Advances in genomics offer promise for earlier detection or prevention of cancer, by personalisation of medical care tailored to an individual’s genomic risk status. However genome sequencing can generate an unprecedented volume of results for the patient to process with potential implications for their families and reproductive choices. This paper describes a protocol for a study (PiGeOn) that aims to explore how patients and their blood relatives experience germline genomic sequencing, to help guide the appropriate future implementation of genome sequencing into routine clinical practice.We have designed a mixed-methods, prospective, cohort sub-study of a germline genomic sequencing study that targets adults with cancer suggestive of a genetic aetiology. One thousand probands and 2000 of their blood relatives will undergo germline genomic sequencing as part of the parent study in Sydney, Australia between 2016 and 2020. Test results are expected within12–15 months of recruitment. For the PiGeOn sub-study, participants will be invited to complete surveys at baseline, three months and twelve months after baseline using self-administered questionnaires, to assess the experience of long waits for results (despite being informed that results may not be returned) and expectations of receiving them. Subsets of both probands and blood relatives will be purposively sampled and invited to participate in three semi-structured qualitative interviews (at baseline and each follow-up) to triangulate the data. Ethical themes identified in the data will be used to inform critical revisions of normative ethical concepts or frameworks.This will be one of the first studies internationally to follow the psychosocial impact on probands and their blood relatives who undergo germline genome sequencing, over time. Study results will inform ongoing ethical debates on issues such as informed consent for genomic sequencing, and informing participants and their relatives of specific results. The study will also provide important outcome data concerning the psychological impact of prolonged waiting for germline genomic sequencing. These data are needed to ensure that when germline genomic sequencing is introduced into standard clinical settings, ethical concepts are embedded, and patients and their relatives are adequately prepared and supported during and after the testing process. Despite substantial therapeutic progress, over one-third of patients diagnosed with cancer will die of their disease . Early rCancer may be regarded as the product of cumulative somatic genetic mutations, with some arising in individuals with a germline variant conferring an increased risk, modified by environmental exposures. It is estimated that the heritable component of common cancers such as colorectal, breast and prostate cancer contribute to 25–50% of their aetiology, , 4. AdvaThe cost and effectiveness of screening and prevention programs in general is dependent on targeting high-risk populations . ClinicaGenomic sequencing to define an individual’s risk may soon offer a universal, acceptable and cost-effective method of personalising medical prevention strategies tailored to genetic status that predicts risk of developing new cancers. This study will focus on GGS. A second study examining the psychosocial and ethical issues and outcomes of tumour genomic profiling for patients with advanced cancer is underway and is described in a separate paper. These two studies comprise the P  I (in) GE (genomic) ON (oncology) Project.GGS determines the complete DNA sequence of an organism’s genome which in humans, includes ~ 20,000 genes . GGS canIndividuals with pathogenic germline variants that signal high risk of a particular disease(s) (and their blood relatives) can be offered more intensive risk management. But GGS will only realize its potential in an effective and ethically appropriate way if patients can understand, manage, and make informed decisions to pursue health recommendations based on genomic results. A number of challenges inherent in GGS present barriers to these outcomes.Although uncertainty pervades medical information, its scope in genomics may be unprecedented . Han et Patient uncertainty and poor tolerance of ambiguity has been found to reduce patient willingness to receive GGS results –17. WhilThe psychological impact of single gene testing has been well studied, particularly in the context of hereditary breast and ovarian cancer, Lynch syndrome and melanoma . The eviThere has also been a question raised regarding whether GGS can deliver behavioural change in cancer care, as several studies of allegedly healthy participants have indicated that this was not always the case following receipt of actionable results –30. MostResearch to date has revealed cautious interest in genomic testing in the general population. Several Australian studies have found limited knowledge about, and little interest in pursuing, direct-to-consumer personal genomic testing amongst the general public,  who haveInterest among cancer patients appears stronger . A US stOverall, there is insufficient understanding of the preferences, attitudes and values of cancer patients who have actually had GGS: their expectations, their experience of uncertainty, or the psychological effects of testing. The Clinical Sequencing Exploratory Research studies  and ClinThis report will outline the first Australian study to collect longitudinal data on cancer patients’ experiences of GGS, and one of the first studies internationally to follow probands and their blood relatives who undergo GGS, over time. The study results will inform ongoing ethical debate and clinical practice development on issues such as protocols for obtaining informed consent for GGS, and informing people and their relatives regarding genomic results. The study will also provide critical outcome data concerning the psychosocial impact of waiting for GGS results on patients and their blood relatives. These data are needed to ensure that when GGS is introduced into routine clinical care, ethical concepts are embedded, and patients and their relatives are adequately prepared and supported during and after the testing process..There is a strong evidence base supporting the use of social cognition models to provide a structured framework for identifying psychosocial and cognitive influences on health behavior including, genetic testing . This stIn this study, protection motivation will ensue from perceived susceptibility to disease risk, and/or fear of cancer progression, as well as participant and proband knowledge of, attitudes to, and value given to, GGS (as a strategy to guide more effective screening and thus reduce the threat of cancer development). Patients who understand GGS to be valuable will be prone to remain satisfied with their decision to undertake GGS, over time. Intolerance of uncertainty may make GGS less attractive and intensify regret, doubt and poor psychological outcomes.evaluate the impact of testing on cancer-related anxiety, patients’ and blood relatives’ perceived value of genomic information, andundertake a critical reflection on all results, with reference to relevant ethical issues and concepts.The aim of this study in people who undergo GGS is to:The primary psychosocial endpoint is: impact of testing on cancer-related distress, measured by change in the Multidimensional Impact of Cancer Risk Assessment (adapted) Scale.The primary attitudinal endpoint is: patients’ and blood relatives’ perceived value of genomic information measured by a hypothetical time trade-off scenario.The primary ethical outcome is: a critical reflection on all results, within reference to a range of normative ethical issues and concepts.Secondary psychosocial /ethical outcomes include significant changes in scores of any of the following: Impacts of Events Scale Hospital Anxiety and Depression Scale; Fear of Cancer Occurrence/Recurrence/Progression; Herth Hope Scale; risk perception; perceived susceptibility to cancer; and perceived likelihood of having a pathogenic variant.Secondary attitudinal outcomes include: understanding of GGS; views on disclosure of genomic information; perceived benefits and drawbacks of GGS.Secondary decisional outcomes include: degree of decisional satisfaction and regret.The Genomic Cancer Medicine Program, funded by the NSW Ministry of Health, Australia, involves the parent study, Genetic Cancer Risk in the Young (Cancer Risk) Study. This study is prospectively recruiting 1000 cancer patients with features suggestive of a genetic aetiology. Two thousand first degree blood relatives of the cancer probands will also be recruited. Both probands and relatives will be offered GGS. The Cancer Risk Study participants will receive results 12–15 months after initial enrolment if actionable results are found, and study participants wish to receive them. Risk management will be offered within a clinical trial setting as part of a separate funded study, the Surveillance in Multi-Organ Cancer prone syndromes study. Patients and blood relatives will be recruited to the Cancer Risk psychosocial study when they give written consent to the Cancer Risk Study. Thus they will have already considered and given consent to GGS but not yet had GGS testing.The current PiGeOn study represents a sub-study of a germline genomic study targeting people with cancers likely to have genetic aetiology.This study is led by the Psycho-Oncology Co-operative Research Group based at the University of Sydney, Australia. The project was funded by a project grant from the National Health and Medical Research Council, Australia, and ethics approval was given by the Human Research Ethics Committee at St Vincent’s Hospital, Sydney.This study is a mixed method, prospective, cohort sub-study of a GGS program recruiting probands with a history suggestive of a genetic aetiology and two of their first-degree blood