needed. By following patient journeys, through interviews and group discussions, we revealed gaps in timeliness, continuity and delivery of care. Generic challenges in transitions between departments were identified in the receipt of critically ill patients, the ability to sustain monitoring and treatment and preparation for any subsequent transition. While the global response to COVID-19 focused initially on providing technologies and training, first ventilators and later oxygen, organisational and procedural challenges seemed largely ignored. Yet, they may even be exacerbated by new technologies. Efforts to improve care for the critically ill patients, which is a complex process, must include a whole system and whole facility view spanning all areas of patients’ care and their transitions and not be focused on a single location providing ‘critical care’. We propose a five-part strategy to support the system changes needed. At the onset of the COVID-19 pandemic critical care was too often narrowly conceptuallised as comprising skilled, technologically supported care delivered in specific hospital locations, as a result the response initially paid insufficient attention to Essential Emergency and Critical Care that is needed in all facilities and across all clinical locations in larger facilitiesEnsuring Essential Emergency and Critical Care is available in all clinical settings requires a change in mindset of managers, who must prioritise essential forms of care, and requires that all healthcare providers and teams develop a form of systemic vigilance and response that becomes central to their work.Achieving such system change will, we propose, require a systems response that we articulate as five key strategies to generate alignment of different actors' efforts combined with sustained, skilled leadership and managementIn the early months of the COVID-19 pandemic, as surges in caseloads of critically ill patients caused by SARS-CoV2 infection threatened to overwhelm high-income country hospitals, a multidisciplinary, collaborative team of researchers was assembled. The team hoped to examine the provision of care for critical illness in Kenya and Tanzania in the face of major concerns about the consequences of fundamental gaps in capacity and care quality in less resourced countries’ hospitals. These had previously been highlighted in areas such as maternal, neonatal and child health, general adult and critical and perioperative care.1–6Given this backdrop, the team was specifically interested to learn lessons of relevance to all hospitals to guide those demanding improvements in care for emergencies and the seriously ill with a particular focus on ‘Essential Emergency and Critical Care’ (EECC).8Our studies in Kenya and Tanzania conducted in late 2020 and in 2021 found that hospitals were generally poorly prepared to cater to the basic needs of those with critical illness.11EECC requires that all staff are able to identify critical illness and provide the most basic immediate care that a critically ill patient would need. In Tanzania, personnel with any relevant training were available in the facilities on average less than 50% of the time.11 14Effective management of critical illness including COVID-19 requires quality care sustained over hours or days. This includes periods spent in emergency departments, during transfers, during waits in diagnostic departments for investigations and sometimes prolonged periods spent in wards. The quality of continuous care, often reliant on nurses, has been much less well examined in low-resource settings, although some prior work suggests inadequacies.9Combining insights from patient journeys, formal interviews and direct observations allowed us to develop a broader conceptual understanding of the process challenges to delivery of quality EECC in Kenyan and Tanzanian hospitals. We propose it is helpful to think of three distinct areas of concern in any hospital location; how it receives, sustains and transfers any patient with critical illness. The receipt of patients into a facility or department was rarely well structured. For example, systems to identify that a patient waiting in a queue has probable sepsis and low blood pressure or other features of incipient or manifest critical illness were absent. Frequently, establishing the patient’s ability to pay was prioritised above such triage. Maintaining vigilance (monitoring and reassessment) even by assessing basic vital signs or monitoring ongoing treatment  was often lacking. Worryingly, this was apparent during extended stays in wards as well as during shorter periods in emergency or other hospital departments. Although this often reflects staff shortages, functional systems for prioritising patients so that time is devoted to reassessment and care of the most ill were rare. Lastly, the flow of patients within and between departments was often problematic and reliant on family members if present. It could lead to long periods within a facility where critically ill patients could deteriorate while waiting to move to the next stage of care.Concerned by the catastrophic potential of COVID-19, countries had to act. With rich countries’ ICUs flooded with patients early in the pandemic, initial global attention focused on how to rapidly scale up advanced respiratory support in low-resource settings.20Recognition that attention had to be paid to apparently basic forms of care for critically ill patients with COVID-19 rather than advanced respiratory support was slow. In our facility surveys, sometimes many months after the onset of the pandemic, access to oxygen remained low.7 25Our team also examined the potential cost-effectiveness of improving care for critical illness in low-resource settings and specifically improving coverage of EECC. These studies suggest that EECC can be both low cost-effective and highly cost-effective. In Tanzania and Kenya, the cost of current advanced critical care conducted in ICUs with specialised staff, facilities and technologies is nearly ten times than that of EECC.30Our findings highlight the foundational quality challenge of delivering the right care, at the right time to the right individual across all parts of hospitals at scale.In summary, our multidisciplinary team found that hospitals in Kenya and Tanzania, as in other low-resource settings, may have some of the essential resources they need to offer EECC. Although each study has limitations, and even a set of studies cannot claim to be a full and accurate representation of complex systems, taken together our work indicates that we need to go beyond considering EECC as a package of life-saving interventions and think of it as a continuous process. Considered this way, EECC is almost a state of mind requiring a form of systemic vigilance and response, while ensuring the care that is most needed is prioritised first. High-income countries have invested over decades in personnel and their skills, improvements in tools and organisational processes. In some places, this even includes dedicated ‘floor level’ management to promote timely action. These system responses are almost completely absent in Kenya and Tanzania. They must be carefully developed alongside efforts to tackle basic infrastructure and resource gaps. Addressing only the problem of technical inputs by offering training and technologies without addressing system challenges seems little more than building sandcastles on a beach and ignoring the reality of the incoming tide.Making wise investments should be a priority of countries and the global community. Reactive responses in the first months of the pandemic resulting in a focus to supply advanced equipment for critical illness were often inappropriate.


