Cocaine is associated with a wide array of complications through a number of different mechanisms. Although the majority of cocaine-related morbidity has been attributed to complications in arterial vasculature, the deleterious impact of venous complications appears to be largely unrepresented in current literature as well as clinical practice despite emerging evidence of the high prevalence and annual incidence of deep vein thrombosis (DVT) in illicit drug users.Our case report illustrates an uncharacteristic presentation of cocaine-related widespread thrombotic cascade involving both arterial and venous circulations causing significant morbidity. The complex pathophysiology of widespread prothrombotic state caused by cocaine includes endothelial damage promoting the increase of fibrinogen and Von Willebrand factor to platelet aggregation and clot formation. It is important to identify the impact cocaine-induced venous thrombosis can mount, especially in the form of potentially fatal complications like pulmonary embolism. Although recent studies have focused on increased incidence and prevalence of venous thrombosis in the setting of cocaine abuse, ours is the first case of a documented pulmonary embolism caused by cocaine-related venous thrombosis. Further studies are needed to identify patients at higher risk for this complication like rare thrombotic disorders. Majority of drug misuse emergency department (ED) visits nationwide have been contributed by cocaine use. Additionally, short term visits involving illicit drugs were documented to have experienced an increase by a tremendous 29% -2.The consumption of cocaine is associated with a wide array of complications through a number of different mechanisms. These adverse effects include a wide range of cardiac , arteriaThis case illustrates an uncharacteristic presentation of cocaine-related widespread thrombotic cascade involving both arterial and venous circulations and causing significant morbidity.A 27-year-old previously healthy male presented to the ED with a new onset of difficulty speaking and facial droop. The patient was last seen normal eight hours prior to the presentation when he went to sleep. On waking up, his brother noticed a slurred speech along with a right facial droop and called the emergency medical services.On arrival, he was afebrile with the following vitals: temperature 98.5 °F, blood pressure 142/97, heart rate 94, and respiratory rate 16. The examination was remarkable for expressive aphasia and right facial droop with no other focal neurological finding. He was alert and oriented to person, place, time and in no acute distress apart from being irritable due to word finding difficulty. History and review of symptoms were limited due to the patient’s aphasia, and writing board was used to illicit limited pertinent history. He denied headache, nausea, vision changes, weakness, chest pain or palpitations. Although initially hesitant, he eventually reported using intravenous heroin on a daily basis and cocaine for the first time on the previous day. Family history, as reported by his mother, was remarkable for death of patient’s father from a myocardial infarction (MI) three months ago, history of MI in his uncle at age of 46, and two spontaneous abortions in paternal aunt who had a known clotting disorder, the specifics of which could not be established.Stat computed tomography (CT) scan of the head showed thrombozed left middle cerebral artery (MCA) along with infarct in the left frontal lobe as well as basal ganglia Figure . HematolComputed tomography angiography (CTA) of head and neck showed complete abrupt occlusion of the proximal left internal carotid artery (ICA) with distal reconstitution at the segment M1 of MCA and an intraluminal thrombus in the M1 segment. An ICA dissection leading to occlusion was possible as per the imaging. A limited bedside echocardiogram was remarkable for severely reduced right ventricle systolic function with akinesia of basal and mid free wall raising concern for right ventricular infarct versus strain from a pulmonary embolism (PE). CTA of chest done due to significant troponinemia and EKG/echo findings revealed bilateral segmental and sub-segmental PE without any acute pulmonary disease Figures -4.In the setting of acute stroke, the patient was deemed not a candidate for tissue plasminogen activator (tPA) or endovascular intervention due to low National Institutes of Health Stroke Scale (NIHSS) scale of 4. At presentation, he was also not considered a candidate for percutaneous coronary intervention or anticoagulation, due to high risk of hemorrhagic conversion of his territorial stroke should he require dual antiplatelet therapy and/or anticoagulation if found to have coronary artery thrombosis.Further management included placing an inferior vena cava filter in setting on PE with contraindication to anticoagulation. Magnetic resonance imaging (MRI) of the brain showed infarction in the left frontal lobe and basal ganglia consistent with patient’s expressive aphasia as well as small areas of deep cortical infarction in the caudate and sub-insular area. A trace area of hemorrhagic transformation was identified in the caudate region. A later Doppler of lower extremities revealed DVT in the left peroneal and posterior tibial veins.Due to limited area of hemorrhagic transformation identified in the caudate region, after the multidisciplinary team’s discussion with the family, a decision was made to start the patient on low dose anticoagulation without a bolus with plan to repeat a CT head over the next 24 hours to monitor for worsening hemorrhagic conversion while being continued on Q1 hour neuro-checks.A following comprehensive echocardiogram with a bubble study next day failed to reveal an intracardiac shunt precluding the possibility of paradoxical thrombosis. While still awaiting the results of hypercoagulable workup and repeat CT head without worsening hemorrhagic conversion, the patient was continued on anticoagulation with intravenous heparin for 10 days before being switched to oral anticoagulant Xarelto. He experienced improvement in word finding ability with every passing day and was beginning to frame two to three-word phrases by the end of 10 days when his care was transitioned to a rehabilitation facility for continued physical, occupational, and speech therapy. Subsequently, hypercoagulability work-up including prothrombin gene mutation, factor V Leiden deficiency, anti-phospholipid antibody (APLA), and anti-thrombin deficiency eventually turned out to be negative.Right from the initial presentation, it was challenging to find a unifying diagnosis for this peculiar constellation of findings including widespread venous and arterial thrombosis causing DVT and PE, carotid dissection, and ischemic stroke. However, the initial leading hypothesis was that an intracardiac shunt, if present, would explain the paradoxical thrombus shower into arterial circulation including coronary arteries as well as ICA. Given the patient's young age with a significant family history of MIs, pregnancy loss and known clotting disorder, the genetic hypercoagulable state was also high on differential; appropriate tests for factor 5 Leiden mutation, APLA, prothrombin gene mutation, and antithrombin were conducted. Although low on differential, there was a cascade of thrombosis caused by intravenous drug abuse leading to arterial as well as venous thrombosis.However, with subsequent comprehensive echocardiogram unrevealing for a patent foramen ovale and negative for common hypercoagulable states, left a few possibilities which could include rare thrombotic states , acute hypercoagulability caused by illicit drug abuse or possibly, a combination of both.Cocaine-related complications, especially in the arterial circulation have been well documented These include cardiac complications such as sudden death, acute myocarditis, dilated cardiomyopathy, life-threatening arrhythmias, myocardial ischemia and infarction, dissections of coronary, aortic, carotid, and arteries. The major cerebrovascular effects of cocaine consist of ischemic and hemorrhagic strokes -7. The mAdditionally, inflammation and atherosclerosis are substantial potentially lethal vascular effects of cocaine use that have acute and chronic systemic impact -13 CocaiIn contrast to arterial thrombosis, the prevalence, incidence, and morbidity associated with cocaine-induced venous thrombosis seem to be overlooked in the current literature. This is likely due to the acuity, higher frequency, and mortality associated with arterial thrombosis. However, as exemplified in our case report, it is important to identify the impact cocaine-induced venous thrombosis can mount, especially in the form of potentially fatal complications like pulmonary embolism. Although ours is the first case of a documented pulmonary embolism caused by cocaine-related venous thrombosis, many recent studies have focused on increased incidence and prevalence of venous thrombosis in the setting of cocaine abuse .Although the mechanisms of clotting used to explain arterial thrombosis can be implicated in venous thrombosis as well, the lower prevalence of the latter prompts further curiosity in elucidating additional factors that may be involved in pathogenesis cocaine-related venous thrombosis; the identification of these factors is crucial for their anticipation and management. Postulated mechanisms include direct toxicity of cocaine to the veins possibly exacerbated by the citric acid used to dissolve the drugs and the local anesthetic property of the cocaine , adulterCocaine can cause not only arterial but also significant venous thrombosis via multiple possible mechanisms and this can contribute to significantly increased morbidity associated with cocaine abuse. As the stride into identifying the most potent risk factors for cocaine-induced venous thrombosis continues, a clinical pearl which can be elucidated from this case is that cocaine abuse in a patient with a family history of clotting disorders can lead to devastating outcomes. Anyone with a personal or family history of thrombotic disorders should be particularly warned of potentially fatal complications of cocaine abuse; and in those presenting with it, it may be befitting to chase rare genetic disorders that still need to be identified to better serve this population.