relatives.Participants will be recruited by the parent study, from incident and prevalent cases at oncology units in Sydney hospitals. All eligible participants will be asked to give consent using established protocols for genetic research, validated by large genetic studies, the Kathleen Cuningham Foundation Consortium for research into Familial Breast cancer  and the Inclusion criteria include having a histologically confirmed malignancy, age 16–40 years at diagnosis, or an individual with > 1 primary cancer diagnosed < 50 yrs. of age or an individual with > 2 primary cancers at any age; first degree relative aged 18 years or over of an individual meeting the above criteria or a cancer affected blood relative aged 18 years or older of an individual meeting the above criteria; willing and able to comply with all study requirements, including timing and/or nature of required assessments; signed, written informed consent to participation in GGS.Exclusion criteria include: inability to understand an English language consent form.As seen in Fig. Patient-reported outcomes will be measured according to the following schedule  outcome measures will be administered at baseline only:This 5-item measure adapted from Hay et al.  assessesAn 8-item, multiple choice, study-developed questionnaire assessing knowledge of the purpose of GGS, likely frequency of informative results, cancers in which informative results are more likely to be found, availability of tailored risk-management or treatment options, and source of genetic knowledge. Scores are summed, with high scores indicating greater knowledge.Three items to assess participants’ views on who should receive GGS if it were clinically available, for comparison with a previous study .A hypothetical time trade-off scenario based on those used in three previous studies , 56, 57.Four Likert-scale items adapted from Tabor et al.,  assessinFour items adapted from Rosenburg et al.,  assessinSeven Likert-scale items developed from the literature to measure the participant’s anticipated likelihood of behavioural change in the event of receiving a positive result for cancer risk. Higher scores indicate stronger intention to change behaviour. This item is included for comparison with a future study. Study-developed.This measure from Kasparian et al.  includesat the first and second follow-ups:The following outcome measures will be administered Nine Likert-scale items and two open-ended questions adapted from an earlier study  assessinThe 15-item Impact of Events Scale (IES)  assessesThe 14-item Hospital Anxiety and Depression Scale (HADS)  compriseTwenty-three items from the Multidimensional Impact of Cancer Risk Assessment  assessinThe 12-item Herth Hope Index (HHI)  measuresThe 5-item Decisional Regret Scale (DRS)  measuresat baseline and all follow-ups:The following outcome measures will be administered Three items where participants indicate perceived likelihood of having a gene fault that increases risk of cancer occurrence or progression from ‘much lower’ (0) to much higher , and alsThe five-item Concerns about Recurrence Questionnaire (CARQ),  adapted The 6-item Satisfaction with Decision (SWD) scale  measuresn = 20–40) and their participating blood relatives (n = 20–40) will be invited to participate in three semi-structured interviews (at baseline and each follow-up). Probands and relatives will be interviewed separately to encourage greater openness. Interviews will explore views on who should be offered GGS, attitudes to disclosure of results, perceived benefits and challenges of GGS and experiences of waiting for results. Both cohorts will be sampled purposively to include a wide variety of experiences, including individuals with and without a cancer diagnosis. Recruitment will continue until data saturation is reached.A subset of probands , approaching uncertainty in GGS and determining when it is appropriate to offer testing; and to whom. The result will be a series of normative positions, supported by empirical data , 71.Mean differences in outcomes will be compared using t-test (continuous) or chi-squared tests (dichotomous). Non-parametric tests will also be used where appropriate. Temporal changes in scales will be investigated by calculating the difference between time-points. Multiple (continuous outcomes) or logistic (dichotomous outcomes) regression will be employed to adjust for the effect of confounders and identify predictors of outcome. The correlation structure between blood relatives or when outcomes are analysed over more than 2 time points will be suitably adjusted for when needed using mixed effects models. Linear mixed models and logistic mixed models will be performed in R: A language and environment for statistical computing employing package nlme for the linear mixed model and lme4 for the logistic mixed model. Assumptions of normality of residuals and homogeneity of variance will be confirmed visually though diagnostic residual plots. Multivariable models will be created with the inclusion of all potential confounders, and those predictors that show little evidence for an association with the outcome in univariate analysis. Backwards elimination followed by forwards addition will be employed to select predicting variables in the final model. Known and identified confounders will be incorporated regardless of their statistical significance. Collinear independent variables will be identified and eliminated.The parent study plans to recruit 3000 probands in total over three years. With a conservative retention and survival estimate of 75%, this is reduced to at fewest 469 patients per cohort. With this sample size estimate, using a significance level of 0.05 this project: has 90% power to estimate the perceived value of genomic information in each time trade-off category of all patients and blood relatives to a margin of error of at most 5%; and estimate other secondary outcomes with adequate precision. Multiple regression on each cohort with at most 24 explanatory variables (including dummy variables) has 90% power to detect significant categorical variables with 5 categories  when that predictor explains greater than 3.6% of the residual variance, or to detect a continuous variable when it explains more than 2.3% of the residual variance.The PiGeOn study will contribute to the literature by identifying the knowledge, values, attitudes and coping strategies of patients and their blood relatives with regards to GGS. The project aims to determine how these, and other factors, predict subsequent cancer-related behaviour and psychosocial outcomes (in association with a future study). This study will also describe these factors’ alignment with current ethical norms. This will be the first Australian study to collect longitudinal data on cancer patients’ experience of GGS, and one of the first studies internationally to follow probands and their blood relatives who undergo GGS, over time.The parent study, the Cancer Risk in the Young Study, will involve participants who have the experience of a prolonged wait time until GGS results are available, and the majority of participants will receive no results (as will be explained during the consent process). Despite being advised that it is unlikely that a pathogenic gene variant will be identified, previous studies involving healthy participants suggest that patients have high expectations for the usefulness of returned results, and may therefore continue to hope for, and possibly worry about, results nonetheless . The timA qualitative component has been included in order to capture and explore the complexity of patient expectations, the experience of uncertainty, their understanding of the implications of testing and their attitudes towards sharing results, which may have familial repercussions. Motivation for testing, information needs and preferences are expected to vary and qualitative research has value in understanding processes.Genomic screening, both tumour and germline, is likely to become widespread in healthcare, and to influence cancer prevention, diagnosis, treatment and risk-management. GGS generates information of unprecedented volume, some of which will have uncertain significance, the meaning of which will change over time. It gives rise to testing methods that are both diagnostic and predictive and lead to implications for communication within families and reproductive choice on a scale which we have previously never had to manage in healthcare. The study results will inform ongoing ethical debate on issues relevant to the large-scale introduction of GGS into the clinical setting, such as protocols for obtaining informed consent for GGS as well as assenting to unknown future research, and informing (or not informing) patients and their relatives regarding genomic results. The study will also assess whether there are psychosocial sequelae following GGS testing, after being informed that results for patients and their blood relatives may not be forthcoming. Results will be linked to further studies to inform understanding of behaviour following receipt of results. These data are needed to ensure that when GGS is commenced as part of routine clinical care, ethical principles are well-established in practice, and patients and their relatives receive sufficient preparation and support before, during and after GGS testing. While this cohort is a selective sample with risk factors suggestive of a germline mutation such as young age of onset or multiple primaries, understanding the influences on behavioral change will also increase the likelihood that the introduction of germline genomic cancer screening for the general population will have the hoped-for positive benefits.Given the scarcity of evidence on responses to actual GGS for cancer, this study is an important and timely step in filling the critical gaps in understanding about the best way to introduce GGS into clinical medicine.