Over the past two decades, laser additive manufacturing technology has evolved rapidly and has been applied in many industrial sectors. However, traditional manufacturing methods such as casting, forging, rolling, and welding are still prevalent around the world . To meetSignificant technical advances have been made in the field of metal powder-based additive manufacturing, and a range of high-performance materials have been successfully developed ,3. HowevThe current Special Issue entitled “Recent Advances in Metal Powder-Based Additive Manufacturing” collects the recent research outcomes addressing the main challenges and aiming to provide possible solutions that may revolutionize the metal powder-based additive manufacturing technology and its applications. It is believed that this Special Issue will provide an innovation platform for researchers in this area to communicate and disseminate their most recent ideas and achievements which will facilitate and support scientists and engineers to continuously make contributions to the field.


Socioeconomic disadvantage is one of the most important predictors of morbidity and premature mortality . ExposurWe used data from 2475 men and 2834 women (aged 35–75 years (mean 52)) in the Swiss population-based cohort CoLaus|PsyCoLaus to test if life course socioeconomic conditions were related to cohort members’ Pace of Aging [Our findings converge with those of the Dunedin Study investigators, who found that lower parental occupation predicted a faster Pace of Aging in a sample of younger adults . Among oSocioeconomic inequalities in the Pace of Aging could be attributed to multiple mechanisms that accumulate across the life course, including differences in access to health care and health-promoting leisure activities, inadequate nutrition, psychosocial stress, and exposure to environmental hazards, as well as direct biological processes relevant to health and longevity. Intervention to promote upward socioeconomic mobility, as well as addressing associated health behaviors, may help slow the pace of aging and increase healthy lifespan for socioeconomically disadvantaged individuals. Interventions to slow aging in at risk individuals may be most effective when delivered by midlife, before aging-related diseases become established.


Plants face a constantly changing environment, requiring fine tuning of their growth and development. Plants have therefore developed numerous mechanisms to cope with environmental stress conditions. One striking example is root response to water deficit. Upon drought (which causes osmotic stress to cells), plants can among other responses alter locally their root system architecture (hydropatterning) or orientate their root growth to optimize water uptake (hydrotropism). They can also modify their hydraulic properties, metabolism and development coordinately at the whole root and plant levels. Upstream of these developmental and physiological changes, plant roots must perceive and transduce signals for water availability. Here, we review current knowledge on plant osmotic perception and discuss how long distance signaling can play a role in signal integration, leading to the great phenotypic plasticity of roots and plant development. Like some prokaryotic and most fungal cells, plant cells are wall-encapsulated. Although it provides important advantages such as a robust exoskeleton and a protection of cells from adverse abiotic or biotic factors, the wall creates a direct restriction for cellular expansion. This important constraint can be overcome by intracellular turgor that in response to cell wall loosening allows growth. The cell turgor, that can be up to 10 bars in certain cell types, is actually built from the osmotic gradient between cell interior and the external media. This gradient triggers a flux of water into the cell that leads to a compensatory hydrostatic pressure, called turgor. Any change in the osmotic gradient by for instance an increase/decrease in water potential in the external media or changes in the internal solute concentration, leads to a direct change in turgor that can modify cell volume and tissue rigidity. To allow localized expansion growth or prevent wilting, cells have therefore to maintain a constant dialog between cell wall mechanical properties, solute concentration and turgor.Whereas plant aerial parts are protected from air drying by specialized waxes deposited on their surface, the root system is in direct contact with its surrounding environment and therefore has to cope with dramatic changes in water potential. Soil is a complex porous medium with marked differences in its composition and structure e.g. air pockets and hydrated soil particles. Thus, root systems are continuously facing contrasted water availabilities during soil exploration. In addition to a local perception of water availability, followed by signal transduction and responses at cellular level, long-distance signals are produced. These so-called systemic signals coordinate responses at the multicellular scale, to modulate integrative traits, such as root system architecture (RSA).In this review, we will focus on recent advances in the field of plant osmotic perception and early signaling in roots. We will also underline how systemic signaling can