Up to half of all patients do not respond to pharmacological treatment as intended. A substantial fraction of these inter-individual differences is due to heritable factors and a growing number of associations between genetic variations and drug response phenotypes have been identified. Importantly, the rapid progress in Next Generation Sequencing technologies in recent years unveiled the true complexity of the genetic landscape in pharmacogenes with tens of thousands of rare genetic variants. As each individual was found to harbor numerous such rare variants they are anticipated to be important contributors to the genetically encoded inter-individual variability in drug effects. The fundamental challenge however is their functional interpretation due to the sheer scale of the problem that renders systematic experimental characterization of these variants currently unfeasible. Here, we review concepts and important progress in the development of computational prediction methods that allow to evaluate the effect of amino acid sequence alterations in drug metabolizing enzymes and transporters. In addition, we discuss recent advances in the interpretation of functional effects of non-coding variants, such as variations in splice sites, regulatory regions and miRNA binding sites. We anticipate that these methodologies will provide a useful toolkit to facilitate the integration of the vast extent of rare genetic variability into drug response predictions in a precision medicine framework. Pin vivo. However, for the functional interpretation of rare variants these approaches suffer from multiple shortcomings:Heterologous expression in cell lines followed by quantitative determination of gene product functionality using appropriate end points is considered as the gold standard strategy to characterize the functional impact of pharmacogenetic variants. Furthermore, epidemiological association studies can provide additional indications about the consequences of genetic variants on drug metabolism related phenotypes These methods are generally low throughput and are not compatible with the interrogation of tens of thousands of variants.Experimental characterizations are time consuming, expensive and require specially trained technical staff, which renders them unsuitable for the rapid functional interpretation of the pharmacogenotype of an individual patient at the point of care.Epidemiological analyses require a sufficient number of patients who carry the allele, which drastically limits their feasibility for rare genetic variant studies.Thus, in the absence of viable experimental strategies, computational prediction methodologies are routinely used to predict the functional impact of genetic variants. Most of these algorithms focus on predicting the functional consequences of variants that result in amino acid substitutions. However, recently much progress has also been made regarding the interpretation of non-coding variants that affect splice sites, promoters, enhancers or miRNA binding sites Figure .Prediction algorithms are generally trained on pathogenic variant sets and most tools base their conclusions, at least in part, on the evolutionary conservation of the respective sequence. Importantly however, pharmacogenes are hallmarked by low evolutionary conservation and are generally not associated with human disease. These peculiarities result is specific problems for the interpretation of pharmacogenetic variants. Here, we provide an updated overview of computational approaches for the functional interpretation of genetic variants, specifically focusing on their suitability for pharmacogenetic predictions. We describe the underlying statistical frameworks and discuss their different bases for decision-making. Furthermore, we highlight important progress particularly in the interpretation of non-coding genetic variability. We conclude that computational tools are essential for the functional interpretation of an individual's pharmacogenotype and that their further improvement constitutes one of the most important frontiers for the clinical implementation of NGS-based genotyping.Genetic variants that result in amino acid substitution, henceforth termed missense variants, can impact the functionality of the respective protein by various mechanisms, including alterations in active sites, structural destabilization due to protein misfolding, perturbations in solvent accessibility or modification of post-translational processing. Each individual harbors 10,000–12,000 missense variants, many of which are rare  that often pivot around 0.9, drastic drops in performance to AUCROC of 0.5–0.75 have been reported on independent, functionally determined human variant datasets , CYP2C19*2 (MAF = 34% in South Asians) and CYP2D6*4 (MAF = 16% in Latinos)  is modified upon point mutations (ΔΔG°) with negative and positive values of ΔΔG° indicating destabilizing and stabilizing mutations, respectively. In recent years a large number of mechanistically diverse approaches have been presented, with machine learning-based strategies being most prevalent. SDM constitutes a statistical potential energy function that can estimate variant effects on protein stability  causing frameshifts, stop-gain and start-lost variants. Some of these variants are clinically relevant and occur with high frequencies in specific populations, including the stop-gain variant Early bioinformatic tools, such as LOFTEE, prioritize truncation variants based on a set of empirical rules, including whether the variant of interest occurs in the last 5% of transcript or whether the truncating allele is the ancestral state . NMD is a conserved translation-dependent mechanism that is responsible for recognizing and eliminating aberrant mRNA transcripts to prevent the production of truncated peptides, thereby playing a critical role in preventing the accumulation of misfolded protein and subsequent initiation of the unfolded protein response (UPR)  , translational efficacy and ribosomal loading. Effects of indels on RBP binding can be evaluated using PinPor, which has been demonstrated to have some success in distinguishing disease-causing and neutral indels  that allow to model key target tissues associated with drug metabolism or safety provide (Ewart et al., Importantly, leveraging this information as guidance for clinical decision-making promises to increase treatment efficacy and reduce the risks of adverse events in carriers of pharmacogenetic variants whose effects have not been experimentally evaluated. Current market analysis estimates suggests that implementation of artificial intelligence into the clinical decision support toolbox might increase average life expectancy in the Western World by 0.2–1.3 years and reduce total health care expenditures by 5–9%, corresponding to 2 trillion to 10 trillion USD globally per year (Bughin et al., In summary, computational prediction methods are essential for the implementation of NGS into clinical decision-making. While much progress has been made and a plethora of conceptually diverse tools is already available, there is a need to develop specialized methods that are optimized for the prediction of variant functionality rather than pathogenicity and are calibrated specifically on pharmacogenetic data. We envision that technological, methodological and analytical advances will soon allow to comprehensively predict variant effects with sufficient accuracy to justify the design of trials in which the clinical value of NGS-guided treatment decisions can be tested in a prospective setting.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.VL is co-founder and owner of HepaPredict AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phalacrocorax aristotelis), we tested the effect of parasitism on maintenance costs as well as the relationship between proxies for RMR and DEE. We found a positive relationship between parasite load and our RMR proxy in females but not males, and no relationship between proxies for RMR and DEE. This provides evidence for increased maintenance costs in individuals with higher parasite loads and suggests the use of an allocation energy management strategy, whereby an increase to RMR creates restrictions on energy allocation to other activities. This is likely to have fitness consequences as energy allocated to immunity is traded off against reproduction. Our findings demonstrate that understanding energy management strategies alongside fitness drivers is central to understanding the mechanisms by which these drivers influence individual fitness.Parasites often prompt sub-lethal costs to their hosts by eliciting immune responses. These costs can be hard to quantify but are crucial to our understanding of the host's ecology. Energy is a fundamental currency to quantify these costs, as energetic trade-offs often exist between key fitness-related processes. Daily energy expenditure (DEE) comprises of resting metabolic rate (RMR) and energy available for activity, which are linked via the energy management strategy of an organism. Parasitism may play a role in the balance between self-maintenance and activity, as immune costs can be expressed in elevated RMR. Therefore, understanding energy use in the presence of parasitism enables mechanistic elucidation of potential parasite costs. Using a gradient of natural parasite load and proxies for RMR and DEE in a wild population of breeding European shags ( Summary: Demonstration of the sub-lethal costs of endo-parasitism for a wild population of seabirds via increased maintenance metabolism. Parasites cause major fitness consequences to a huge array of taxa . Often, Energy can be a limited resource, and individuals are required to allocate energy to the demands of competing life-history traits to maximize fitness . TherefoParasitism may play a central role in the relationship between self-maintenance and activity because a primary predicted cost of parasitism is increasing RMR, through immune-associated costs . Hosts sUnderstanding the impacts of natural parasite burdens on individual metabolic rate is crucial, with experimental studies suggesting that endo-parasites and elevated immune function incur significant costs to hosts . HoweverPhalacrocorax aristotelis (Linnaeus 1761)] to understand the effect of endo-parasites on maintenance costs as well as the relationship between maintenance and total energy expenditure under high energetic demands during the breeding period. Specifically, we investigate two questions: (1) Does parasite load relate to RMR? We expect that individuals with higher parasite loads will have higher immune costs and that this will be reflected in elevated RMR. (2) Does DEE relate to RMR and what can this tell us about the allocation between energy to reproduction and self-maintenance of this species? We predict that individuals will be energetically constrained by rapidly growing chicks during the chick-rearing period under the allocation model and therefore any increase in RMR will not result in an increase in DEE.Here, we measure proxies of both DEE and RMR in individuals with known natural endo-parasite loads in a free-living population of breeding European shags  using bater see . Thus, aater see .All models were fitted separately for males and females due to non-independence of nest pairs. To answer the first question of whether parasite load is related to elevated RMR, we modelled T3 concentration using linear mixed effects models (LMMs). Parasite load, brood size (number of chicks), brood age (age of the oldest chick in the brood at the time of sampling), year, and adult age and body mass were explanatory variables and we controlled for variation between birds and repeated sampling by including individual as a random factor. We fitted models containing all combinations of the fixed effects, and interactions between parasite load and brood age and parasite load and brood size  using LMMs. Adult age, adult body mass, brood age (at the time of sampling), brood size and year were also included as explanatory variables . We conthttp://www.R-project.org/) and model selection for both sets of models was based on Akaike's information criterion (AIC) (All models were fitted using the lme4 package in R (on (AIC) .A total of 87  individuals were