Editorial on the Research TopicUpdates and New Concepts in Regulation of Proinflammatory Gene Expression by Steroid HormonesInflammatory reactions and their associated molecules contribute to the progression and persistence of several human pathologies. Modern medicine developed successful pharmacological strategies to inhibit or decrease inflammation. Glucocorticoids (GCs) acting on their cognate nuclear receptors (GRs) represent the leading strategy for almost completely shut down inflammation, but the side effects are of major concern regarding long-term use of steroidal anti-inflammatory drugs. Significant advances in the field of molecular biology helped to clarify the “genomic” effects mediated by GRs on DNA transcription in immune cells, establishing a new paradigm of inflammatory response regulation. Recently, our knowledge reached a much higher level of complexity, which could not be completely translated yet to the medicine field. In order to uncover how endogenous GCs and other steroidal molecules, including sex hormones, naturally shapes the immune response, researchers have explored the ways some nuclear receptors modulate gene expression programs involved in proinflammatory signaling. This research topic invited articles that focused on highlighting the specific effects of steroid hormones on inflammatory signaling of a particular biological system or describing recent advances on the current knowledge in terms of signal mediation or mechanism.Moss and Jaffe). Blunted MR signaling seems to be beneficial during brain injury by avoiding exaggerated immune cells responses. MR binds to CORT with higher affinity than Aldo and increases the complexity of neuroinflammation regulation in the brain of stressed animals; a condition that elevates substantially the levels of GCs. GR behaves paradoxically during prolonged and disturbed GC levels and seems to cooperate with a proinflammatory effect of GCs in the brain (Duque Ede and Munhoz). Neuroinflammation is recognized as an important component associated with the progression of various neurodegenerative diseases. Dietary interventions, such as excessive calorie intake and intermittent fasting, modulate GCs and sex hormones and could impact the overall gene expression profile of proinflammatory molecules in the brain during injury or neurodegenerative diseases. While the former statement remains to be verified in its totality, it is well established that estrogen and testosterone modulates gene expression in stimulated immune cells . Although it was not possible to include all variety of steroidal hormones in this research topic, the contribution relating cardiotonic steroids (CTS) and the modulation of inflammation is noteworthy. Unlike most of the actions of the aforementioned hormones, CTS bind to and modulate Na+, K+-ATPase (NKA pump) and do not modulated transcription through nuclear receptors . Only very recently an unusual interplay between CD36, NKA, and Toll-like receptor 4 revealed the link between CTS and nuclear factor NF-κB activation, a key transcription factor of proinflammatory genes (The GC corticosterone (CORT) can be further metabolized to aldosterone (Aldo) in the adrenal cortex, a closely related molecule that signals through mineralocorticoid receptor (MR). It is well established that Aldo/MR plays an important role in blood pressure regulation through sodium retention at the kidney level. Growing evidences point toward a different scenario for MR effects on vascular cells and macrophages, including the increased levels of proinflammatory molecules and leukocyte inflammation. This effect can contribute to the pathogenesis of atherosclerosis and hypertension . GR interference on transcription activity can operate through different modalities, including tethering and changes in the chromatin state. In fact, GR tethering is the basis of the generation of a new pharmacological interference strategy in inflammation that employs selective glucocorticoid receptor agonists. Diverse mechanisms have been proposed to explain GR effects on proinflammatory transcription, which can include time-dependent dynamics . Finally, GR can operate non-canonically, and interferes with inflammation via “non-genomic” mechanisms through second messengers or via a mitochondrial GR . The many faces of GR actions on proinflammatory transcription are still under debate and recent findings reveals unpredicted mechanisms, such as rapid and prolonged chromatin decompaction promoted by the nuclear receptor  is an example of the first case. GILZ interferes with proinflammatory signaling in leukocytes at transcription factor level, which is well documented in lymphocytes (receptor . Future All authors have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Meeting climate change targets to limit global warming to 2°C requires rapid and large reductions in demand for products that most contribute to greenhouse gas (GHG) emissions. These include production of bulk materials (e.g. steel and cement), energy supply (e.g. fossil fuels) and animal source foods (particularly ruminants and their products). Effective strategies to meet these targets require transformative changes in supply as well as demand, involving changes in economic, political and legal systems at local, national and international levels, building on evidence from many disciplines. This paper outlines contributions from behavioural science in reducing demand. Grounded in dual-process models of human behaviour (involving non-conscious and conscious processes) this paper considers first why interventions aimed at changing population values towards the environment are usually insufficient or unnecessary for reducing demand although they may be important in increasing public acceptability of policies that could reduce demand. It then outlines two sets of evidence from behavioural science towards effective systems-based strategies, to identify interventions likely to be effective at: (i) reducing demand for products that contribute most to GHG emissions, mainly targeting non-conscious processes and (ii) increasing public acceptability for policy changes to enable these interventions, targeting conscious processes.This article is part of the themed issue ‘Material demand reduction’. Effective strategies for timely reduction of the demand leading to these damaging levels of production and supply require systems thinking involving contributions from many disciplines. Evidence needs to be generated for multiple interventions implemented at different points in transformed supply and demand chains within the complex systems that comprise the multi-dimensional environments in which we live. The kinds of transformations being called for include action in seven dimensions  , a circuThe focus of this paper is upon one contribution to these envisaged transformations, namely that of changing behaviour across populations to reduce consumption to a sustainable level. Drawing upon evidence for reducing over-consumption of products that harm health  this paper considers, first, the types of interventions likely to be effective at reducing demand for products that contribute most to carbon and equivalent emissions, and second, how public acceptability for these interventions might be increased to enhance their chances of being incorporated into policy.2.(a)Much of the research published to date on sustainable consumption has focused on interventions targeting individual behaviour change  with a p(b)‘Essentially, all models are wrong but some are useful.’ )(i)Targets for change need to be those that have the potential to make the largest impact on reducing carbon and equivalent emissions. For example, there has been more research and policy interest in reducing food waste than on reducing meat consumption, the former contributing an estimated 1–2% to emissions and the latter an estimated 50% . Similar(ii)Reviews. The strength of evidence for interventions to achieve sustainable behaviours would be increased by more systematic literature reviews including formal evidence synthesis where possible to estimate the effect sizes of interventions. While reviews exist, these have mainly used unsystematic methods, with results synthesized narratively. (An example of the type of review needed is one on the use of feedback to reduce energy consumption [http://training.cochrane.org/handbook).(a) sumption .) WithouExperiments. The second type of study that is key to increasing the strength of evidence in this area is the field experiment, i.e. a comparison between two or more groups that differ only in the intervention with allocation to groups determined if at all possible by randomization. While such designs are increasingly evident in research on interventions for sustainable behaviour, good quality experiments are scarce.(b) (iii)Three ways in which experimental studies would be improved concern outcome measures, study populations and study power. Outcomes need to be objectively measured behaviours and not self-reported behaviour change or intentions to change behaviour, both of which are subject to bias including over-estimation of change. Study populations need to be representative of general populations. While proof-of-concept or pilot studies can usefully rely on convenience samples such as students, responses from these and other highly educated populations can differ markedly from general population responses to the same intervention (e.g. ). In add(e)Having identified key drivers and effective interventions, effective implementation of many of them will be through policies that require regulation ,43. ThisThe next section considers how public acceptability of interventions to change behaviour might be increased to overcome these barriers to progress.3.Implementing many of the interventions described in the previous sections will require regulation. This will probably include increasing prices and restricting marketing of products and activities with high carbon and equivalent emissions such as animal source foods, cement, aluminium and plastic materials, car and plane travel. Mandated labelling schemes might also be introduced. Other interventions include capping the proportion of non-renewal energy that companies can supply.Such changes will meet with strong opposition from industries that stand to lose. In health contexts, the tobacco, alcohol and food industries have lobbied and framed debates on regulation often with great success to the detriment of population health ,45–48. SPublic acceptability is a critical consideration for policy-makers. Public acceptability also goes some way towards addressing ethical concerns about targeting non-conscious processes to change behaviour . ParadoxOne possible resolution to this paradox of public acceptability generally being lower for more effective interventions and higher for ones that are less effective stems from the observation that public perception of intervention effectiveness is one of the most reliable predictors of acceptability –56, withIn the context of environmentally sustainable behaviours, willingness to change one of five sets of behaviours, including driving less and eating less meat, increased with perceived effectiveness of the action . A seriePublic acceptability of interventions might therefore be increased by effective communication of the impact of interventions targeting physical and economic environments to reduce carbon emissions. Building on existing research on personal risk communication ,62, the There are two further factors that may be important to target in increasing policy acceptability. The first factor is people's understanding of the non-conscious nature of much of human behaviour. For example, the more people attributed over-consumption to the physical environment in which the behaviour occurs, but not free will, the greater their support for policy interventions to tackle obesity . The secIn addition to communications aimed at increasing public acceptability, a range of other interventions has been described for increasing public demand for policy action. These include interventions aimed at establishing social movements involving non-government organizations with the resources to be able to act including countering industry interests that conflict with policy goals . Two casLegislation to implement a sugary drinks tax in Mexico is an example of a successful change of policy with preliminary evaluation showing the expected outcomes on reduced consumption of sugary drinks . With fu4.Huge and immediate changes are needed to reduce demand for environmentally unsustainable products in order to meet a climate change target of 2°C. Towards this a step change is needed in the number and quality of behavioural experiments evaluating the impact of altering cues in physical environments to achieve sustainable behaviours. Evidence on effective interventions is generally insufficient for their implementation. Developing research on increasing public acceptability of effective interventions including economic ones could raise the chances that policy-makers act in line with the growing evidence for achieving sustainable behaviours to meet the unprecedented global challenge from climate change.