integrate local signals to modulate RSA.Although being searched for many years, the exact nature of the signals that allow plants to perceive changes in water availability is not clearly defined yet. At the cellular level, mechanics e.g. cell wall/membrane tension, but also cell or subcellular volumes are directly impacted by changes in external osmolarity Fig. A and B. Saccharomyces cerevisiae, perception is made by a two-component histidine kinase system, where recognition of the stimulus leads to kinase activation and phosphorylation of a histidine residue  with members involved in hormonal signaling such as ethylene  association with lipids is an important determinant for the stabilization of the closed conformation and therefore plays a key role in channel sensitivity to tension  gene family represent the closest homologs of bacterial MscS  was cloned by complementing the lethal mutation of Physcomitrella patens genome are localized in the tonoplast and control cell growth and cytoplasmic calcium oscillation  was therefore identified from an EMS mutagenized population. OSCA1.1 encodes for a hyperosmolality-gated calcium-permeable channel, which is responsible for calcium signaling, but also regulation of root growth, stomata closure and transpiration upon osmotic stimulation , a close homolog of OSCA1, is also responsible for osmotic-dependent calcium influx when expressed in Chinese hamster ovary (CHO) cells -dependent stomatal closure , Wall Associated Kinases (WAK), Lectin Receptor-Kinase (LRK), Proline-rich Extensin-like Receptor Kinases (PERKs), and formins have been shown to interact with the cell wall  CrRLK was isolated from a genetic screen for suppression of the short hypocotyl and ectopic lignin phenotype of the cell wall synthase mutant cesa6prc1–1  such as MIK2 has been associated with changes in rosette dry weight in response to mild salt . This LRR kinase was identified from a reverse genetic screen for impaired ectopic lignin deposition under treatment with isoxaben, an inhibitor of cellulose deposition  is another CrRLK that participates in cell wall sensing. Loss-of-function studies have revealed that FER is involved in many processes such as cell elongation, root hair development, responses to hormones, nutrition and plant defense , which perform plastid localized potassium antiport, are needed to maintain plastid ion homeostasis. In corresponding loss of function plants, plastids were swollen and had impaired calcium influx after osmotic stimulation   Fig. . These d2O2) is facilitated by certain aquaporins localized at the PM (PIP1–4)  accumulate minutes after cell stimulation  was found to be necessary and sufficient to induce osmotically-induced ROS production  and glr3.3 glr3.6 (glutamate receptor-like protein)  or signaling mutant (abi1) indicating an ABA independent signal  and transporters. Transient depolarization and repolarization events with set amplitude, velocity and duration or variation of potential can convey key information about the strength and distance of the stimulus that triggered such electrical signals, within and between plant organs. These signals rely on fluxes of ions including K+, Cl−, H+ or Ca2+, the latter being the most documented. Electrical signaling has been reported to be involved in the response to heat, cold, touch, salt, flooding, hypoxia, osmotic stress, and drought  was identified from a forward genetic screen for impaired ROS-induced Ca2+ waves . This releases CaDrought, high salinity or temperature dramatically modify the metabolic profiles of plants  form the most recently discovered class of phytohormones. ABA and strigolactones share a carotenoid precursor, from which strigolactone synthesis proceeds through a partially known series of enzymes to produce bioactive strigolactones , aims at locally irrigating a limited part of the root system while the other part of the root system faces water deficit  after local dehydration of the root system  is a process where two liquids can be separated into non-miscible phases depending on concentration and which can be modulated by physico chemical alterations of the system . Thus, it is difficult to integrate all available information to build a systemic signal network for plant response to heterogeneous water distribution. Furthermore, many of the signal molecules and mechanisms identified under homogeneous water deficit condition have not been or very partially investigated under heterogeneous water distribution.Accordingly, little is known about the nature, the temporality and the function of sensing and local and systemic responses to heterogeneous water availability. We believe that studying rapid and long term responses to local water deficit in model species and under controlled conditions should lead to breakthroughs in the identification of the molecules and of their interactions that trigger plant acclimation responses. Identifying the main actors that trigger, prime or coordinate plant responses to water availability will provide a first step toward improving the efficiency and coordination of these responses. In the long term, these studies will allow identifying novel breeding targets to enhance crop tolerance to drought and develop new varieties that are well adapted to water saving irrigation strategies.