blood sampled and endoscoped over 3 years (of which 27 were sampled in more than one year). Whilst the sample size in 2015 is clearly smaller than the other years, removal of these data had no impact on the conclusions of the analyses and were therefore included. The best-supported model for females showed a positive relationship between parasite load and T3 concentration, an effect of year, and an interaction between brood age and parasite load in that there was a more positive relationship between parasite load and T3 when broods were younger see . When yeDEE was estimated in a subset of the birds  to gain insight into the relationship between RMR and DEE. The best-supported model for females was the null model with no relationship found between T3 and estimated DEE see . For malMeasuring both natural parasite burdens and metabolic rates in free-ranging animals is challenging. In particular, estimating metabolic rate requires the use of proxies, the potential limitations of which have been extensively documented e.g. . Our stuWe found no relationship between our proxy for RMR and parasitism in males despite the strong relationship found in females. Males are hypothesized to experience the immunosuppressive qualities of testosterone ; thus, tThis study is the first to use T3 concentration as a proxy for RMR in the context of parasitism effects. This enables us to gain greater insight into the energy use of free-ranging animals and complements our previous findings and other approaches. It is likely that energy management is not fixed across time but varies according to particular conditions, as seen in At the scale of a lifetime, life-history theory predicts that trade-offs between reproduction and self-maintenance change with age. As the probability of future reproduction declines with age, it is predicted that resources will be increasingly allocated to current reproduction rather than maintenance . There iWe found no evidence for a relationship between proxies for RMR and DEE, indicating that DEE does not vary with maintenance costs. Despite parasite-induced behavioural costs, European shag estimated DEE does not correlate with parasite load , suggestIt is unclear why species vary in their energy management strategy, although, during energetically demanding periods, the capacity to increase expenditure can impose energy limits, in terms of foraging constraints, making an allocation strategy more likely . Under l


Sepsis is life-threatening organ dysfunction due to dysregulated response to infection. Patients with sepsis exhibit wide heterogeneity stemming from genetic, molecular, and clinical factors as well as differences in pathogens, creating challenges for the development of effective treatments. Several gaps in knowledge also contribute: (i) biomarkers that identify patients likely to benefit from specific treatments are unknown; (ii) therapeutic dose and duration is often poorly understood; and (iii) short-term mortality, a common outcome measure, is frequently criticized for being insensitive. To date, the majority of sepsis trials use traditional design features, and have largely failed to identify new treatments with incremental benefit over standard of care. Traditional trials are also frequently conducted as part of a drug evaluation process that is segmented into several phases, each requiring separate trials, with a long time delay from inception through design and execution to incorporation of results into clinical practice. By contrast, adaptive clinical trial designs facilitate the evaluation of several candidate treatments simultaneously, learn from emergent discoveries during the course of the trial, and can be structured efficiently to lead to more timely conclusions compared to traditional trial designs. Adoption of new treatments in clinical practice can be accelerated if these trials are incorporated in electronic health records as part of a learning health system. In this review, we discuss challenges in the evaluation of treatments for sepsis, and explore potential benefits and weaknesses of recent advances in adaptive trial methodologies to address these challenges. Sepsis is a leading cause of critical illness and mortality globally , 2. It iSeveral preclinical models suggest that sepsis results from a disproportionate pro-inflammatory response to infection. However, several clinical trials of anti-inflammatory agents in relatively broad patient populations were disappointing . Subsequin silico, benefit was observed after considering genetic and microbiological factors  and markers that predict treatment response . This distinction between prognostic and predictive markers is critical. For example, in a trial of anti-TNF, a group of patients with high levels of IL-6 had a higher mortality rate, but not a higher drug response, suggesting prognostic but not predictive utility as a biomarker . Promoted by encouraging preclinical data, a phase 2 safety study was conducted and showed a promising trend toward increased survival in the treated group , but not during analyses. Alternatively, Bayesian statistical approaches provide a formal mathematical mechanism for combining prior and current information for use in the design, conduct, and final analysis stages of the trial . AdaptivThe Bayesian paradigm provides a natural foundation for statistical tools utilized in many adaptive designs, which involve iteratively updating or “adapting” information gathered during the trial . One sucImplementation of RAR could benefit sepsis trials in several ways. First, it would enable the study of multiple drugs simultaneously in a phase 2 trial, increasing the chances that at least one drug being tested will improve outcomes while reducing the time and costs needed to evaluate them individually by “learning” which ones are superior during the phase 2 trial and will have high likelihood of success in future phase 3 trials. The use of RAR instead of fixed randomization ratios underscores a focus on identifying the best-performing arm, instead of expending resources to rank all arms from worst to best performance. Second, instead of using RAR to assign patients to different arms, phase 2 adaptive trials could test different dosing and/or duration strategies for a single drug to better inform the optimal treatment strategy for phase 3 testing. This approach was implemented in SEPSIS-ACT, an adaptive trial of selepressin dosing strategies in adults with septic shock . In thisOften there is interest in a variety of drugs as well as identifying potential sub-populations within which the drugs are most effective. In a traditional enrichment design, randomization is simply limited to patients with a specific biomarker profile known to be predictive of treatment response. However, we may not know which patient groups may benefit the most from a treatment in sepsis. Using adaptive trial methodologies, it is possible to incorporate putative predictive biomarkers to “learn” the optimal biomarker profile in the case that a meaningful underlying HTE exists .In one approach, the RAR algorithm is used in an adaptive platform trial (see below) of several drugs, where patients are categorized into several candidate predictive biomarker strata before randomization. In the BATTLE I trial, non-small cell lung cancer patients were classified into four candidate strata defined by genomic and expression markers before being randomized to one of four drug regimens. Separately for each stratum, the RAR weights were adjusted as data accumulated to favor assignment of drugs with higher within-stratum response rates . The resAn alternative enrichment approach allows for more flexibility in the scenario where candidate predictive biomarkers have not been identified. In this framework, the optimal target population for the experimental treatment is adaptively learned and estimated as a function of baseline covariates , 36. SucWhile underlying drug response strata may exist and may be delineated by putative biomarkers, demonstrating this may be difficult in scenarios where treatment effects are relatively homogeneous or when the overall treatment effect is small. Thus, adaptive enrichment strategies present a potential advantage by incorporating mechanisms to adapt to the presence of HTE if evidence for it mounts over the course of the trial.The traditional drug evaluation pipeline is usually segmented into several phases, each involving a brand new trial. To streamline this process and reduce associated time and costs, a number of designs have been developed that combine multiple phases into a single trial, including several within the Bayesian adaptive framework . This applatform for testing experimental treatments in a perpetual manner via a common master protocol, by dropping treatments lacking efficiency and adding new treatments going into the future. They are able to incorporate several design features of adaptive trials, such as RAR, biomarker enrichment, and seamless transitioning, often all in the same design. Currently there are platform trials enrolling patients in oncology  due to treatment correspond with changes in new long-term endpoints such as quality-adjusted life-years at 6 months.Randomized, embedded, multifactorial adaptive platform (REMAP) trials utilize all of the features of a perpetual adaptive platform trials like I-SPY 2 or GBM-AGILE, the key distinction being that a REMAP trial is executed directly within clinical practice through the electronic medical record [EMR ]. A key Most REMAP trials determine the effectiveness of various treatments used in routine clinical care but in a randomized setting. An example is the REMAP-CAP trial being conducted in patients with community-acquired pneumonia severe enough to be admitted to an intensive care unit . Upon suAdaptive designs may have many promising features for future trials in sepsis, but they also come with their own challenges. Statistical models and the exploration of operating characteristics are complex and simulation-intensive. Selection of potential trial trajectories is especially important during the simulation process, as an overly narrow set of scenarios may lead researchers to fail to understand the consequences of their design choices. A range of alternative trajectories should be explored by varying important simulation parameters, for example: choice of Bayesian prior (assuming a Bayesian model is used); choice of data model; underlying treatment effects for each arm; proportions of patients within subtypes; accrual rates; and others. For designs relying on simulation-based outcome metrics such as the predictive probability of success, failure to explore sensitivity to modeling assumptions may pose risk to future patients, e.g., if drugs are erroneously selected for graduation. In designs considering many subgroups or combinations of drugs, careful consideration must be taken to craft statistical models with only the necessary complexity to preserve statistical power. In addition, the benefits to trial efficiency of periodically updating RAR weights are somewhat dependent on patient accrual rates; if accrual of data occurs faster compared to ideal updating time, RAR updates may occur based on incomplete follow-up. The complexity of these designs also creates difficulties communicating their features to important stakeholders who may be unfamiliar with them, such as funding agencies, institutional review boards, patients, research journals, and clinicians.Many studies employing Bayesian adaptive trial designs have already led to promising discoveries in several diseases, including the identification of two promising candidate drugs in breast cancer. These studies leverage several key features of adaptive designs, including RAR, enrichment methods, seamless transitioning between trial phases, perpetual platforms for ongoing evaluation of candidate treatments, integration within the EMR, and others. These features are well suited to address the many challenges presented by complex, heterogeneous diseases, yet are rarely utilized in sepsis. Adoption of these designs may aid in the efficient identification of promising treatments for sepsis.All authors contributed to the editing and preparation of this 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.