We congratulate Endo and co-authors on their retrospective registry-based analysis comparing closed-chest cardiopulmonary resuscitation (CCCPR) and open-chest cardiopulmonary resuscitation (OCCPR) in cardiac arrest patients following blunt trauma  and woulOCCPR is generally deemed futile in blunt trauma patients whenever cardiopulmonary resuscitation (CPR) is ongoing for more than 10 minutes without the patient showing any signs of life . Among pA further confounding element is that OCCPR is initiated in most of the cases for logistic reasons after initial treatment by CCCPR. During this initial window period a substantial proportion of potentially salvageable patients might achieve return over spontaneous circulation (ROSC) . Early RThe results would be more consistent if duration of CCCPR before OCCPR were known. This would allow exclusion from the analysis of cases where ROSC occurred before the average duration of CCCPR at OCCPR initiation.While acknowledging that the utilization of an instrumental variable (IV) might be used to control for unmeasurable confounders in non-randomized experiments, its choice should nevertheless respect the fundamental assumption that the IV is not directly associated with the outcome variable . The IV In our opinion OCCPR in blunt trauma cannot be rigorously associated with reduced rates of hospital discharge and of 24-h survival following Emergency Department admission based on the available evidence. We would like to thank Malinverni et al. for their comments regarding our recent article . We had Information on the duration of CCCPR before the implementation of OCCPR was lacking in the Japan Trauma Data Bank, and could be considered as an unmeasured confounder. However, we performed subgroup analysis according to the timings of cardiac arrest  and pre-hospital transport time, which were considered to be greatly associated with CCCPR duration before OCCPR, and could be surrogate variables. Interestingly, these subgroup analyses did not show significant interactions and did not have any trends for favorable/unfavorable outcomes, indirectly suggesting that the impact of CCCPR duration before OCCPR on in-hospital survival was limited. As for the concern regarding the IV we used, to our knowledge, the volume–outcome relationships regarding OCCPR have not been reported previously, and these relationships do not always exist in surgical procedures . It is cWe would like to emphasize that we did not evaluate the effectiveness of emergency department thoracotomy (EDT), but only a procedure of OCCPR that is a part of EDT, which includes hemostasis of intra-thoracic organs and/or aortic cross-clumping. EDT is occasionally also performed in patients without cardiac arrest. Several reports have described cases where patient survival was dependent on EDT including OCCPR ; we haveHowever, we should recognize that the evidence supporting the effectiveness of OCCPR remains scarce, at least among blunt trauma patients as a whole, and we have to conduct further studies to assess a limited specific population who benefit from EDT including OCCPR.


Cortical sensory systems are active with rich patterns of activity during sleep and under light anesthesia. Remarkably, this activity shares many characteristics with those present when the awake brain responds to sensory stimuli. We review two specific forms of such activity: slow-wave activity (SWA) in the adult brain and spindle bursts in developing brain. SWA is composed of 0.5–4 Hz resting potential fluctuations. Although these fluctuations synchronize wide regions of cortex, recent large-scale imaging has shown spatial details of their distribution that reflect underlying cortical structural projections and networks. These networks are regulated, as prior awake experiences alter both the spatial and temporal features of SWA in subsequent sleep. Activity patterns of the immature brain, however, are very different from those of the adult. SWA is absent, and the dominant pattern is spindle bursts, intermittent high frequency oscillations superimposed on slower depolarizations within sensory cortices. These bursts are driven by intrinsic brain activity, which act to generate peripheral inputs, for example via limb twitches. They are present within developing sensory cortex before they are mature enough to exhibit directed movements and respond to external stimuli. Like in the adult, these patterns resemble those evoked by sensory stimulation when awake. It is suggested that spindle-burst activity is generated purposefully by the developing nervous system as a proxy for true external stimuli. While the sleep-related functions of both slow-wave and spindle-burst activity may not be entirely clear, they reflect robust regulated phenomena which can engage select wide-spread cortical circuits. These circuits are similar to those activated during sensory processing and volitional events. We highlight these two patterns of brain activity because both are prominent and well-studied forms of spontaneous activity that will yield valuable insights into brain function in the coming years. A key feature noted by early investigators of brain activity was that its pattern depends on behavioral state. The most dramatic shift in behavior occurs when falling asleep, when the brain becomes much less responsive to the external world and focuses inwards on an internal one. Sleep would suggest a brain at rest, although it remains active with a structured set of ongoing spontaneous activity patterns that do not rely on external sensory inputs. Less is known about this spontaneous activity compared to activity evoked by external stimulation given the challenges of recording and interpreting it. In contrast to events related to sensory inputs or motor outputs, ongoing spontaneous activity has no fixed point to which it can be referenced. Factors that influence it are less clear and thus harder to experimentally control. Most challenging, though, has been the lack of a clear and consistent framework through which to examine it Raichle, .Converging lines of evidence show spontaneous brain activity during sleep reflects the communication of brain regions to process past experiences and mediate learning and memory in the adult brain , a 0.5–4 Hz oscillation in the sleeping adult brain which synchronizes sensory, motor and association cortices in non-REM (NREM) sleep  genes associated with synaptic potentiation  are decreased during sleep  in the clinical setting (André et al., spindle bursts in the research setting. Khazipov et al. (An important developmental milestone occurs at 28 weeks post-conception, when the human EEG remains discontinuous, but a prominent pattern of short 5–25 Hz bursts super-imposed on a slower 0.5–2 Hz background emerges during active sleep. The dominance of this pattern is illustrated by its many descriptions in the early literature (Khazipov and Luhmann, v et al.  were theAnalogous results were also shown in preterm human infants; the clinically well-known delta brush pattern in the somatosensory cortex follows spontaneous hand and foot movements (Milh et al., Although our understanding of the function of cortical activity in sleep during infancy remains limited, there are clues that spindle bursts play important developmental roles. Limb twitches during development have a clear instructive role to the nervous system. This was shown directly by manipulating the sensory feedback that arrives following a spontaneous twitch in 2-week old rat pups (Pearson et al., It is well established that, although the gross patterning of the somatosensory cortex may occur independently of activity, refinement relies on activity in the sensorimotor system Pallas, . In partThe role of ascending sensory information depends on functional NMDA receptors in the cortex (Iwasato et al., In summary, cortical development depends on activity, which refines neural connections via NMDA-receptor mediated by Hebbian mechanisms. One source of this activity is via ascending sensory inputs, and the spindle bursts generated by these inputs also depend on NMDA receptors. An intriguing possibility that follows is that spindle bursts are an essential pattern of activity that underlies the prolific activity dependent plasticity of early life, perhaps by synchronizing related groups of pre- and post-synaptic neurons (Mohajerani and Cherubini, In this review article, we have discussed two forms of spontaneous cortical activity present during sleep within sensory systems. In the adult, SWA is a prominent pattern activity seen during sleep or light sedation. Previously, it was thought to be a global phenomenon that synchronizes large swathes or cortex and sub-cortical regions. However, as new ideas about its role in scaling synapses within local systems have emerged, evidence has concurrently been gathered to show SWA has localized and regional features that reflect underlying functional cortical circuits. In the developing brain, spindle bursts are the dominant feature of sleep activity. In contrast to the adult brain, they rely only minimally on intrinsic brain circuitry and depend on ascending sensory inputs, which themselves are spontaneously generated by the developing nervous system. The precise roles for spindle bursts remain unclear, but their contribution in synaptogenesis and circuit connectivity is unquestionable.We do not, however, claim any particular functional links between these two forms of activity. In fact, the independence of these patterns is a key message regarding the spontaneous activity of the brain. We believe it is neither noise nor a wasteful consequence of brain structure; it is an useful activity, purposefully generated, and used for specific and definable purposes.DAM and MHM wrote the manuscript, material in this manuscript was used in DAM’s PhD thesis who was supervised by THM. THM edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre . We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD. DMD) that leads to a lack of dystrophin protein production, and results in muscle degeneration and premature death of the patients (reviewed in  . Ou. Ou19]. In conclusion, we have developed a semi-automated and high throughput method for acquiring an entire muscle section and quantifying dystrophin expression for each myofibre within that section. We demonstrate that this method is robust and operator-independent, due to the fully automated acquisition and Definiens algorithm analysis. We tested this new technique on muscle sections of paediatric control, DMD and BMD patients and we showed that the method was able to distinguish even small differences in dystrophin expression. Furthermore, we demonstrated that each dystrophin distribution curve representing either controls or DMD/BMD samples are composed by normally distributed data. Therefore, dystrophin is differentially expressed in the myofibre population within one muscle, underpinning an inherent biological variability which we describe here for the first time. The basis and consequences of this variablility are yet to be elucidated.