Bariatric surgery is one of the most effective interventions for treating obesity and weight-related complications. Since the early days of bariatric surgery, several innovations in surgical technique and periprocedural management have improved overall outcomes and reduced the risk of short and long-term complications. Bariatric surgery continues to be the treatment of choice for many patients with medically complicated obesity in whom lifestyle interventions, with or without anti-obesity medications, have been insufficient.Weight regain is a recognized complication of bariatric surgery that affects about 20 to 25% of patients and is more prevalent after the second postoperative year . Weight Ames et al.). Recognizing problematic eating behaviors and the complexity of eating behaviors offers an opportunity to tailor a behavioral treatment strategy that is effective for the patient.‘Behavioral Interventions to Attenuate Driven Overeating and Weight Regain after Bariatric Surgery’ focuses on identifying and classifying eating behaviors after bariatric surgery that lead to over-consumption of calories  This is an area of immense growth, and as more patients undergo bariatric surgery and the number of available anti-obesity medications increases, we will learn more about the efficacy, safety, and scope of these medications for this understudied patient population.Anti-obesity medications are effective therapies for treating post-bariatric weight regain. In ‘Pharmacologic Management of Weight Regain Following Bariatric Surgery‘, the authors discuss the evidence supporting the use of anti-obesity medications for weight regain in patients who have undergone bariatric surgery . This review highlights short and long-term weight loss outcomes and safety data associated with these emerging procedures. As with other treatment modalities, endoscopic therapies show superior outcomes when paired with a multidisciplinary approach to weight regain, which reinforces the importance of a comprehensive treatment strategy for patients with weight regain after bariatric surgery.‘Endoscopic Management of Weight Recurrence Following Bariatric Surgery’ highlights the role of minimally invasive, endoscopic options for managing weight regain in select patients . Understanding the contributors to weight bias internalization can help improve the patient-provider relationship and may reduce delays in patients with weight regain seeking care.A patient’s lived experience with obesity, including their interactions with the healthcare system, influences how and whether they choose to seek treatment for weight regain following bariatric surgery. This is complicated by the high level of attrition to follow-up after bariatric surgery, which may be impacted by experienced weight stigma and bias. In ‘The Role of Weight Stigma in Weight Regain in Bariatric Surgery’ the authors discuss the critical role of societal messaging, as well as messaging within the medical community towards postsurgical patients that can affect adherence and follow-up (Bariatric surgery is a safe, effective, and durable therapy for obesity and its complications. Beyond improving health, bariatric surgery also increases quality of life and life expectancy. However, obesity is a chronic disease and bariatric surgery is not curative. Significant weight regain is a reality for a small proportion of patients who undergo bariatric surgery and may be challenging to treat. The optimal management of post-bariatric weight regain acknowledges the complexity of the contributing physiological, behavioral, and anatomical factors, and that a multidisciplinary team approach can increase the likelihood of optimizing weight, health outcomes, and quality of life.AS: Writing – review & editing. MS: Writing – original draft. JA: Writing – review & editing.


Adolescents with attention-deficit/hyperactivity disorder (ADHD) are at elevated risk of a range of difficulties, among which emotion regulation, peer and co-occurring mental health problems are prominent challenges. To better support adolescents with ADHD, ecologically valid interventions that can be embedded in daily life to target the most proximal antecedents of these challenges are needed. Ecological momentary assessment (EMA) designs are ideally suited to meeting this need.In the mental health in the moment ADHD study, we will use an EMA design to capture the daily life experiences of approximately 120 adolescents aged 11–14 years with a clinical diagnosis of ADHD and the same number of age-matched and gender-matched peers without a diagnosis of ADHD. We will combine this with comprehensive information gathered from online surveys. Analysing the data using techniques such as dynamic structural equation modelling, we will examine, among other research questions, the role of emotion regulation and peer problems in mediating the links between characteristics of ADHD and commonly co-occurring outcomes such as anxiety, depression and conduct problems. The results can help inform interventions to support improved peer functioning and emotion regulation for adolescents with ADHD.This study received a favourable ethical opinion through the National Health Service ethical review board and the University of Edinburgh PPLS Research Ethics panel. The results will be disseminated through journal publications, conferences and seminar presentations and to relevant stakeholders, such as those with ADHD, their families and clinicians. This study will use an ecological momentary assessment (EMA) design to collect high temporal resolution, ecologically value daily life experience data from young people with attention-deficit/hyperactivity disorder.It will use cutting edge statistical analysis techniques to disentangle different aspects of emotional functioning in daily life to identify key intervention targets.A limitation is that data will be collected only over a short period, therefore, developmental changes will not be captured.Further, EMA methods may be vulnerable to low response rates, careless responding or dropout and the reliance on self-reports can be associated with reporting biases.et alAttention-deficit/hyperactivity disorder (ADHD) symptoms may increase the risk of experiencing a range of difficulties in adolescenceDifficulties with emotion regulation among children and adolescents with ADHD are well documented.1Emotion dysregulation often exists alongside and has been proposed to contribute to peer difficulties such as bullying victimisation and perpetration, peer aggression, rejection, dislike and conflict among adolescents with ADHD.9Emotion dysregulation and peer problems are also thought to contribute to the development of co-occurring mental health and behavioural