Cardiovascular disease is a leading cause of morbidity and mortality in the Western world and represents an enormous global health burden. Significant advances have been made in the conservative, medical and surgical management across the range of cardiovascular diseases however the inflammatory components of these diseases have traditionally been neglected. Inflammation is certainly a key component of atherosclerosis, a chronic inflammatory condition, but it is at least correlative and predictive of risk in many other aspects of cardiovascular medicine ranging from heart failure to outcomes following reperfusion strategies. Inflammation therefore represents significant potential for future risk stratification of patients as well as offering new therapeutic targets across cardiovascular medicine. This review explores the role of inflammation in several of the major aspects of cardiovascular medicine focusing on current and possible future examples of the targeting of inflammation in prognosis and therapy. It concludes that future directions of cardiovascular research and clinical practice should seek to identify cohorts of patients with a significant inflammatory component to their cardiovascular condition or reaction to cardiovascular intervention. These patients might benefit from therapeutic strategies mounted against the inflammatory components implicated in their condition. Cardiovascular medicine has been a notable success of the twentieth century with the development of a variety of effective therapies . In the Further to this, we now have a growing volume of experimental evidence that inflammation has roles in heart failure, myocardial infarction (MI) and responses to cardiac interventions ,5,6. InfThis review will explore the role of inflammation in the clinical management of atherosclerosis and myocardial infarction; as well as that of heart failure which is a major consequence of these conditions. It will also address the impact of inflammation in the reperfusion strategies used in these conditions, namely CABG and PCI. Finally, it will suggest possible future directions in each of these topics. In future, we envisage an approach that risk stratifies patients according to inflammatory biomarkers and utilises new therapies specifically targeting inflammatory processes unique to cardiovascular disease.Atherosclerosis is a chronic inflammatory condition affecting the medium- and large-sized arteries, characterised by the progressive development of lesions consisting of lipid, fibrosis, and inflammatory cell infiltrate within the tunica media . The proAtherosclerosis has seen intense research activity targeting reductions in C-reactive protein (CRP) as a result of retrospective analysis of primary prevention trials with statins which found that the benefits of statins were not solely due to their reduction of low-density lipoprotein (LDL) ,16. InstThere is now a proposed ‘residual inflammation’ in certain individuals that accounts for cardiovascular risk in the presence of normal or low LDL. Notably, the JUPITER trial  specifically demonstrated that at-risk individuals with elevated high-sensitivity CRP (hs-CRP) but normal cholesterol can significantly reduce their risk of MI and stroke by taking statins . More re++ CD16+ monocyte population [lo/Ly6Chi monocyte population previously discussed [Other pro-inflammatory substances have also been explored: IL-6 administration exacerbates atherosclerosis in mice and it has been shown to be elevated in those with stable cardiovascular disease and is an independent predictor of cardiovascular events . Fibrinopulation , this poiscussed . It is eiscussed ,24. The iscussed ,26. Eveniscussed .The chronic nature of atherosclerosis coupled with the sheer proportion of the population who have a subclinical form makes potent anti-inflammatory therapies with numerous side effects, corticosteroids for example, undesirable. Therefore, the challenge has been to develop specific immunosuppressive or immunomodulatory therapies with minimal side effects. Given the role of oxidized LDL in promoting macrophage activation and inflammation within atherosclerotic lesions , specifiPhospholipase 2 (PLA2) inhibitors similarly reduce levels of oxidized LDL and so were speculated to beneficial in atherosclerosis. These agents displayed a capacity to reduce inflammatory biomarkers in phase II trials  however −/− CCR2−/− and ApoE−/− CCR5−/− murine models [Most recently, the phase III CANTOS study (Canakinumab ANti-inflammatory Thrombosis Outcome Study) assessed the use of canakinumab  in post-MI patients who have a CRP >3 mg/L at the time of recruitment . The rese models ,35 or the models ,37. WhilMI describes the death of cardiac tissue due to prolonged ischaemia (>20 min) through the occlusion of a coronary vessel, usually by lumen-reducing atherosclerotic plaques and subsequent thrombus formation . It is o++ CD16+ monocyte population (previously discussed) after infarction [Similarly, IL-6 and TNFα have predictive value for mortality and future coronary events following acute myocardial infarction . In the farction  which hafarction . Based oThe role of immune cells in scar formation and resolution in MI makes the development of any anti-inflammatory therapies difficult. Such therapy would have to carefully modulate the immune system to prevent excess inflammation whilst allowing wound healing. Nevertheless, several therapies have been tried in this field. Some of the most potent anti-inflammatory agents available are glucocorticoids and, despite concerns, they have been used in several clinical trials of patients following acute MI. A recent meta-analysis demonstrated a 26% relative risk reduction in mortality although this effect was not seen when analysis only included larger studies . ConversThe complement cascade has also been investigated as a potential target given its early activation following myocardial infarction and several agents have shown promising results in preclinical studies. Pexelizumab, a monoclonal antibody against C5, demonstrated a significant decrease in mortality in a phase II clinical trial  however In patients who have suffered with clinical atherosclerosis and MI, reperfusion is a frequently used therapy . In thosThis is particularly the case with CABG which not only has the systemic inflammatory response associated with major surgery but the added inflammatory stimulus of cardio-pulmonary bypass (CPB) in on-pump surgery. CPB now has a well-documented inflammatory response through a variety of proposed mechanisms . Indeed,Several RCTs have focused on improving the extra-corporeal circuits and whilst they show clinical benefit, it is not however consistently linked to a correlated reduction in inflammation . There hIn addition, there have also been several pilot studies utilising a variety of anti-inflammatory strategies in cardiac surgery including delivery of Nitrous Oxide  and neutOne of the most serious consequences of atherosclerosis and MI, particularly in the absence or failure of reperfusion, is the development of heart failure. Heart failure is defined as inadequate cardiac function and affects 1–2% of the adult population. It is associated with a poor prognosis with 1-year mortality at 30–40% . Since 1Long term animal studies have also been able to demonstrate the capacity of TNF to induce deleterious left ventricular remodelling . Indeed,There is a clear and established role for the use of inflammatory biomarkers in heart failure. CRP has been shown to elevated in more severe grades of heart failure and can independently predict morbidity and mortality in heart failure  and TNF ++ CD16+ monocyte population. Recently, this population was shown to be significantly increased in patients with congestive heart failure and the level of this population correlates to the severity of heart failure, left ventricular ejection fraction (LVEF) and pro-brain natriuretic peptide levels [In similarity to subclinical atherosclerosis and MI, there is now also an implicated role for the CD14e levels . This poWith the apparent contribution of TNF to heart failure pathophysiology, it has emerged as a distinct target in clinical trials. Pentoxifylline and thalidomide have both been shown to reduce TNF production as well as other pro-inflammatory mediators and in several small clinical trials have demonstrated a capacity to improve LVEF and symptoms in patients with heart failure ,85 thougFurther to this, etanercept has also been trialled in the RECOVER (Research into Etanercept CytOkine Antagonism in VentriculaR dysfunction) and RENAISSANCE (Randomised Etanercept North American Strategy to Study Antagonism of CytokinEs) studies  with a combined total of 1500 heart failure patients. These studies, however, failed to show a benefit of etanercept on a variety of primary outcomes and the RENAISSANCE trial specifically demonstrated a significant worsening of heart failure in patients . InflixiDexamethasone has also been trialled in idiopathic dilated cardiomyopathy patients with heart failure and was able to demonstrate no significant benefits over placebo . StatinsFinally, methotrexate has also been trialled in heart failure patients due to its ability to reduce heart failure rates in patients with rheumatoid arthritis . The METIn conclusion, there is a prominent role for inflammation in several aspects of cardiovascular medicine from atherosclerosis and one of its major consequences in MI. Inflammation is involved in the reperfusion strategies used to address these conditions as well as heart failure, one of the major consequences of MI and failed reperfusion.This review demonstrates groups of patients who have a significant inflammatory component contributing to their cardiovascular disease. These patients can often be detected with the use of current inflammatory biomarkers with several more promising and robust biomarkers on the horizon. This stratification has the potential to improve prognostic accuracy and identify distinct patient groups who might benefit from anti-inflammatory strategies. There is evidence already that such strategies are of benefit in cardiovascular medicine and there exists the potential to expand both the scope and efficacy of such strategies.In future, we will hopefully be able to identify ever more specific targets to the inflammatory component of a patient’s cardiovascular disease which in turn will allow specifically tailored therapies that may prove to be of benefit. This is in stark contrast to the blunt anti-inflammatory therapies discussed in this review. With all of this in mind, the links between basic, translational and clinical research will prove more important than ever. Indeed, this review already demonstrates how quickly these therapeutics have gone from the bench side to clinic. If we are to have success, then the relevant research scientists will need to closely follow these clinical trials in order to refine their hypotheses. In turn, clinical scientists will need to keep abreast of developments in translational research in order to identify and trial promising therapeutics sooner rather than later.