After decades-old efforts to nudge consumers towards healthier lifestyles through dietary guidelines, diet-related diseases are on the rise. In addition, a growing share of U.S. consumers proactively chooses nutritional supplements as an alternative preventative way of maintaining good health, a $25.5 billion industry in the United States. This paper investigates possible linkages between the economics of consumer supplement choices and the relationship to important dietary and health outcomes. We use National Health and Nutrition Examination Survey (NHANES) data to estimate the impact of nutritional supplements intake on respondent’s body weight outcomes, controlling for diet quality.: The focus of this article is to determine whether nutritional supplements takers differ from non-takers with regard to their health outcomes when controlling for differences in diet quality, based on individual Healthy Eating Index (HEI-2010) score. The analysis applies treatment effects estimators that account for the selection bias and endogeneity of self-reported behavior and diet-health outcomes. The analysis demonstrates a negative association between supplement intake and BMI but no significant effect on an individual’s diet quality. Our findings suggest that individuals proactively invest into their health by taking nutritional supplements instead of improving diet quality through more nutritious food choices. Our results provide important contributions to the literature on a key food policy issue. Knowledge of the determinants of supplement demand in the context of strong diet-health trends should also be helpful to stakeholders in the U.S. produce sector in their competition over consumer market share. Despite the proven health benefits of a diet rich in fruits and vegetables –6, the aHowever, while the interplay of appropriate food choices, nutrient intakes and physical exercise in consumer health behavior and outcomes –14 is weThis article provides an important research contribution by estimating the relationship between health behavior and its potential linkage to dietary quality outcome measures, utilizing the case of nutritional supplements intake. Our objectives are to identify and quantify (1) determinants of nutritional supplements intake decisions (2) whether and to what extent supplement takers and non-takers differ with regard to diet-health outcomes (e.g. Body Mass Index (BMI)) when differences in diet quality  are controlled for, and (3) whether and to what extent supplement takers and non-takers differ in diet quality (HEI) outcomes when differences in BMI are controlled for.Previous studies acknowledge the interdependence of health behavior, dietary choices and health outcomes in terms of their short- and long-term public health impacts –23. HoweThe analysis in this article builds on , to our PSM has emerged as a popular approach in the estimation of causal treatment effects in economic analyses. Given the reliance of the diet-health behavioral literature on cross-sectional observational data, such as NHANES, the analysis of treatment effects is often complicated by non-linear relationships and limited dependent outcome variables that are possibly endogenous. Compared against established analytical techniques including fixed effects models , HeckmanFrom a policy standpoint, it is important to understand what factors drive consumers’ compliance with nutritional recommendations  and whatEconomists have long been interested in the study of the interdependencies between dietary choices, nutrition and health outcomes in terms of their short- and long-term impacts on diet patterns and public health outcomes . Becker’A diet that follows the recommendations of the 2010 DGAs could be considered as an investment into an individual’s health stock and consuming the recommended amount of fresh fruits and vegetables as an investment in health. If an individual substitutes or complements the fruit and vegetable intake with nutritional supplements, the latter would constitute a similar investment in health capital, given that supplements may contribute to the overall utility derived from good health. Consumption choices such as smoking, alcohol intake, lack of exercise, and poor dietary patterns could accelerate the depletion rate of a person’s health stock. The depletion of the health stock beyond a certain threshold is associated with a higher probability of early death.There are many intertemporal utility functions that could serve as a theoretical model for our analysis, such as the one developed by –34. The In this study, the nature of the NHANES data and the specific research questions make it even more difficult to find suitable instruments. For these reasons, common IV approaches are deemed less suitable. PSM, originally developed by Rosenbaum and Rubin (1983), has enjoyed increasing popularity in empirical studies of situations where the effect and outcome of a specific treatment is of interest ,40–41. ID represents the intake of nutritional supplements , and X is a vector of pre-treatment characteristics (e.g. gender). If the health outcomes are Y0i and Y1i for non-takers and supplement takers, respectively, then the treatment effect for an individual ‘i’ can be written as:The rationale behind the PSM approach is to assess the effect of receiving treatment from a pool of treated and non-treated individuals. In this article, consumers who took nutritional supplements during the past 30 days will be referred to as the treatment group (supplement takers) and those who did not consume any supplements will form the control group (non-takers). We define nutritional supplement intake following  as any sThe propensity score can be estimated with any standard probability model. The population average treatment effect (ATE) and the average effect of treatment on the treated (ATT) are the two commonly cited parameters of interest in literature and are given by:Y(1) and Y(0) are the two possible outcomes with and without supplement intake. The parameter of interest is the average treatment effect on the treated (ATT), because it gives the difference between expected outcome values of supplement takers and non-takers. Estimating the average treatment effect on the treated is only possible under certain assumptions, because the counterfactual is not observed. Several assumptions need to hold in order to obtain reliable treatment effects using PSM.X, irrespective of their treatment status. This ensures that treatment is random and takers and non-takers are observationally random.p(X) is the propensity score. This implies that all variables that influence treatment assignment and potential outcomes simultaneously have to be observed by the researcher.The first assumption is balancing the pre-treatment variables on a given propensity score ,41,49. TThe next assumption is usually referred to as ‘unconfoundedness’ or ‘conditional independence’ assumption (CIA) ,49–50.This assumption implies that potential outcomes are not dependent on treatment. In other words, variables that can affect both treatment and potential outcomes concurrently have to be observed by the researcher. Another assumption is that of ‘overlap’ ,49 givenX (e.g. income level) are assumed to have an equal chance of being part of the treatment or control group. Once the above assumptions are satisfied, the propensity score of the ATT can then be estimated reliably. To further validate whether our selections models meet the assumption of conditional independence, in other words, whether and to what extend unobserved variables in the treatment selection model may bias the estimates of subsequent treatment effects we perform Rosenbaum bounds tests [This assumption ensures that individuals with the same characteristics ds tests . The bouds tests .The analysis in this article employs data from the 2007–2008 National Health and Nutrition Examination Survey (NHANES) , diet quality and health indicators (outcomes), demographics, and different relevant lifestyle determinants (HEI) is a tool used to measure the diets of Americans against the DGAs. The HEI is composed of twelve sub-components such as HEI Total Fruits, HEI Total Vegetables, HEI Greens & Beans, which carry individual scores that add up to hundred to give the Total HEI. A higher HEI score indicates a diet of higher quality. Using the code written by [The Healthy Eating Index   standardThe focus of this article is to determine whether nutritional supplements takers differ from non-takers with regard to their health outcomes when controlling for differences in diet quality. Supplements are assumed to contribute to an individual’s utility derived from good health and are inputs to the person’s health production function. The factors associated with diet-health behavior and specifically nutritional supplements intake decisions are diet quality, health indicators, demographics, and lifestyle. In order to identify and quantify the determinants of supplement intake decisions, the PSM model in  was estidiabetes and blood pressure [pressure –57, othepressure ,55. In aage, ethnicity, a higher level of education, and a higher household income. These results conform to previous research [males are 59% less likely to take nutritional supplements compared to their female counterparts. This finding confirms the results of previous studies [male and supplement intake suggests that females might be more concerned about diet behavior. Our findings suggest that ethnic heritage seems to play an important role in determining selection into the treatment group. In comparison to African American individuals, individuals of other races are more likely take nutritional supplements.research ,59–61,65 studies ,59,62,65household size on nutritional supplements intake suggests that members of larger households may not consume supplements, given budgetary constraints [education may be in a more informed position to take control of their health. Participants who fall in the highest income group, often correlated with higher educational attainment have the greatest propensity (69%) to take supplements, which confirms an income and educational gradient in that nutritional supplements intake decisions reported in previous studies [The negative effect of straints . Consume studies ,65.food stamp recipients are 22% less likely to take nutritional supplements compared to other respondents. Food stamps may not be used for the purchase of vitamins and supplements [Our results for the lifestyle category show that plements . Our ressmokers are 17% less likely to take nutritional supplements as compared to non-smokers. This negative relationship may indicate that smokers are less concerned about their health. Our explanation for the sign change in the smoker variable indicates that smokers tend to be less concerned about their health. However, alcohol and taking nutritional supplements. Previous research shows that the health impact of alcohol on diet quality is ambiguous [As has been commonly found in previous related literature ,65,67–69mbiguous .Individuals who exhibited active lifestyles are 49% more likely to take nutritional supplements. This is consistent with findings from previous literature ,69,72–73In order to deepen the PSM analysis, we used different matching algorithms to build on the estimated PSM model in order to determine whether regular consumers of nutritional supplements may display