challenges associated with ADHD symptoms. For example, previous research in children and adolescents has found that peer issues and emotion regulation mediate the associations between ADHD symptoms and internalising problems such as anxiety and depression21 22However, the daily life mechanisms underpinning these links and—correspondingly—the most proximal targets for intervention remain poorly understood, particularly in the critical period of adolescence. Ecological momentary assessment (EMA) has proven valuable in child and adolescent mental health research for capturing symptoms and related experiences in near real time and in the flow of daily life and can thus inform more ecologically embedded intervention approaches.24 25There have, however, been only a handful EMA studies of young people with ADHD during the critical period of adolescence.These initial findings point to the value of further exploring, using EMA, the daily life manifestation and impacts of emotion regulation and peer interactions in adolescents with ADHD. For example, as well as seeking to extend these findings to examine a wider range of emotion regulation and peer issues, it will be valuable to examine factors that may impact these domains, their interconnections, and their links to mental health and behavioural outcomes. This will provide illumination on further modifiable targets for intervention, such as physical activity, improved sleep, secure attachment to caregivers and positive teacher–student relationships.We will thus use an EMA design to illuminate how, in the flow of their daily lives, adolescents with ADHD may experience difficulties with emotion regulation and peer problems, how these may contribute to co-occurring mental health and behavioural problems. We will also examine other potential modifiable targets for intervention that may impact emotion regulation, peer problems, mental health and behaviour as well as their links among adolescents with ADHD . Core research questions are:Which aspects of emotion regulation differ between adolescents with and without ADHD, for example, negative or positive emotional lability, emotional reactivity, emotional inertia, emotion regulation strategy use and emotion regulation strategy success?Which aspects of emotion regulation mediate the association between ADHD diagnosis/symptoms and peer problems in daily life?Do emotion regulation and peer issues mediate the links between ADHD diagnosis/symptoms and anxiety, depression and conduct problems?Exploratory analyses may be used to answer a range of additional questions such as exploration of moderation of the links between ADHD diagnosis/symptoms and outcomes to identify other potential intervention targets.Participants are expected to include approximately 120 adolescents aged 11–14 years with a clinical diagnosis of ADHD and 120 matched controls. The clinical sample will be recruited via Child and Adolescent Mental Health Services (CAMHS) in two primary sites in the UK (one in Scotland and one in England) and community partners. In CAMHS, the recruitment procedure will vary from service to service; however, in general participants will be recruited at medication clinics and through ongoing casework.A matched control  will be selected for each participant with a clinical diagnosis of ADHD from a larger pool of participants taking part in adolescent mental health studies (co-)led by the first author. To maximise relevance to community-ascertained samples, minor modifications to the protocol will be made, detailed in the Measures section.Inclusion criteria for the clinical sample will be: aged 11–14 years, a formal clinical diagnosis of ADHD (parent-reported in the case of the youth recruited via the community), the ability to provide informed assent and parental informed consent to participate and the ability to independently complete online surveys and EMA protocols. There will be no explicit exclusion criteria based on co-occurring neurodevelopmental conditions as ADHD commonly co-occurs with other neurodevelopmental conditionsAdolescents with ADHD and parents of adolescents with ADHD are the most important stakeholders in this project. Their views have informed the project design, including the current protocol and will continue to inform the project and the dissemination of its outputs. Young person and parent advisors were recruited via the ADHD charities and organisations with whom our team has collaborative links. Examples of major changes made to the protocol based on their input included extending the inclusion criteria to include lower ages, reducing the length of the surveys, including more positively worded and fewer negatively worded items, removing elements of the project that would have involved location tracking and wearable technology, adding a measure of rejection sensitivity and embedding more fun and educational elements into the study, such as providing ‘ADHD facts’ at the end of each completed EMA survey.Data collection is projected to begin in August 2023 and be completed in 12 months. 34–36EMA surveys. They will be provided with instructions on how to take part and offered on-boarding support via a video call with study staff. Participants will use their own smartphones where possible; however, to avoid selection biases and promote inclusion, study smartphones will be available. An email helpline will also be available for participants to receive help on any issues they encounter.On completion of the online intake survey, adolescent participants will initiate the EMA data collection will take place over a 2-week period and involve five measures per day, all administered via a smartphone application. Four of these measures will be delivered at quasirandom intervals (random within four blocks) and include identical measures of momentary experiences. The fifth will be a brief ‘daily diary’ that captures concepts that vary over slightly longer timescales . All measures will be designed to take less than 2 min as long questionnaires are associated with increased burden and lower compliance and our young person advisory group (YPAG)/parent advisory group (PAG) emphasised the importance of minimising burden.37end-of study survey. Here, they will complete repeat measures of a subset of the questions administered in the original survey, as well as a brief set of questions capturing their participation experiences. Full questionnaires are provided in At the end of the 2-week EMA period, participants will complete an online 10.1136/bmjopen-2023-077222.supp1Supplementary dataThe measures that adolescents will complete over the course of the study are presented in The measures that parents will complete over the course of the study are presented in Prior