ApoE ] gene) and environmental factors  can alter the risk of AD.Alzheimer’s disease (AD) is a progressive brain disease that results in a dementia accompanied by severe changes in cognitive ability, perception, and mood. Although the precise cause of the disease remains elusive, clusters of specific genetic factors   and in wOn the other hand, studies that correlated prior estrogen usage with the incidence of AD have reported mixed findings—some reporting that estrogen is beneficial, others finding that estrogen is detrimental  may not be an effective replacement strategy.The contradictory outcomes from the prospective studies and the clinical trials suggest that estrogen use is likely to be prophylactic rather than therapeutic. This hypothesis is supported by experimental evidence in animals which shows that prior treatment with estrogen attenuates forebrain injury caused by inadequate blood supply   and by bLifestyle factors such as heavy alcohol consumption may increase the risk for AD among both genders and in females in particular. Although women, on average drink less than men, start drinking later, and consume less alcohol, experimental evidence indicates that females may be more vulnerable to alcohol-induced neurodegeneration than males. Female alcoholics perform worse on tests of recall and psychomotor speed than male alcoholics with similar drinking histories . Brain iFurthermore, on other measures of alcohol-related brain damage, such as ventricle-to-brain-volume ratios  and grayNot all studies, however, have shown that females are more vulnerable to alcohol-induced toxicity than males. For example, Heavy alcohol consumption leads to memory deficits. Research shows that the relationship between alcohol use and dementia appears to follow a J-shaped curve rather than a linear pattern  .Recent evidence indicates that the nonlinear relationship between alcohol and cognitive deficits may be gender specific . DrinkinFemales’ greater alcohol bioavailability may be associated with alcohol-induced damage to hormone synthesis and metabolism. Alcohol reduces estrogen release during the period preceding ovulation  in the rat . In humaThe effects of moderate alcohol consumption on estrogen levels may be related to reproductive status. In premenopausal women, moderate alcohol consumption (one drink per day) increases estrogen levels, which is thought to be a possible mechanism by which alcohol may increase the risk of breast cancer. However, in post-menopausal women these data are contradictory, with some studies reporting that alcohol is associated with an increase in estradiol and others reporting no change (summarized in If decreased estrogen exposure potentiates the risk for AD, chronic consumption of alcohol by women may increase the severity or onset of AD. To directly test these hypotheses, longitudinal studies of cognitive changes in heavy and moderate drinkers need to be coupled with regular hormonal profiles and other indicators of estrogen status such as reproductive history and bone density.


The goal of this work is to consider physiological fluctuations in brain oxygen levels and its changes induced by opioid drugs. This review article presents, as a comprehensive story, the most important findings obtained in our laboratory by using high-speed amperometry with oxygen sensors in awake, freely moving rats; most of these findings were separately published elsewhere. First, we show that oxygen levels in the nucleus accumbens (NAc) phasically increase following exposure to natural arousing stimuli. Since accumbal neurons are excited by arousing stimuli and NAc oxygen levels increase following glutamate (GLU) microinjections in the NAc, local neural activation with subsequent cerebral vasodilation appears to mediate the rapid oxygen increases induced by arousing stimuli. While it is established that intra-cerebral entry of oxygen depends on brain metabolism, physiological increases in NAc oxygen occurred more rapidly than increases in metabolic activity as assessed by intra-brain heat production. Therefore, due to neural activation and the subsequent rise in local cerebral blood flow (CBF), the brain receives more oxygen in advance of its metabolic requirement, thus preventing potential metabolic deficits. In contrast to arousing stimuli, three opioid drugs tested  decrease oxygen levels. As confirmed by our recordings in the subcutaneous space, a densely vascularized location with no metabolic activity of its own, these decreases result from respiratory depression with subsequent fall in blood oxygen levels. While respiratory depression was evident for all tested drugs, heroin was ~6-fold more potent than oxycodone, and fentanyl was 10-20-fold more potent than heroin. Changes in brain oxygen induced by respiratory depression appear to be independent of local vascular and blood flow responses, which are triggered, via neuro-vascular coupling, by the neuronal effects of opioid drugs. The brain is one of the heaviest consumers of oxygen in the body Siesjo,  and oxygThe goal of this work is to consider physiological fluctuations in brain oxygen levels and their changes induced by opioid drugs that directly affect the CNS and respiration. This work is based primarily on series of our recent experiments obtained by using oxygen microsensors coupled with high-speed amperometry in freely-moving rats. Although this technology cannot provide information on oxygen consumption resulting from brain metabolic activity, it is a valid tool to examine physiological and drug-induced fluctuations in brain oxygen in discrete brain areas and other bodily locations. To better understand the mechanisms governing oxygen entry into brain tissue under different experimental conditions, we also consider our data on fluctuations in brain glucose, another critical metabolic parameter. Like oxygen, glucose arrives to the brain from arterial blood based on its concentration gradient via GLUT-1 transporters  coupled with high-speed amperometry in awake, freely moving rats  time resolution, we found that all arousing stimuli significantly increased NAc oxygen levels . However, oxygen increases occurred more rapidly, more strongly, and peaked at an earlier time than changes in brain temperature. Oxygen levels surged during the first minute of stimulation, when brain temperature had only begun to increase and was still close to baseline. Then, oxygen levels continued to increase during the next 2–3 min and showed a strong direct correlation with brain temperature. This correlation disappeared from ~8 min after stimulus onset, when both parameters began to decrease toward baseline.As shown in Figure Although brain temperature is an important homeostatic parameter and its changes are related to metabolic activity, changes in brain temperature also depend on the temperature of arterial blood. Since the temporal muscle is a non-locomotor muscle and it receives its arterial blood supply from the carotid artery like the brain does, the difference between brain and muscle temperature  eliminates the influence of arterial blood temperature on brain temperature, thus allowing us to reveal the component of the temperature response that is related to intra-brain heat production resulting from metabolic brain activation  for the entire analysis interval apart from the short time intervals following the stimulus onset .To mirror such a real-world scenario, in which an individual believing that he or she is consuming a standard dose of heroin is actually consuming a heroin sample laced with a smaller amount of fentanyl, we examined changes in NAc oxygen induced by heroin (0.4 mg/kg) contaminated with 10% fentanyl (0.04 mg/kg). These doses of both drugs are much lower than the LD50 assessed in rats  and they increased in magnitude and duration with increases in drug doses. This hypoxic effect results from respiratory depression with subsequent decreases in blood oxygen levels. This effect is typical to all opioid drugs at high doses, and it appears to be independent of local vascular and blood flow responses occurring due to the neuronal effects of these drugs. Robust decreases in oxygen induced by large-dose opioid drugs in the NAc appear to be generalized to other brain structures as tested by our control recordings in the basolateral amygdala, a distantly located brain structures involved in regulating different brain functions (Solis 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.


However, their actual role in the cellular biochemical cascade that eventually affects the fungal growth morphology, such as mycelial pellet formation, is unclear. This article intends to open up the detailed study on the possible correlative effect of cAMP on the morphological and physiological growth aspects of filamentous fungi, with special emphasis on the industrial metabolite production.The effect of second messenger  Culture factors resulting in evenly distributed small pellets in the liquid medium gave rise to a higher pigment accumulation in the biomass compared to mycelial and big clumps. Since the production of secondary metabolites, for example, pigments has been shown to be a stress‐related response . Also, mutation of a putative histidine kinase gene (dcc‐1) results in enhanced conidiation. Interestingly, this effect was reversed by exogenous cAMP addition, indicating DCC‐1 to be another part of the signaling pathway that promotes cAMP production . In this study, lovastatin and pigment production was downregulated when an excess amount of glucose was available in the medium, in connection with cAMP production , resulting in lower levels of intracellular cAMP have been observed to enhance pigment production compared to the wild‐type cells . Hence, it could be hypothesized  formation from filamentous fungi with an industrial favorable growth morphology (pellet formation). Molecular‐level studies in line with the factors or responses controlling the cAMP levels in the fungal cell could be interesting aspects in the industrial scale studies for commercial metabolite production from filamentous fungi.https://urn:nbn:se:hb:diva-674 (PhD thesis).https://urn:nbn:se:hb:diva-12436 (PhD thesis).The authors declare that they have no conflict of interests.


