The NVU regulates material exchange between the bloodstream and the brain parenchyma, and its dysfunction is a primary or secondary cause of many cerebrovascular and neurodegenerative disorders. As such, there are substantial research thrusts in academia and industry toward building NVU models that mimic endogenous organization and function, which could be used to better understand disease mechanisms and assess drug efficacy. Human pluripotent stem cells, which can self-renew indefinitely and differentiate to almost any cell type in the body, are attractive for these models because they can provide a limitless source of individual cells from the NVU. In addition, human-induced pluripotent stem cells (iPSCs) offer the opportunity to build NVU models with an explicit genetic background and in the context of disease susceptibility. Herein, we review how iPSCs are being used to model neurovascular and neurodegenerative diseases, with particular focus on contributions of the BBB, and discuss existing technologies and emerging opportunities to merge these iPSC progenies with biomaterials platforms to create complex NVU systems that recreate the  The blood–brain barrier (BBB) maintains central nervous system (CNS) homeostasis by strictly regulating transport of ions, small molecules, proteins, and cells between the bloodstream and CNS   as a new cell source for modeling BBB. iPSCs are characterized by their ability to proliferate indefinitely and differentiate into any cell type of the human body   . The reproducibility of the differentiation procedure has been subsequently confirmed in several publications all demonstrating BBB identity through elevated TEER and other molecular characterizations  for approximately 4 days  helps induce BBB properties , which are vascular malformations found predominantly in the CNS that cause hemorrhagic stroke, are caused by the loss of function mutations in three genes that form an intracellular adaptor protein complex , and amyotrophic lateral sclerosis (ALS); whether this dysfunction causes the degeneration or is secondary to the diseases and simply exacerbates their progression remains to be determined. iPSCs represent a potential route for deconstructing neurovascular changes in these disorders, especially given the progress in differentiating other NVU cell types including neurons, microglia, pericytes, and astrocytes  carrying a protective PICALM allele exhibit increased amyloid transport  in the extracellular space and buildup of hyperphosphorylated tau fibers in the cytoplasm of neurons Selkoe, . Geneticin vitro  exhibit elevated α-synuclein protein expression, thus recapitulating the in vivo phenotype . Given the more significant tightness of iPSC-derived BMECs, accumulation and/or transport of α-synuclein, and its influence on barrier function, could be examined in a more physiologically relevant model system. This is particularly relevant given recent findings that α-synuclein assemblies can cross the BBB in vivo . Academia has made significant strides in screening both existing and newly developed drugs within iPSC-derived neurological models  or glass substrates, which, while conducive to long-term cell culture, fail to recapitulate the complex 3D microenvironment of natural tissue. Scaffolds fabricated from hydrogel matrices are appealing for modeling the NVU, in that they offer a more physiologically representative platform in terms of stiffness, architecture, degradability, and a means by which to allow more natural interactions with surrounding cell populations  through spatially controlled cross-linkage of photosensitive prepolymer solution by targeted light exposure, delivered by computer-controlled digital micro-mirror assemblies or through a 2D “mask” in a layer-by layer fashion  is the most common method for assessing barrier strength in 2D cultures, but obtaining reliable resistance measurements from endothelial lumens lining perfused channel networks within 3D hydrogel scaffolds is not realistic (Srinivasan et al., in vivo vessel structures. Valuable insights into neurological diseases have already been reported using iPSC-based model systems, and it is expected that these models will improve further when combined with novel biomaterial scaffolds into full NVU constructs. Once built, these complex in vitro models are poised to provide relevant clinical knowledge regarding debilitating cerebrovascular diseases and ultimately facilitate the next generation of therapeutic interventions.Major strides have been made toward building BBB models that take advantage of human iPSC technology. In addition, ever-better microfluidics platforms and perfusable hydrogels are being developed to provide three-dimensional architectures that better mimic All authors contributed to the preparation of this review.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.