improved health outcomes, as measured by their BMI. Thus, we aimed at quantifying whether and to what extent supplement takers and non-takers differ in BMI outcomes when variations in diet quality (HEI) are controlled for. We used the factors discussed in 2The results in The significant difference in BMI between nutritional supplements takers and non-takers is striking, because the components of the HEI-2010 did not have a significant effect on the selection into the treatment group. Our results expand the findings of previous studies that have found inconclusive results . AccordiBMI and some college into our model.In order to quantify whether and to what extent supplement takers and non-takers differ in diet quality (HEI) outcomes when differences in BMI are controlled for, we repeated the matching procedure while controlling for differences in BMI. BMI has a significant negative relationship on selection into the treatment group. Previous research has documented the negative relationship between BMI and nutritional supplements intake [The introduction of another education variable resulted in all of the education variables becoming significant at explaining the selection into the treatment of group of being a nutritional supplements taker. s intake ,69,72–75HEI Total, HEI Total Vegetables and HEI Total Fruits) due to the known relationship between fruit and vegetables intake and obesity. We calculated ATTs to determine whether significant differences exist between supplement takers and non-takers in terms of HEI. Furthermore, we selected three sub-component scores of the HEI-2010  and obesity status as measured by BMI. Thus, consumers of diet supplements do show a lower BMI compared to non-takers. Given decreasing intake levels of fruits and vegetables, it is important to determine the role nutritional supplements play in health behavior and in determining diet quality outcomes.Indeed, we find that nutritional supplements intake may have a positive effect on diet quality of supplement consumers, which in turn may affect diet health outcome indicators, such as the BMI. However, the effect of supplement intake as an explicit health behavior does not significantly alter the levels of overall diet quality or any of its important sub-components observed in this study. This finding expands the results commonly found in consumer stated preference surveys on diet-health in that a direct linkage between preventative health behaviors and the consumption of fruits and vegetables exists .However, the estimated differences in obesity and diet quality outcomes between nutritional supplement takers and no-takers should not be interpreted as the causal effect of supplementation. Instead, the binary supplement intake variable in the PSM analysis represents a proxy of individual’s (supplement taker’s) health behaviour, which results in significant differences in the chosen outcomes when compared against a control (non-taker) population.2, when all other observable characteristics between supplement takers and non-takers are controlled for. We also found that supplement takers are likely to be white, highly educated, of higher household income, non-smokers and of overall higher health status.The results of the analysis suggest that several health indicators, demographics, and lifestyle variables significantly affect the selection into the treatment group of nutritional supplement takers. Nutritional supplement intake is positively associated with a significantly lower BMI of above 1kg/ (body height in meters)The findings reveal that food stamp recipients and lower income households do not take nutritional supplements, even though these two groups may be especially at-risk groups of failing to meet recommended intake levels for major fresh food categories (e.g. fresh fruits and vegetables). On the other hand, individuals of normal weight  and individuals who consume more fruits were found to proactively hedge against health risks by consuming nutritional supplements a preventative measure for maintaining good health. One way to encourage consumption of nutritional supplements among at-risk groups would be to establish a health policy on consumption, especially with regards to fruits and vegetables and nutritional supplements, in order to target specific at-risk populations.Given the increasing importance of individuals’ dietary choices to consumer diet-health and public policy in the United States, accurate estimates of existing behaviors and their impacts on relevant health outcomes have become essential tools for the purpose of policy guidance. Moreover, greater general awareness of diet-health issues and trends toward proactive health behaviors have increased consumers’ demand for more products with identifiable health benefits . This trend towards “nutritionism”  demands Finally, a key component in the quest for improving food policies is the improvement in analytical methods aimed at eliminating the self-selection bias and resulting mismeasurement commonly associated with working with cross-sectional observational data, such as NHANES. The econometric analysis carried out in this article contributes to the discussion regarding whether consuming supplements leads to positive diet-health outcomes. Appropriate methods such as treatment effects estimators (e.g. PSM) can provide more reliable insights into an individual’s diet and health behavior, which will provide the prerequisite for effective and efficient public policies.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


A sexual history offers an opportunity to identify risk factors for contracting sexually transmitted infections and to promote safe sexual practice, thus is an important skill for healthcare professionals regardless of specialty. However, despite medical students and clinicians alike agreeing that taking a sexual history is an important skill, many feel ill-prepared to take one ,2. We prEmbarrassment, fear of being insensitive and cultural differences can make sexual history-taking a challenge for any medical student or clinician . This apAsked to take our first sexual histories in a clinic, we found it difficult to phrase sensitive questions and were worried about embarrassing ourselves . Hesitance led us to omit certain topics entirely. On reflection, although we feel that taking histories under supervision is an integral part of student training, additional techniques implemented in other aspects of our clinical education may allow us to make more of this opportunity. For example, performing intimate examinations on patient-experts made us more confident prior to seeing patients and the use of group role-play has allowed us to identify examples of good practice and potential pitfalls when having difficult discussions. An initiative combining group work, role-play and clinical attachments was found to encourage discussion on attitudes and assumptions surrounding sexual behaviour . FurtherFollowing on from student feedback, our medical school is introducing simulated teaching sessions into our sexual health curriculum. We hope that establishing this form of teaching has the potential to shape a generation of medical students more competent and comfortable in identifying sexual health issues.


A trend towards the abandonment of obtaining pure culture isolates in frontline laboratories is at a crossroads with the ability of public health agencies to perform their basic mandate of foodborne disease surveillance and response. The implementation of culture-independent diagnostic tests (CIDTs) including nucleic acid and antigen-based assays for acute gastroenteritis is leaving public health agencies without laboratory evidence to link clinical cases to each other and to food or environmental substances. This limits the efficacy of public health epidemiology and surveillance as well as outbreak detection and investigation. Foodborne outbreaks have the potential to remain undetected or have insufficient evidence to support source attribution and may inadvertently increase the incidence of foodborne diseases. Next-generation sequencing of pure culture isolates in clinical microbiology laboratories has the potential to revolutionize the fields of food safety and public health. Metagenomics and other ‘omics’ disciplines could provide the solution to a cultureless future in clinical microbiology, food safety and public health. Data mining of information obtained from metagenomics assays can be particularly useful for the identification of clinical causative agents or foodborne contamination, detection of AMR and/or virulence factors, in addition to providing high-resolution subtyping data. Thus, metagenomics assays may provide a universal test for clinical diagnostics, foodborne pathogen detection, subtyping and investigation. This information has the potential to reform the field of enteric disease diagnostics and surveillance and also infectious diseases as a whole. The aim of this review will be to present the current state of CIDTs in diagnostic and public health laboratories as they relate to foodborne illness and food safety. Moreover, we will also discuss the diagnostic and subtyping utility and concomitant bias limitations of metagenomics and comparable detection techniques in clinical microbiology, food and public health laboratories. Early advances in the discipline of metagenomics, however, have indicated noteworthy challenges. Through forthcoming improvements in sequencing technology and analytical pipelines among others, we anticipate that within the next decade, detection and characterization of pathogens via metagenomics-based workflows will be implemented in routine usage in diagnostic and public health laboratories. Clostridium perfringens (11%), Campylobacter spp. (8%), and non-typhoidal Salmonella spp. (5%). According to Health Canada, approximately 2.4 million cases or 60% of foodborne illness are attributed to unknown causes versus only 1.6 million cases or 40% causatively linked to 30 recognized foodborne microbes which include bacteria, viruses and parasites  and antigen-based tests  among others. The extensive adaptation of CIDTs to clinical and food settings is largely reliant on inherent advantages over traditional culture-dependent diagnostic tests; use of CIDTs offers a considerably faster TAT which is crucial for (i) clinical decision-making and decreasing the unnecessary use of broad-spectrum or ineffective antimicrobials, (ii) early outbreak detection and control, and (iii) food industry release or recall of products. Moreover, most conventional CIDTs require less technical expertise and in the long-term may offer a more cost effective alternative.Campylobacter spp.  thus eliminating the requirement for pure culture. Metagenomics and similar techniques traditionally have been applied to interrogate microbiomes of a particular ecological niche through sequencing of all nucleic acids recovered from a sample . MoreoveFigure 2. Herein, we will review the current state of CIDTs as they relate to foodborne illness and food safety. We will furthermore discuss the diagnostic utility of metagenomics and comparable detection techniques in clinical microbiology, food and public health laboratories.Laboratory methods that detect and identify pathogens serve two critical functions: clinical decision-making  and public health decision-making (population level). While traditional culture-based methods meet