There is an enormous interest in identifying the causes of psychiatric disorders but there are considerable challenges in identifying which risks are genuinely causal. Traditionally risk factors have been inferred from observational designs. However, association with psychiatric outcome does not equate to causation. There are a number of threats that clinicians and researchers face in making causal inferences from traditional observational designs because adversities or exposures are not randomly allocated to individuals. Natural experiments provide an alternative strategy to randomized controlled trials as they take advantage of situations whereby links between exposure and other variables are separated by naturally occurring events or situations. In this review, we describe a growing range of different types of natural experiment and highlight that there is a greater confidence about findings where there is a convergence of findings across different designs. For example, exposure to hostile parenting is consistently found to be associated with conduct problems using different natural experiment designs providing support for this being a causal risk factor. Different genetically informative designs have repeatedly found that exposure to negative life events and being bullied are linked to later depression. However, for exposure to prenatal cigarette smoking, while findings from natural experiment designs are consistent with a causal effect on offspring lower birth weight, they do not support the hypothesis that intra-uterine cigarette smoking has a causal effect on attention-deficit/hyperactivity disorder and conduct problems and emerging findings highlight caution about inferring causal effects on bipolar disorder and schizophrenia. Psychiatric disorders have a complex etiology; influenced by multiple genetic as well as environmental risk factors. Although most are heritable, in the shorter term, environmental factors are more tractable to modification. If environmental exposures are causal, then their modification should lead to improvements in population health or the psychiatric disorder being treated  suggested that this was not the case  design  Prenatal exposures. As they share the same mother, they become of special interest when they have been differentially exposed to prenatal factors. For example, taking the example of maternal smoking in pregnancy and ADHD, eight studies of discordant sibling pairs have now found that the siblings who were unexposed to smoking in utero showed elevated levels of ADHD  Assessing later adversities using a sibling pair design and its extension, the co-relative study. The discordant sibling pair design and its extension involving pairs of relatives from the same generation such as half-siblings and cousins have also been used to assess causal links between adolescent and adult exposures and psychiatric disorders. For example, the observed association between cannabis use and schizophrenia has been well-established. However, the causal relationship could be subject to question given that those who are at elevated familial or genetic liability or with prodromal symptoms could be more likely to use cannabis . In one large, Swedish study, the authors used an extended sibling pair design to investigate the causal relationship between cannabis and schizophrenia .Twin designs utilize the fact that monozygotic (MZ) twins share on average 100% of their genes (DNA sequence) and dizygotic (DZ) twins share on average 50% of their genome.The twin design allows variation in any given measure to be partitioned into genetic and environmental variance. Where both exposure and psychiatric outcome are assessed, ideally longitudinally to avoid the problem of reverse causation, the association between exposure (e.g. life events) and outcome (e.g. depression) can be decomposed into genetic and environmental components. As the genetic covariance between exposure and outcome is explicitly modelled, essentially the genetic confound is removed. Here, the investigator is interested in whether there is an environmental link that remains between the exposure and outcome. This design has been invaluable in demonstrating a number of potentially causal environmental risk factors for psychopathology.et al., For example, family and twin studies of depression in childhood, adolescence, and adult life have observed a familial and genetic contribution to life events, mainly those that are person-dependent (e.g. losing a job) rather than ones that are independent (death of a relative), as well as to depression  show differences in a given outcome (e.g. depression).et al., et al., For example, in the UK E-risk twin study of 7–10 years old, 110 MZ twin pairs who were discordant for bullying victimization were assessed . Maternal expressed emotion was found to be environmentally associated with later teacher-reported behavioral problems.In another longitudinal MZ discordant twin study  design allow investigation of cross-generational links between parent and offspring psychopathology or parentally provided exposures and offspring outcomes. It takes advantage of the fact that the offspring of MZ and DZ twins are socially cousins  but the MZ twin offspring are genetically half siblings.et al., et al., et al., et al., et al., This type of design, for example, has been used to assess the cross-generational transmission of depression. In an Australian study of twins, their spouse, and offspring, environmental factors were found to explain the link between parents and offspring depression even when accounting for depression in spouses  utilizes the random assortment of parental genotypes to offspring during meiosis. Here a genetic variant that is robustly associated with the exposure is used as the instrumental variable and provided certain assumptions are met should provide a means of controlling for confounding and reverse causation see . As moreIt is crucial that genuinely causal influences on psychopathology are identified if interventions and policies are going to be effective. In recent years, findings relevant to psychiatry have emerged from different natural experiment designs and some are consistent across different designs; this strengthens causal inference. For example, hostile parenting affects antisocial behavior and RCTs uphold this causal inference. Genetically informative studies converge in favor of life events and victimization being environmentally linked with depression and environmental cross-generational transmission for depression. However, although smoking cessation programs for pregnant women are clearly a priority as cigarette smoke is detrimental to offspring physical health, the natural experiment designs suggest these will not be a useful means for preventing ADHD or antisocial behavior. So do natural experiments have an important future in the study of mental disorders? The answer is a firm yes.


Previous researchers have reported that aerobic exercise improves cognition in older adults; however, few researchers have examined the role of arousal on improvements in cognition after exercise. The purpose of this study was to understand how changes in arousal acutely affect changes in cognitive performance after a single session of light compared to moderate intensity aerobic exercise. Cognitively normal older adults (N = 34) were enrolled in a randomized controlled trial where they were asked to complete the N-back task with faces, a cognitive task used to test working memory, in an fMRI scanner. On separate days, the task was completed before and 15 to 20 minutes after light and moderate intensity exercise. An intervention was also completed, but our question focuses on the acute effects of exercise rather than training. Arousal was measured before and after exercise through a questionnaire and a direct measure of physiological activation of the sympathetic nervous system with galvanic skin response (GSR). On average, resting GSRs decreased from pre- to post-exercise scan; however, the change was not statistically significant. The decrease in arousal after light exercise indicated that older adults had decreased sympathetic activity after both light and moderate intensity exercise. By contrast, N-back task performance improved most after moderate compared to light intensity exercise. Together, evidence that sympathetic activity tended to decrease generally for both intensities, whereas cognitive improvements were more specific, suggests that changes in arousal at rest were not a critical factor connecting exercise and improved working memory in this study.


Psychological stress is recognized as a major modifiable risk factor for adult non-communicable disease (NCD) that includes depression, type 2 diabetes mellitus, and cardiovascular disease. Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity resulting in either exaggerated or blunted cortisol responses, and altered autonomic cardiovascular control have been thought to underlie this association. The developmental origins hypothesis proposes that impaired nutrition during fetal and early postnatal growth is associated with a higher NCD risk later in life. Maternal nutrients are vital for fetal growth and development, and both maternal undernutrition and over nutrition as in the case of gestational diabetes are associated with higher NCD risk markers in the offspring. Recent studies suggest that fetal exposure to maternal nutritional imbalances may permanently alter cortisol and cardio-sympathetic stress-responsiveness, which may link early life nutrition with adult disease risk. A few recent studies that examined the association between low birth weight as a marker of fetal undernutrition and stress response in humans showed that lower birth weight was associated with an altered HPA axis and cardiovascular sympathetic nervous system responses to stress in adults as well as in children. In addition, altered stress responses in relation to gestational diabetes have been noted. In this paper, we present available evidence from India for the association between maternal nutrition and offspring stress responsiveness against the backdrop of global evidence, and discuss its role in the escalating NCD rates in this population. We also discuss the scope for future studies in India and other transitioning countries. There has been a steady increase in type 2 diabetes, cardiovascular disease, and other non-communicable diseases (NCD) including depression in the world, but this increase is pronounced in transitioning countries such as India . This maIt is suggested that a number of biological, environmental, and social factors during different stages of life course may confer variations in stress reactivity among different individuals, and thus susceptibility to future development of diseases . Fetal HThe Developmental Origins of Health and Disease (DOHaD) hypothesis proposes that impaired nutrition during critical stages of fetal development results in permanent changes in structure and function of key physiological systems (programming) . The resThe fetal programming hypothesis was initially described by David Barker and colleagues, who showed among UK adults that the prevalence of type 2 diabetes and coronary heart disease was higher in individuals with lower birth weights , 15. SimRaikkonen et al., adult men who had lower birth size  and slower postnatal growth during the first 2 years of life had poorer cognitive abilities in late adulthood  and at 1 year  with a higher prevalence of coronary heart disease . A recendulthood .in utero were more likely to be obese and had a higher prevalence of cardio-metabolic disease and depression . Findings from subsequent studies were inconsistent. However, a meta-analysis of published studies among Caucasian populations until 2004 showed a significant inverse association between birth weight and cortisol concentrations (∼25 nmol/L per kg increase in birth weight)  . The cor weight) . These rKajantie et al. demonstrated among older adults from the Helsinki Birth Cohort that birth weight had an inverse “U” shaped association where both lower and higher birth weights were associated with lower cortisol response to stress. This study suggested that intra-uterine conditions may program not only hyper-responsiveness, but also blunted HPA axis activity  