to the main data collection, the protocol will be piloted in a sample of n=10 adolescents (5 with and five without ADHD). Further modifications may be made to the protocol after piloting.Data will be analysed using techniques such as dynamic structural equation modelling (DSEM).https://osf.io/pvrma/ includes tailored simulations for this study. Taken together, our choice of n=120 per group balances feasibility with statistical precision and includes buffer for missing data and attrition.Our sample size (n=120 adolescents with ADHD and n=120 without) draws on several sample size determination approaches. It is partly based on a resource constraint approach, that is, we will maximise the sample size attainable within the resources available.Ethical review for the collection of the data from the clinically ascertained sample will follow a three-stage process in which sponsorship will be sought from ACCORD, followed by ethical review by the National Health Service ethical review board, and finally, research and development approval. Ethical review for the collection of data based on community and school recruitment will be by the University of Edinburgh PPLS Research Ethics panel. The results will be disseminated through journal publications, conferences and seminar presentations and to relevant stakeholders, such as those with ADHD, their families and clinicians.The current protocol describes the planned design for a combined traditional and EMA survey studyThe study is motivated by a need for new data to inform more ecologically valid interventions that can provide effective improvements to adolescents’ emotional and social functioning in their daily lives.Our findings may be particularly valuable for informing smartphone-based interventions.Our findings can also help accelerate progress in the application of EMA methodologies in populations of adolescents with ADHD. To date, despite their promise, very few such studies have been conducted.et al and Babinski and Welkie56Though a large body of research has investigated optimal design features for EMA in general,


Preventing the rise in obesity is a global public health priority. Neighbourhood environments can help or undermine people’s efforts to manage their weight, depending on availability of nutritious and nutrient-poor ‘discretionary’ foods. The proportion of household food budgets spent on eating outside the home is increasing. To inform nutrition policy at a local level, an objective assessment of the nutritional quality of foods and beverages on food service menus that is context-specific is needed. This study describes the development and piloting of the Menu Assessment Scoring Tool (MAST), used to assess the nutritional quality of food service menus in Australia. The MAST is a desk-based tool designed to objectively assess availability of nutrient-poor and absence of nutritious food and beverages on food service menus. A risk assessment approach was applied, using the best available evidence in an iterative way. MAST scores for 30 food service outlets in one Local Government Authority in Perth, Western Australia highlight opportunities for improvements. MAST is the first tool of its kind in Australia to assess the nutritional quality of food service menus. It was practical and feasible to use by public health nutritionists/dietitians and can be adapted to suit other settings or countries. Obesity is a common, complex, chronic, relapsing, diet-related condition . Global Public health interventions designed to reduce and prevent obesity include food and nutrition policies to improve population diets , as healEating behaviour is influenced by multi-level factors, including individual , social , environmental , and macro-level  . A rangeThe food environment is multifactorial, including food access, provision, and price, as well as how food is labelled and marketed . The terFood service outlets  are important considerations for nutrition policy because they contribute an increasing proportion of total dietary intake in many countries, as more people are ‘eating out’ or ‘ordering in’ ready-to-eat meals or snacks. Australians purchased an average of 2.73 meals from outside of the home each week in 2018 . A thirdMost of the existing tools to assess the impact of food service outlets on food environments and dietary health  require The Portuguese Kids’ Menu Healthy Score (KIMEHS) was developed as a desk-based tool and provides an easy and fast way to assess children’s menus for consistency with the Mediterranean diet . KIMEHS The East Metropolitan Health Service (EMHS), based in metropolitan Perth, is one of four publicly-funded area health services within Western Australian (WA). EMHS provides tertiary, secondary, specialist, and community and population health services that aim to maintain and improve the health of one-third of the WA population . The EMHAs part of the implementation of the EMHS Obesity Prevention Strategy, the Food Outlet Dietary Risk assessment tool (FODR) was developed to objectively assess and score the dietary risk of all consumer-facing food outlets present in EMHS neighbourhood food environments. It assigns scores for six public health nutrition attributes: availability of nutrient-poor foods; availability of nutritious foods; acceptability and appeal; accessibility; type of business operation; and complex food outlet considerations . A risk This study aims to describe the development and piloting of the Menu Assessment Scoring Tool (MAST), to assess the nutritional quality of food service menus in Australia. MAST was designed using a risk assessment approach , to be un = 4136) were identified [EMHS staff collected registered food business data from all 13 Local Government Authorities (LGAs) in 2018. A database of 6963 food businesses was constructed by the research team, and a classification framework applied . Food buMAST was developed to be fit-for-purpose in the Australian context, applying the following principles during its development: (1) menu items were classified as either nutritious  or nutrDetails provided in the Australian Guide to Healthy Eating  and the The Australian Guide to Health Eating recommends enjoying a wide variety of nutritious foods from five food groups every day, including: grain  foods; vegetables and legumes/beans; fruit; milk, yoghurt, cheese and alternatives; and lean meats and poultry, fish, eggs and alternatives. Plenty of water is recommended, and small amounts of healthy fats and oils can be used when preparing meals . DiscretLack of consistency in classifying foods as ‘discretionary’ has been identified among Australian health professionals, food industry, policy makers, and consumers . TherefoThe MAST scoring system was adapted