both of these needs simultaneously, CIDTs are exclusively designed to improve clinical decision making only. This leaves critical public health activities at great risk. A summary of techniques used in primary diagnostic labs is illustrated in Traditional culture-based methods for detecting enteric pathogens in biological specimens, food processing environments or foods is dependent on the growth of viable and culturable microbes; biochemical tests and subtyping are further needed to confirm microbe identification and to establish genetic linkage, respectively. Though sensitive, relatively easy and inexpensive, culture-based methods are laborious and ineffective for non-culturable or fastidious microbes. The diagnostic potential of CIDTs to circumvent the demanding task of culturing and subtyping pathogens is a widely attractive alternative.Campylobacter (92%), Shigella (284%), Salmonella (247%), and STEC  up to 22 microbes. Commercially available assays differ in their microbial targets detecting distinct subsets of bacteria, viruses and/or parasites. Second, the various assays have different TAT – most ranging from 1 to 4 h – though even the most time-intensive assays (8 h) are considerably faster than stool culturing which can take 2–5 days dependent on the microbe being cultured. Third, multiplex panels retain the ability to detect polymicrobial infections, which is particularly relevant in clinical settings. Lastly, multiplex panels are highly sensitive (90–100%) and specific (98%) .Next-generation sequencing and other high-throughput laboratory techniques are circumventing laborious testing by active replacement or complement to traditional microbiological and molecular tests for identifying, typing and characterizing pathogens. An increase in read-length, output and quality of short-read NGS technologies has made it possible to apply WGS as a genomic surveillance system of foodborne diseases and outbreak management . In mostSalmonella enterica serovars such as S. enteritidis have been challenging to resolve using PFGE (the gold standard typing method). For some clonal microbes, molecular typing methods have proven unable to accurately discriminate genetically distinct isolates. As an example, in past outbreak investigations, highly clonal Listeria monocytogenes isolates  in a technique referred to as shotgun metagenomics . Though cpn60 . As such, these analyses are often performed by high-throughput targeted-amplicon sequencing of a universal phylogenetically informative genetic marker; the 16S rRNA gene is most commonly used though other markers are similarly able to discriminate between prokaryotes including cpn60 , rpoB . Features differentiating the two methods include targeted microbial community, associated costs and computational and technical expertise requirements among others. Thus, the decision to use one approach versus the other should be made with careful consideration and will be highly dependent on the research and/or diagnostic goals and hypotheses in question (Table 2). In this regard, targeted-amplicon assays are more apt for describing a specific group of microbes  whereas a shotgun metagenomics approach is more suitable for characterizing the entirety of microbial DNA in a given sample limited by sequencing technology used and sample matrix with high host DNA-containing material.While the science community has persistently been using the term metagenomics interchangeably to describe both high-throughput targeted-amplicon and shotgun metagenomics studies to profile microbial populations, it is important to make a clear distinction between both methodologies. Targeted-amplicon and shotgun metagenomics sequencing each present with advantages and disadvantages (L. monocytogenes1. This is attributed to how foods are categorized based on the ability to support (or not), the priority assigned to high-risk foods and the ability of the pathogen to survive during the shelf life of the food1. Moreover, the target pathogen is often in such low numbers that in the presence of the background microbiota, enrichment is required – and culturing is the most effective strategy . If however, a high-sensitivity metagenomics methodology, capable of reliably detecting foodborne pathogens in samples with high-levels of background microbiota were developed, the time for outbreak recognition, causative agent sub-typing, secondary analyses for virulence and AMR genes and source attribution would be reduced . However, even after suitable techniques are developed, widespread use of CIDT as a replacement for culture-based techniques will still face significant challenges in food regulation and industry. International regulatory agencies with stakes in food safety such as Health Canada, US FDA, French agency for Food, Environmental and Occupational Health Safety (ANSES) are still in the initial phases of accepting WGS results as a replacement for more traditional techniques for outbreak investigations2. Some countries though, are much further along in this process than others  of supporting growth  may be elucidated through the use of WGS. Metagenomics has been used to help determine aspects related to enrichment , suggesting that an alternate pre-enrichment media should be developed for cilantro testing . Sanger sequencing of norovirus requires several PCR rounds to obtain usable information, and still only yields partial genome sequences . The metatranscriptomics approach has the advantage of being rapid and further, a de novo assembly can be obtained. This approach is thorough and additionally, allows for the presence of other co-infections to be detected from the same sequence data (Kudoa septempunctata) was the likely cause of the outbreak , which is unsurprising given the challenges associated with culturing most viruses. Throughout the last decade, viral metagenomics (viromics) has been used as a tool to diagnose cases of acute gastroenteritis and further, to determine etiological causation retrospectively in gastroenteritis outbreaks . This teCampylobacter jejuni sequences from a stool sample obtained when the patient was symptomatic versus no sequences identified in a sample obtained 3 months post-illness. This investigation represents one of the first proof-of-concept studies to be conducted in a clinical diagnostic setting.An early study tested the capability of shotgun metagenomics sequencing to detect bacterial pathogens during a case of acute gastroenteritis following culture based diagnostics where no candidate pathogens were identified . This caLeptospiraceae family and mapped most closely to the pathogenic Leptospira borgpetersenii genome. The patient was therefore treated for neuroleptospirosis with intravenous penicillin G and markedly improved. In this context, treatment was initiated on the basis of metagenomics evidence and congruent clinical presentation prior to the completion of validated confirmatory testing. Five months post-treatment, targeted PCR and Sanger sequencing identified L. santarosai as the infectious agent. This case highlights various advantages and limitations associated with diagnostic metagenomics that will therefore be discussed in the following section.A relatively recent case study emphasizes the clinical relevance of metagenomics as a diagnostic assay . BrieflyBrucella spp. detection) and directed towards appropriate antibiotic therapy ultimately leading to a favorable patient outcome  and NGS techniques (16S rRNA targeted-amplicon and shotgun metagenomics) for nfection . IntriguWe mentioned previously that high-throughput targeted-amplicon sequencing is at the moment largely research based. A recent study described the usefulness of 16S rRNA targeted-amplicon sequencing in an exploratory setting . This stDientamoeba fragilis. While the role of D. fragilis in causing gastroenteritis remains unclear it is suggested that in the absence of other enteric pathogens that this parasite could be considered a causative agent  in pediatric samples. A more recent study applied several diagnostic techniques  to four diarrheal samples as a means to detect multiple pathogens simultaneously  in the human gastrointestinal tract, in addition to elucidating genomic epidemiology. ve agent . This staneously . Metagenorldwide  and in aa malayi .L. borgpetersenii; confirmatory testing revealed the causative agent to be L. santarosai. In this context, query sequence homology to a reference sequence should not be misconstrued as causation. Third, the exceptionally short TAT led to appropriate therapeutic management and thus clinically actionable results, which corresponds to our next discussion point – perhaps most importantly is the developmental need for analysis tools and bioinformatics pipelines modernized for the requirements of diagnostic laboratories. For example, tools for pathogen detection must provide analytical results while concurrently minimizing analysis time to allow for reasonable diagnostic TAT. Though several analyses software or platforms for microbial identification are available  metagenomics data is overwhelmed by the abundance of host DNA. Considerations should be given to samples where eukaryotic cell content is expected to be high . Similarly, variable host DNA content will be found in samples such as stool dependent on the consistency and quality . Studies have attempted to address the issue of contaminants  and hostWhile we frequently refer to metagenomics as an unbiased methodology, there are biases that need to be considered to ensure that all microbes of interest are captured adequately in the sequencing output. As an example, interrogating the DNA or RNA virome using a metagenomics approach  requires complex wet-laboratory procedures that are considerably distinct from that of a sample workup for a general metagenomics assay of ‘all’ microbes. Viruses have significantly smaller genomes as compared to prokaryotes or eukaryotes, thus their genomic content represents a microscopic fraction of total DNA or RNA in a given sample . Moreove7–109 for enterotoxigenic E. coli, L. monocytogenes is 103 and Salmonella spp. is only 4–50  is 10nly 4–50 . Achievinly 4–50 . The autMetagenomics has already proven an effective complement to conventional diagnostics in complex cases and outbreaks  though mMetagenomics sequencing of a clinical or environmental sample could offer a universal test for pathogen detection, clinical diagnosis, as well as a subtyping test for routine surveillance activities. Surveillance for foodborne infections historically has required the culturing of an isolate in order to perform the appropriate genotypic or phenotypic characterization, yielding a phenotype or genetic fingerprint capable of making detecting and resolving outbreaks possible. By tracking foodborne pathogens along the farm to fork to clinical specimen continuum, it is possible to monitor trends over time, track which foods are capable or implicated in causing illnesses and detecting outbreaks.5  has relied on these for nearly two decades but is presently transitioning to the use of WGS. The other national surveillance systems dedicated to foodborne disease in Canada, the National Enteric Surveillance Program6  and FoodNet Canada7  are also transitioning from molecular subtyping to WGS.Similar to trends occurring in infectious disease diagnostics, techniques for subtyping have enhanced the ability to distinguish between epidemiologically linked