and indices of later neuro-endocrinal stress responses. Furthermore, initial evidence suggests sex-specific programming effects. In a systematic review of the early programming effects on HPA axis authors concluded that there seems to be an increased vulnerability among females particularly in terms of HPA axis reactivity . Other iIndia and other south Asian countries are experiencing a phenomenal rise in the prevalence of type 2 diabetes, cardiovascular disease, and other NCDs including depression. Nearly 80 million people in India alone are expected to develop type 2 diabetes by the year 2030 . Some inThe only studies in India on programming of stress responses as a risk factor for NCDs come from two birth cohorts in Mysore, India. In preliminary work in Mysore, morning cortisol concentrations were measured in a cohort of 500 adults of 40–60 years of age born in one maternity hospital . This stThe above findings were subsequently replicated among the younger participants of Mysore Parthenon cohort . SimilarThe above findings prompted the researchers in India to explore the association between maternal nutritional status and offspring stress responses using a dynamic stress paradigm. The only reported study of this association tested the role of intrauterine overnutrition related to maternal GDM in altered offspring stress responses . This stWhen the participants were ∼13.5 years of age, the Parthenon study examined their cortisol and cardiovascular responses to acute stress induced by the TSST for children . For theAutonomic nervous system (ANS) is an important component of bodily stress response system and the HPA axis and ANS are considered as complimentary systems . The ANSThus, longitudinal studies are required to fully understand the early programming effects on fetal and childhood ANS modulation and its implications for later development of NCDs.conceptus will have an impact on the optimal fetal growth. It was suggested that when there is a poor nutritional supply, the growing fetus adapts to adverse conditions by prioritizing the growth of the brain, which is vital for survival, thus compromising the growth and functions of “less important” insulin sensitive organs like the pancreas, liver, and skeletal muscles (A number of mechanisms have been proposed to underlie the early-nutritional programming of NCDs. The growing fetus depends on the mother for its nutritional needs, and it is not surprising, therefore, that any alteration in the maternal nutritional status or its supply to the  muscles . This pr muscles .Early epigenetic modifications may be another mechanism for early life programming. Epigenetic changes are heritable changes in gene expression without altering DNA sequence. These are established early during fetal growth and are influenced by environmental factors including maternal nutrition and metabolic status . EpigeneThe above pathways may themselves also explain the link between maternal nutritional status and altered stress reactivity. Impaired intrauterine nutrition has been thought to induce permanent changes in the regulation and set point of several hormonal systems . In partMechanism for the association between maternal overnutrition and stress responses is not clear. Animal model studies have shown permanent changes in hypothalamic structure and function in relation to maternal diabetes ; the stiin utero. Research from India and elsewhere suggest that this phenomenon may be a major triggering factor in the rapid escalation in the prevalence of type 2 diabetes and other NCDs in the country. Rapid urbanization in the country has introduced its own challenges in terms of energy-rich diets, decreased scope for physical activity, and increased life stresses, particularly among youth. This may further exacerbate the NCD and adverse mental health situation in the country (In India, maternal undernutrition and specific nutritional deficiencies may underlie the high prevalence of intra-uterine growth retardation and childhood undernourishment . On top  country , 99.in utero.In this context, studying stress reactivity as mechanism linking early life nutrition with later development of NCDs, particularly in adolescents and young adults, may provide vital opportunities to intervene during key life course periods. However, despite a growing consensus on the importance of stress systems for later disease development, relationship of maternal nutritional status to offspring stress responses is yet to be clearly elucidated. Maternal nutrients, particularly B-vitamins involved in one-carbon metabolism are known to be vital co-factors in many neuro-developmental and gene methylation processes , 101. FiThese objectives form the premise for a recently launched multi-centric study in India . This prIn conclusion, it is apparent that, although animal studies have shown consistent association between impaired maternal nutritional status and offspring stress reactivity, research on programming effects among humans is in its early stages. Both animal studies as well as limited evidence in humans suggest that exposure to both undernutrition and overnutrition during fetal development may bring about adverse changes in stress response systems and functioning. This warrants that future studies on stress programming need to focus on both these aspects. Majority of the studies in humans have used birth weight as a proxy for fetal nutrition. However, birth weight is a crude indicator of fetal growth retardation, and does not give a complete estimation of the effects of fetal under nutrition on different physiological systems. Thus measuring offspring stress responses in relation to maternal nutritional status per se will give more objective measures of this association. However, to the best of our knowledge no such studies are available in humans currently, and this is definitely as area for future research. Moreover, causal relationships cannot be inferred by the observational studies and hence prenatal nutritional intervention studies may be an important step forward in this context. More studies adopting a similar approach from different parts of the world may provide robust evidence for the importance of adequate maternal nutrition in shaping optimal stress reactivity throughout life course.Both the authors conceptualized the paper, contributed to the drafting and revising of the manuscript, and read and approved the final content.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.


The use of implementation strategies is an active and purposive approach to translate research findings into routine clinical care. The Expert Recommendations for Implementing Change (ERIC) identified and defined discrete implementation strategies, and Proctor and colleagues have made recommendations for specifying operationalization of each strategy. We use empirical data to test how the ERIC taxonomy applies to a large dissemination and implementation initiative aimed at taking cardiac prevention to scale in primary care practice.EvidenceNOW is an Agency for Healthcare Research and Quality initiative that funded seven cooperatives across seven regions in the USA. Cooperatives implemented multi-component interventions to improve heart health and build quality improvement capacity, and used a range of implementation strategies to foster practice change. We used ERIC to identify cooperatives’ implementation strategies and specified the actor, action, target, dose, temporality, justification, and expected outcome for each. We mapped and compiled a matrix of the specified ERIC strategies across the cooperatives, and used consensus to resolve mapping differences. We then grouped implementation strategies by outcomes and justifications, which led to insights regarding the use of and linkages between ERIC strategies in real-world scale-up efforts.Thirty-three ERIC strategies were used by cooperatives. We identified a range of revisions to the ERIC taxonomy to improve the practical application of these strategies. These proposed changes include revisions to four strategy names and 12 definitions. We suggest adding three new strategies because they encapsulate distinct actions that were not described in the existing ERIC taxonomy. In addition, we organized ERIC implementation strategies into four functional groupings based on the way we observed them being applied in practice. These groupings show how ERIC strategies are, out of necessity, interconnected, to achieve the work involved in rapidly taking evidence to scale.Findings of our work suggest revisions to the ERIC implementation strategies to reflect their utilization in real-work dissemination and implementation efforts. The functional groupings of the ERIC implementation strategies that emerged from on-the-ground implementers will help guide others in choosing among and linking multiple implementation strategies when planning small- and large-scale implementation efforts.NCT02560428).Registered as Observational Study at www.clinicaltrials.gov (The online version of this article (10.1186/s13012-019-0876-4) contains supplementary material, which is available to authorized users. It is well recognized that an active and purposive approach is required to translate research findings into routine clinical care. Methods used to disseminate and implement research findings into clinical practice are termed implementation strategies,  and intePowell and colleagues sought to address the problem of multiple names and definitions for implementation strategies in 2012 . ThroughERIC has made an important theoretical contribution to identifying a broad range of implementation strategies and developing a common nomenclature with definitions. An important next step toward advancing this work is to test and refine the taxonomy based on the use of these implementation strategies in on-the-ground implementation efforts; Proctor and colleagues  recognizResearchers who applied the ERIC and Proctor frameworks to real-world implementation efforts recommended additional implementation strategies for the ERIC taxonomy . Others This paper reports a study we conducted to apply the ERIC taxonomy and the Proctor reporting recommendations to multiple large-scale studies. We use empirical data collected during active implementation with an intent of further refining ERIC, if changes were identified as necessary.The setting for this study is the EvidenceNOW initiative  funded bN = 1721). Practices were smaller (< 10 clinicians) and varied with respect to ownership and geographic location. Accomplishing the goal of large-scale rapid dissemination and implementation in less than 3 years required a large workforce and use of a range of implementation strategies.EvidenceNOW funded seven regional cooperatives that spanned 12 states . For details on the regions and states, see the EvidenceNOW website  and Ono We reviewed and triangulated four sources of qualitative data from years one and two of the EvidenceNOW initiative to identify and define the cooperatives’ implementation strategies. First, we reviewed the cooperatives’ study proposals, which provided an outline of broad, overarching implementation strategies. Second, we examined cooperative-level documents, including training tools and conceptual models, to further specify content of these overarching strategies. We also analyzed facilitator training documents, which provided insights into specific activities and implementation strategies that facilitators would use to support practices. For example, templates for facilitators to document and track their work provided detailed descriptions of activities that facilitators may perform. The third source of data were entries written by cooperative team members on the ESCALATES interactive online diary . The onlAll field notes were prepared soon after the site visit. Interviews followed a semi-structured guide and were professionally transcribed, checked for accuracy, and de-identified. Field notes, interviews, and other documents were entered into Atlas.ti for data management and analysis.We used an iterative and inductive analytic approach  to identNext, four of the authors  independently mapped each action to a strategy in the ERIC compilation  based onNext, we created a consolidated cross-cooperative matrix that organized implementation strategies based