from the work of Tavares et al. (2021), which recognised that food service outlets supportive of healthy eating make nutritious choices available and limit nutrient-poor choices . The HeaUsing the risk assessment approach, MAST assigns a penalty point for the availability of each nutrient-poor food category, and for the absence of each corresponding nutritious food category. When one meal meets the definition of each nutrient-poor food category, one penalty point is allocated. When there are no meals available that meet the definition of each corresponding nutritious food category, one penalty point is allocated. There is no change to the penalty points allocated when more than one meal meets the definition of each nutrient-poor or corresponding nutritious food category. The scores assigned by MAST range from 0% to 100%. The best-case scenario produces a score of 0% which indicates there are no nutrient-poor food groups present on the menu, only nutritious food groups. The worst-case scenario products a score of 100% which indicates there are nutrient-poor food groups present on the menu, but no nutritious food groups .Categorical risk  is not assigned by the MAST score for food service menus. This is because the MAST score contributes two of the six components of the Australian FODR tool  which was developed to assess and score the dietary risk of all consumer-facing food outlets present in EMHS neighbourhood food environments.® , UberEats® ), company websites, company social media platforms ), company mobile phone apps, or user-generated review websites ). To determine whether the menus available at food service outlets differed across online locations, a sample of five major chain food service outlets which sold a range of foods were identified for comparison. Chains were a focus for comparison because they have the most influence over neighbourhood food environments due to contributing a high proportion of food service sales, an approach recommended by the International Network for Food and Obesity/non-communicable diseases Research, Monitoring and Action Support (INFORMAS) [®, Meunlog®  Deliveroo®, and Doordash®   and used in the analysis. In total, 26 menus were collected in October 2022 for comparison as follows: 5 from UberEats®, 4 from Menulog®, 4 from Deliveroo®, 4 from Doordash®, 4 from company websites, and 5 from company mobile phone apps. A standard location in the EMHS geographic catchment was used across all online delivery platforms, websites, and apps. All five chains received identical MAST scores for each of the menus available online. Based on this comparison, the research team agreed that using one online delivery platform as the default for obtaining food service menus would provide robust MAST scores.Some food service outlets provided menus in multiple online locations, including one or more online delivery platforms  . The fivOne LGA agreed to provide an updated database of registered food businesses in 2021, which formed the setting for pilot testing of MAST. The data were cleaned and classified and included 24 food retail and 38 food service outlets.Online searches were conducted to locate menus for each of the 38 food service outlets. Online delivery platforms were used as the primary source of menus, supplemented by food outlet websites and social media. Two food service outlets appeared to have closed, no recent menus could be located for a further six, and 30 menus were obtained.The menus were imported into NVivo  and saved in a predetermined filing system. A MAST coding framework was developed and applied to each menu, which identified availability of six nutrient-poor food categories (which include 15 nutrient-poor food groups), and five nutritious food categories (which include 12 nutritious food groups). NVivo served as a storage platform for web captures of menus and visual records of the assessment decision making process.For menu items that could be tailored by the customer, e.g., by adding a topping or changing the side dish, the default option was used. For menus with meal promotional packages or ‘meal deals’ the item was analysed together and not split into components. Menu items for children  were included if listed on the main menu. MAST assigns a penalty point for each nutrient-poor food category available, so only one item per food category was coded in NVivo to indicate presence. MAST assigns a penalty point for each corresponding nutritious food category that is absent, so only one item per food category was coded in NVivo if available.® . REDCap® housed the MAST tool and functioned as the quantitative data capture application. The presence or absence of each food group was entered into REDCap® for each menu, using a MAST survey to assign scores as previously described, and the software then calculated the final score  to accompany the main dish ; cafes selling a wide range of teas and coffees but a small number of food offerings; fast food outlets with menu items that could be tailored with a choice of toppings or fillings; and specialty food service outlets such as ice cream and gelato shops. The MAST scores were reviewed to see what signals were given to food outlet operators for improvement.Initial testing of the scoring system found that food outlets with nutrient-poor products from only one or two food categories  would be advantaged over food outlets with more diverse menus. This was because they were allocated fewer penalty points due to the absence of multiple nutrient-poor food categories, despite selling few if any nutritious menu items. Based on this feedback, the final MAST scoring system was applied in three stages. Step one is to assign penalty points for the availability of each of six nutrient-poor food categories, and for the absence of each of five corresponding nutritious food categories, with the total used as the numerator for the MAST score . Step two is to identify how many of the food categories received a penalty point, which is used as the denominator for the MAST score. In step three, the numerator is divided by the denominator and multiplied by 100 to give a percentage. This method produces more appropriate signals for improvement to a wide range of food service outlets.The final 27 food groups defined in MAST are mapped to six food categories of vegetables, fruits, grain , meat and alternatives, dairy and alternatives, and beverages and miscellaneous; and further defined as either nutritious  or nutrient-poor  to reflect differences in ingredients and methods of preparation .Water was not included as a nutritious food group, because tap water is freely available in all Australian food outlets as a cultural norm. Alcoholic beverages were not included as a nutrient-poor