isolates from identical microbial species hence improving outbreak detection, surveillance and the overall understanding of microbial epidemiology. Molecular subtyping techniques in routine use include PFGE and MLVA; PulseNet CanadaS. enterica serotype Montevideo outbreak  however, are reliant upon the presence of isolates. Techniques used in public health surveillance for disease tracking and subtyping therefore necessitate adaptation to the culture-independent trend.Rapid infectious disease diagnostics would particularly benefit from the ability to directly subtype pathogens from complex clinical specimens. While subtyping methods such as targeted-amplicon sequencing, FISH  and repeEffective detection of outbreaks, particularly broadly disseminated outbreaks caused by the commercial distribution of contaminated foods is largely dependent on subtyping isolates from a sizeable proportion of cases. As diagnostic laboratories are in the process of shifting from diagnostic tests (culture-based or WGS) yielding isolates to CIDTs , it is essential to maintain a system whereby subtyping can still be performed on a large percentage of positive specimens.8 to ensure that isolation is attempted or that positive CIDT specimens are retained – Canada is in the process of developing analogous guidelines.With changing diagnostic practices, several options to maintain the capability of foodborne surveillance, outbreak detection and source attribution are possible. First, clinical (and food) microbiology laboratories could perform reflex culturing of biological specimens (or food) that test positive via CIDTs such that positive isolates can still be submitted to public health laboratories for subtyping or other culture-based tests such as WGS . Second,A limited number of conventional growth-based methods for AST have persisted in routine usage throughout the transformation of diagnostic microbiology. Included among these are disk diffusion  strategies and broth microdilutions. The latter of which has achieved gold standard status; thus, novel AST methods are compared to the efficacy of broth microdilutions from development through clinical trial. At present, AST is either accomplished via the above-listed conventional manual methods or growth-dependent automated AST systems including the Vitek System , Avantage Test System , Phoenix  and others, all of which are based on broth microdilution testing . More reSalmonella  and tetracycline  resistance whereas macrolide  resistance was variable ranging from 3.7 to 11.1% of samples. It was also shown that WGS of isolates predicted AMR phenotypes with high accuracy. A recent study explored the utility of shotgun metagenomics to detect MDR pediatric bacterial infections, specifically, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and MDR Enterobacteriaceae (Numerous studies have applied shotgun metagenomics sequencing to detect AMR genes. The f 1 gene . The moseriaceae . The stuE. coli). The transformant library is then assayed for AMR by culturing on selective media – persistent AMR genes are sequenced and annotated. Therefore, this method is advantageous, ascribed to the possibility to determine genotypic and phenotypic traits.Functional metagenomics on the other hand is an exceedingly powerful technique attributed to its capacity to discover novel and highly divergent AMR genes . This meAs NGS-based methodologies like WGS are proving useful in clinical and public health laboratories, we expect that other advanced sequencing techniques will similarly be effective in amalgamating molecular AST and phenotypic susceptibilities thereby allowing for a complete bypass of microbial culture. In this regard, NGS supplemented with metatranscriptomics or proteomics can be more informative as it allows for description of both gene presence and expression . A combiSalmonella spp. and Campylobacter spp., as well as virulence profiles in for example STEC. At the Canadian public health level, AMR surveillance is conducted through the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS)9 in combination with FoodNet. Importantly, though AMR in a clinical setting is largely associated with the utilization of antimicrobials for the treatment of infections, their use in agri-food production is also known to contribute to the resistant microbe pool. Resistant bacteria are associated with more severe disease  thus creating the ability to detect any and all pathogens  from various biological specimens or food products. Through forthcoming improvements related to required technical expertise, throughput and cost-effectiveness of sequencing combined with enhanced and streamlined laboratory and bioinformatics pipelines, metagenomics will likely have a predominant role in the diagnostic and public health laboratory. We expect an automated metagenomics pipeline will complement and may even replace several methods currently employed in the diagnostic laboratory while concurrently providing additional information such as AMR, virulence, and genomic epidemiology. It is apparent that the functionality of diagnostic metagenomics has been established in research settings and further, in detecting etiological culprits of unidentified illnesses and outbreaks. We anticipate that within the next decade, detection and characterization of pathogens via metagenomics-based workflows will be implemented in routine usage in diagnostic and public health laboratories.The prospective use of diagnostic metagenomics and comparable techniques offers an assumption-free workflow (though biases are present with each methodology; NK and AR developed the concept for the manuscript. JF, NK, JR, FP, and AR wrote the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adult hippocampal neurogenesis (AHN) in mammals peaks in early postnatal/juvenile periods and is strongly down-regulated thereafter. Depending on species, it may disappear rapidly in adult individuals, or persist at very low levels for a lifetime. Commonly, higher levels of AHN in mammals are thought to provide mental flexibility allowing for adapting to new ecological niches. But why does natural selection not prevent down-regulation of AHN, and why should a rudimentary proliferation rate in humans provide reproductive fitness even for aged individuals? The problem is compounded by species-specific behavioral manifestations of hippocampal functions that depend on brain size and ecological niches. Moreover, in laboratory rodents, proliferation levels of AHN and behavioral covariates appear unpredictable and context-sensitive. Conversely, one might ask why evolutionary mechanisms tolerate in nearly all mammals a certain level of early postnatal or subadult AHN. Specifically, the hypothesis of cognitive flexibility appears odd in species in which AHN is massively reduced in early infancy such as in humans. I suggest that early but not late AHN plays a hidden role in developing randomly different epigenetic personality traits in local populations. Such traits may counteract or enhance natural selection of the underlying genetic architecture—a process known as genetic assimilation.de novo  from which neuroblasts migrate rostrally to the olfactory bulb  and from a secondary proliferation zone in the dentate gyrus, the subgranular zone (SGZ). The ongoing postnatal proliferation there is denoted as “adult” hippocampal neurogenesis (AHN). Molecular markers for migration and differentiation are often not correlated with basic levels of AHN in many species. For example, doublecortin (DCX) is a reasonable proxy for estimating proliferation rates in mice and rats. In other species such markers persist for long periods after the cessation of proliferation or appear even generated  Amrein, . TherefoThe evolutionary role of AHN in mammals is not understood. The most straightforward explanation shared by many has been offered by Kempermann , 2016 whTo answer this, one would need to know what behaviors and abilities are clearly correlated with hippocampal structural traits including AHN. Perhaps surprisingly, the situation is not clear since even laboratory rodents show a bewildering variety of hippocampus-dependent behaviors, mostly ignored by AHN research. For one, they include natural behaviors such as food burrowing and nest building  correlates genetically and individually with behavioral traits, not all of them being considered as “hippocampal”  has a negligible impact on adult on human hippocampal functions. Nowadays, this view has become almost heretical. But one might rightly ask why a putative beneficial structural trait has been curtailed in humans and not increased by natural selection. At present there is no answer. A conceptually related question is why natural selection has (regrettably) not increased the intelligence quotient in humans from an average of 100 to one of 140 points. Obviously, reproductive fitness—a benchmark of evolution—is not strongly correlated with cognitive abilities. Its reproductive advantage is counteracted by competing traits such as physical attractiveness and, among males, physical strength and aggression, not infrequently at the disadvantage of courting academics. For human AHN, the reasons are less obvious. Possibly, the emergence of highly excitable young neurons may interfere with the orchestrated development of other granule cell functions (Drew et al., On the other hand, comparative analysis across mammals Amrein,  suggestsThe problem in humans is to find a useful function for early postnatal neurogenesis during a period of reduced cognition. This function ought to provide a target for natural selection in adults in order to maintain this developmental trait. One might argue that hippocampal neurogenesis in such an early postnatal stage could help in organizing the development of forebrain circuitry underlying cognitive processing, e.g., the emergence of language. But this should have consequences for adult behavior many years later. Yet it remains questionable whether individual differences in language development do have an impact on evolutionary relevant adult behavior.On the other hand, personality traits such as risk-taking behavior or food preferences must have been obvious targets for rapid natural selection during human evolution, specifically in small populations. In larger populations, genetic variation balances competing traits, permitting rapid natural selection of carriers fitting into a changed environment. However, in smaller groups natural selection, for example for timidity, might rapidly eliminate genetic variation supporting risk-taking behavior. This can reduce the ability of genetically adapting to new environments suddenly favoring risk-takers.Loss of evolutionary plasticity due to natural selection is a common problem for small populations in many species. A possible mechanism counteracting such processes is the early development of personality traits in absence of genetic variation. The unpredictable occurrence of correlations between AHN and personality traits in isogenic rodents suggests that such personality traits emerge randomly. For example, sucrose preference shows strong genetic variability in mice and humans (Reed et al., This idea is in line with recent findings of an elegant study using a chemogenetic approach to inactivate selectively perinatally and postnatally born olfactory neurons in mice (Muthusamy et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.