upon their practical application. We pile-sorted each implementation strategy into functionally similar groups based on their justification and expected outcomes. We reviewed and labeled each grouping and considered the cooperatives’ practical applications of strategies within and across the groupings, recognizing interdependencies and differences, such as “actors”  expertise versus cooperative-employed data experts).. Recommended refinements include changes in strategy names for four strategies and changes in definitions for 12 strategies. Additionally, we propose adding three new distinct strategies which we identified as being used in practices among the cooperatives: assess and redesign workflow, create online learning communities, and engage community resources. Definitions for these strategies are provided in Table implementation facilitation instead of ERIC’s original name of facilitation).The 266 actions identified across the seven cooperatives mapped to 33 of the 73 ERIC implementation strategies. We propose refinements for 13 strategies. Table We grouped the 33 ERIC strategies used by the cooperatives into four functional groupings: (1) build health information technology to support data-informed quality improvement (QI), 2) build QI capacity and improve outcomes, (3) enhance clinician and practice member knowledge, and 4) build community connections and patient involvement. Tables  build QI build coaudit and provide feedback strategy to support data-informed QI. This strategy relied on region- and practice-level HIT infrastructure to deliver trusted data to practices at regular intervals throughout intervention. Audit functions  were necessary for feedback functions , which in turn, were necessary to inform QI efforts. For instance, generated data were used to identify quality gaps and help to identify opportunities on which to focus improvement efforts. These data were also used to monitor the impact that changes had on ABCS outcomes. Audit and provide feedback, thus, was a key strategy to improve ABCS outcomes.Multiple implementation strategies are needed to achieve data-informed QI. A critical aspect of the cooperatives’ efforts to rapidly disseminate and implement evidence into practice was helping practices gain access to data to support data-informed QI functions  and to develop dashboards—available through centralized portals —to provide practices with quality reports needed to support audit and provide feedback functions. At times, HIT experts (or an HIT-trained facilitator) worked with practices to change how the practice members documented information from appointments and other sources within their EHR to ensure valid and complete data were extracted (change records systems). This step was necessary to get good quality data to a centralized data warehouse, and for the warehouse then to provide the application and cleaned data to support quality reports, which were provided back to practices. This was necessary because many practices did not have the capability to generate robust and useable reports within their own setting. The local technical assistance HIT experts provided were extended to assisting practices with translating information from reports to actionable opportunities for improvement related to the generated performance reports and monitoring of progress over time.On the ground, up to seven additional ERIC implementation strategies were necessary to accomplish building the infrastructure and capacity needed to implement and sustain identify and prepare champions); organized and facilitated meetings (organize implementation team and team meetings); assessed and offered suggestions for revising clinical work flows (assess and redesign workflow); assessed HIT needs and data set-up (assess for readiness and identify barriers and facilitators); discussed and identified an improvement plan (develop an implementation blueprint); assisted with the change process and supported the change efforts including pre-visit planning and outreach (implementation facilitation); and helped revise plans based upon results of change cycles. As reported above, practice facilitators were also involved in helping set up HIT infrastructure.Practice facilitators used a wide range of implementation strategies to build QI capacity and improve outcomes . Cooperatives employed a range of education strategies and more than one strategy might be used by a cooperative. These also included implementation strategies aimed at strengthening peer exchange of best practices to build stronger, sustained supportive professional learning communities. In addition, we identified one new implementation strategy used by EvidenceNOW cooperatives—create online learning communities. Given the great distances some cooperatives covered to reach practices, online learning techniques were tested to improve connections between practices for distance learning .Several implementation strategies were employed to enhance clinician and practice member knowledge of evidence-based guidelines and interventions designed to improve ABCS outcomes, measures to assess outcomes, and QI methods  to implement and sustain use of audit and provide feedback.We used the naming and definitions provided by the ERIC taxonomy  of impleengage community resources, create online learning communities, and assess and redesign workflow. Each of these strategies encapsulates distinct actions that were not identified in the existing ERIC taxonomy and are likely to be used in a broad range of other settings. Other researchers have also recommended new strategies: obtain worker feedback about implementation plan and plan for outcome evaluation [Cooperatives documented 266 actions mapped to 33 of 73 total ERIC strategies. This number of strategies is consistent with other published studies that have reported a range of 16–59 strategies used in implementation studies , 15, 16.aluation . Thus, aAudit and provide feedback was a critical implementation strategy used across cooperatives. Audit and provide feedback consists of two distinct yet interrelated concepts: audit, the assessment of performance often compared to a specific target or benchmark; and feedback, communication of the performance. Data used to measure and assess performance must be accurate and credible [. Published trials typically only describe the feedback function within audit and provide feedback when reporting findings. In a commentary on an updated Cochrane review of audit and provide feedback, the authors list eight characteristics that contribute to variability in effectiveness across studies; all eight characteristics pertain to feedback with no mention of the role and quality of the audit component [credible –25. Manyomponent . Other pomponent  or proviaudit and provide feedback and to then engage in data-informed QI to improve ABCS outcomes. In the ERIC taxonomy, these seven strategies are spread across four different clusters. We group these HIT strategies based on their shared justification and expected outcomes as this better reflects how these strategies may need to be sequenced to accomplish the intended outcome. Five of the seven strategies to build HIT infrastructure were rated as low-feasibility within the ERIC taxonomy [audit and provide feedback functionality beyond this initiative and because of its focus and substantial funding, EvidenceNOW makes a unique contribution to the ERIC taxonomy by providing insight into what is needed to build the infrastructure to support audit and provide feedback functions.The EvidenceNOW cooperatives described seven strategies that were needed in differing combinations, depending on the local context, to build the necessary HIT infrastructure to support taxonomy  largely implementation facilitation, the core strategy described by all seven cooperatives. Implementation facilitation was used to support practices in accomplishing their QI goals on the path to improving ABCS outcomes; it involves performing interrelated and complex roles and skillfully applying diverse strategies in a flexible and dynamic manner to meet the local needs and priorities of each primary care practice [implementation facilitation). In EvidenceNOW, practice facilitators provided nearly all of the external support to practices. The facilitators, including facilitators with HIT skill, were responsible for performing, either solely or in conjunction with other actors, 27 of the 33 strategies, spanning all four overarching functions within EvidenceNOW. Using this broad array of strategies requires diverse and sophisticated skills including interpersonal , technical (including adept use of EHR platforms), organizational, communication, leadership, and pedagogical skills [A second clear grouping of strategies is those aimed at helping practices build QI capacity and improve ABCS outcomes, supported foundationally by practice –31. Thusl skills , 32, 33.Regrouping the ERIC strategies from the current nine clusters to a clustering that is more pragmatic may make the ERIC taxonomy easier for practitioners to apply and might be helpful in implementation planning. We intentionally developed these groupings to better reflect how strategies were employed across the diverse contexts of EvidenceNOW. Leeman and colleagues have also proposed an alternative organizing structure for ERIC, proposing five groups, based upon actor and target for each strategy . They suIn our analysis, we specified further each cooperative’s implementation strategies following Proctor and colleagues’ recommendations. Specifying actor highlighted the broad array of strategies performed by the practice facilitators. A priori specification of dose and temporality, however, was less useful for EvidenceNOW. Most strategies were designed to be used “as needed” in response to local needs of practices. On the one hand, this was a unifying theme across the cooperatives, and on the other hand, specifying these dimensions for each individual strategy was unnecessary. However, the cooperatives did track the activities of their facilitators at varying levels of detail; analysis of this data is outside the scope of this current work. Other researchers have highlighted the challenges with specifying dose: it is resource-intensive to specify and track often because of its ambiguity , 15. HowData were collected early in the cooperatives’ implementation phase; they reflected strategies that were included or just started to be employed during the implementation phase of each of the cooperatives’ respective large-scale multi-component interventions. Some cooperatives may have under-reported their strategies because they may have been tacit or intuitive and thus not explicitly reported. This limitation was mitigated through online diary postings by cooperative team members including practice facilitators and early site visits where we observed practice facilitators and others who were delivering implementation strategies, rather than relying exclusively on cooperatives’ self-reported data. The cooperatives did track dose and temporality of strategies used , but these data were nearly impossible to harmonize across the cooperatives because of the varying level of detail and inconsistencies within how dose and temporality were tracked. It is important to note that this work includes strategies that were specified by cooperatives to provide external support to primary care practices; it does not include micro-level strategies used within individual practices.Based on empirical experiences across seven large cooperatives participating in the AHRQ-funded large-scale EvidenceNOW initiative, we recommend refinements to the ERIC list of strategies, which if adopted, would better align ERIC with real-world implementation needs and improve its utility for implementers and researchers. Combined use of the refined ERIC taxonomy and the specifications suggested by Proctor et al. add transparency and thus promote replicability by identifying, creating common nomenclature, and harmonizing a potentially diverse array of implementation strategies used by multiple large multi-site dissemination and implementation initiatives such as EvidenceNOW.Additional file 1:Example from a single cooperative’s table of interventions. (DOCX 23 kb)Additional file 2:ERIC strategy names and definitions. (DOCX 52 kb)