food group, because the availability of alcohol is assessed separately in the overall FODR tool, via assessment of the type of liquor licence granted. It was therefore omitted from MAST to avoid duplication. If necessary, MAST may be modified for other settings to include water as a nutritious food group and alcoholic beverages as a nutrient-poor food group in the beverages and miscellaneous food category. For each food group, a definition and examples or exclusions are provided .MAST scores for 30 food service outlets present within the food environment of one LGA in the EMHS geographic catchment are summarised below .The mean MAST score across all 30 food service outlets was 71% (range 29–100%). Almost all food service outlets (24/30) had a MAST score of 56% or higher, indicating the menus were high risk and poor nutritional quality. The mean MAST scores for each type of food service outlet were: restaurants 56% (range 43–67%); cafes and coffee shops 56% (range 36–78%); pubs, hotels, and taverns 69% (range 57–78%); and fast food and takeaway 81% (29–100%). Only one food service outlet, which was classified as fast food or takeaway, had a MAST score below 30%, indicating few nutrient-poor food items were available on the menu. Five food service outlets, all classified as fast food or takeaway, had a MAST score of 100% indicating no nutritious menu items were available.MAST was developed as an objective tool for assessing the nutritional quality of food service menus in Australia, using a risk assessment approach. To ensure MAST was fit-for-purpose in the local context, a number of principles were identified as important during its development and pilot tested. They informed refinements to MAST, which were made using a collaborative and iterative process by the research team of qualified public health nutritionists and dietitians.The first principle identified was to classify menu items as either nutritious or nutrient-poor, consistent with national guidelines, to attribute dietary risk. Food classification requires the skills and expertise of public health nutritionists or dietitians , howeverThe second principle used in the development of MAST was to adopt a risk assessment approach, using the best available evidence . Menu it® survey for scoring added to the time taken for assessment but ensured consistency and transparency. Verification of coding between different EMHS staff could be undertaken, and changes made during the iterative development process. It was also envisaged that the documents saved during the process of deriving MAST scores could be used to provide tailored feedback to food service outlets seeking to improve menu nutritional quality in future. Therefore, the process adopted for MAST was considered time efficient and robust.The third principle that was essential for feasibility of applying MAST to EMHS neighbourhood food environments, was for the desk-based tool to be quick and easy to use. Using menus that were sourced online reduced the time needed to undertake site visits, which have been reported to take up to 40 min per outlet when using the NEMS-R . Also, aThe fourth principle for developing MAST was for face validity in the scores assigned by the research team. Face validity was assessed by identifying whether MAST appeared to do the job it aimed to do, from the users’ perspectives . MAST waThe fifth principle that was identified as important when developing MAST was to signal areas for improvement to food service outlet operators. The way the MAST score is calculated means that when one meal meets the definition of a nutritious food category penalty points are not allocated. In contrast, if numerous meals meet the definition of a nutrient-poor food category only one penalty point is allocated. Therefore, the strongest signal to food service outlet operators is to add nutritious meals to the menu rather than to reduce the number of nutrient-poor meals, although both changes are recommended. An Australian study which used an adapted NEMS-R to assess 28 rural food service outlets recommended urgent action to introduce and promote nutritious meals, as the outlets were dominated by nutrient-poor foods . People Strengths of MAST include the ability to use the objective results to create tailored reports for food service operators willing to make changes to menus. The results can highlight where menus are supportive of healthy eating, and areas for improvement. A toolkit of suggestions can be constructed based on EMHS staff experience of supporting implementation of a mandated food and nutrition policy across health service sites . A qualiThe study also has some limitations. Food service outlets change over time, and menus also change depending on seasonality, ingredient cost and availability, and customer preferences. MAST assessment can be repeated over time but will not capture all of these ad hoc changes. MAST is used to assess nutritional quality of menus using only the information provided. Therefore, cooking methods which include adding significant amounts of fat, sugar and salt may not be identified. MAST does not include assessment of prices, promotions, or portion size. Sourcing menus from online food delivery platforms means there may be some differences in the offer available, compared to visiting the physical food service outlet. This is more likely to occur for smaller and independently operated food service outlets, who may have less capacity to ensure online information is updated regularly. The indicative MAST score for food service menus does not incorporate the comprehensive assessments used in other settings with mandated government policies, such as the Healthy Options WA Food and Nutrition Policy in hospitals . MAST waThe new MAST is the first tool of its kind in Australia to assess the nutritional quality of a wide range of food service menus. MAST was used to accurately and objectively assess availability of nutritious and nutrient-poor food on food service menus. MAST was practical and feasible to use by qualified public health nutritionists and dietitians and can be adapted to suit other settings or countries. Measuring availability of nutrient-poor food and absence of nutritious food contributes two of six components of the Australian FODR tool which assesses the dietary risk of consumer-facing food outlets. Pilot testing MAST with one LGA within Perth, WA identified the typically poor nutritional quality of menus. Findings indicate the challenges to eating healthily faced by people who are managing their weight. Assessing the nutritional quality of menus from neighbourhood food environments across the EMHS geographic catchment using MAST will inform local policy responses addressing obesity.