This paper presents a review of theoretical and empirical work on repetition suppression in the context of predictive coding. Predictive coding is a neurobiologically plausible scheme explaining how biological systems might perform perceptual inference and learning. From this perspective, repetition suppression is a manifestation of minimising prediction error through adaptive changes in predictions about the content and precision of sensory inputs. Simulations of artificial neural hierarchies provide a principled way of understanding how repetition suppression – at different time scales – can be explained in terms of inference and learning implemented under predictive coding. This formulation of repetition suppression is supported by results of numerous empirical studies of repetition suppression and its contextual determinants. Typically, repeated stimuli evoke neural activity with amplitudes smaller than responses to novel stimuli. Although repetition suppression is often portrayed as an expression of relatively simple mechanisms, such as neural fatigue , its depIn the following, we will review modelling and experimental work on repetition suppression in the setting of predictive coding. First, we introduce the predictive coding framework and portray neuronal message passing in terms of descending predictions, ascending prediction errors, and modulatory precision. We will then show how the predictive coding scheme can be mapped onto a canonical cortical microcircuit. The subsequent section will focus on explaining the dynamics of repetition suppression using simulations and computational modelling of empirical data. This will be followed by a review of empirical studies on repetition suppression and its context sensitivity, with a special focus on the crucial role of predictions and precision in modulating the effects of stimulus repetition.2surprise  over time, biological systems should continually minimise their surprise about sensory states. Mathematically, this is equivalent to maximising the Bayesian evidence for their model of sensory inputs, also known as Bayesian filtering. Predictive coding , biological systems have to minimise the excursions or entropy of their interoceptive and exteroceptive states. Since entropy is the average of e coding  is a popMinimising surprise – or maximising model evidence – lies at the heart of the free energy principle, where free energy provides a proxy for surprise that, under simplifying assumptions, can be reduced to prediction error . This meThe ability of the brain to infer the causes of its sensations rests upon the presence of statistical structure or contingencies in the environment. These contingencies can be embodied within a generative model describing the hierarchical and dynamic statistics of the external world:v denote causes representing (hidden) causes , while x denote states of the world mediating the influence of that cause on sensory signals . Because these dynamics follow stereotyped trajectories over time, they endow the model with memory. In equations above, tilde is used to augment the variables with their generalised coordinates of motion, i.e.,In the equations above, xi) as a nonlinear function f of causes and states themselves. Here D is a block-matrix derivative operator, with identity matrices on its first leading-diagonal. Eq. i-1 as a nonlinear function g of hidden causes and states at the level above. Random fluctuations in hidden causes and states are denoted by Eq. Since the brain does not have direct access to the causes and states in the external world, it can only infer the most likely values under its generative model: mathematically, these values are expectations. In other words, the generative model maps from causes to sensory consequences, while perception solves the  inverse problem which is to map from sensations to their underlying causes. An inversion of hierarchical dynamic models can be cast in terms of a hierarchical message passing scheme also known as predictive coding:This message passing suggests two distinct populations of neurons: one encoding the trajectory of the expectations  of hidden causes fi)( and gi) units talk to each other. In brief, the equations suggest that error-units receive messages from populations in the same hierarchical level and the level above, while state-units are driven by error-units in the same level and the level below. The prediction errors from the same level As mentioned above, prediction errors are weighted by their precision (or inverse variance). This allows for a dissociation of the magnitude of prediction error from its reliability – for example, in a noisy or volatile environment, continuous signalling of large prediction errors should not (necessarily) lead to large updates of expectations, since prediction errors will be very imprecise. Conversely, even minor deviations of sensory inputs from the descending predictions can lead to large updates of the conditional expectations if prediction errors are very precise. Crucially, precision itself has to minimise surprise (about the amplitude of prediction errors). Precision can therefore be manipulated exogenously; e.g., by changing the contrast or statistics  of the stimulus, or endogenously; e.g., by attending to a particular sensory stream or changing the contextual expectancy of sequential stimuli. In other words, endogenous attention anticipates precise sensory information (or prediction errors). As we will see, the notion of precision will be key for interpreting some of the empirical findings showing that repetition suppression can be modulated by contextual factors such as attention.3The notion that the brain implements a predictive coding scheme in its cytoarchitecture  has recenonlinear function [Eqs. Ascending and descending connections have also been shown to have distinct spectral profiles. Extensive work in the macaque visual system has demonstrated spectral asymmetries between superficial and deep cortical layers. Within cortical areas, activity in supragranular layers has been linked to local synchronisation of gamma-band oscillations, while neurons in infragranular layers typically synchronize in lower frequency bands such as alpha and beta . Similarion Eqs. , which cMapping the predictive coding scheme onto a canonical cortical microcircuit has important consequences. Since in the predictive coding scheme the descending predictions are subtracted from expectations in lower levels to form prediction errors, descending connections from deep pyramidal cells should inhibit activity in hierarchically lower areas , possibl4Many known characteristics of repetition suppression emerge in simulations of predictive coding. This has been previously shown in a formal model of an artificial brain perceiving sequences of sensory events, simulated using attractor dynamics  Fig. 3)Fig. 3). Here, this architecture was used to model an artificial songbird, assuming a simplified functional anatomy of birdsong generation and perception. The artificial agent's goal was to categorize three trajectories of sensory inputs corresponding to three birdsongs. A predictive coding scheme was used to model perceptual inference and categorisation. In perceptual inference, after a few hundred milliseconds of listening to any of the three birdsongs, the simulated neural activity corresponding to descending predictions about sensory input recovers the trajectory of the input actually presented to the agent. In perceptual categorisation, the expectations of hidden causes (that controlled the trajectories) at a higher level disambiguated or categorised the three songs. This provides an example of how sequences of sensory events unfolding in time can be predicted and mapped to more abstract locations in perceptual space, using predictive coding.deviant, while its subsequent repetitions gradually become a sensory standard. Predictive coding of these sensory events  revealed that the simulated prediction errors at the first (lowest) level peak at around 100 msec after stimulus onset, corresponding to the classical N1 component, while prediction errors at the second level peak at 150–250 msec after stimulus onset, mimicking the typical latency of mismatch negativity and consistent with its neural generators relying on hierarchically higher areas than those of the N1 component  of event-related potentials (ERPs) in humans . A rovinIn the DCM, the effect of stimulus repetition was decomposed into two time courses: a monotonic exponential decay function of stimulus repetition, mimicking slow cumulative effects, and a phasic (gamma-density) function peaking after the first repetition . These tThe two modelling approaches – one based on simulations of an artificial brain, one applied to empirical data – provide converging evidence for the notion that repetition suppression can be formulated in terms of predictive coding. With each stimulus repetition, the model of the sensory contingencies is gradually optimised, as encoded in changes to descending predictions. The descending predictions play a dual role, resolving the prediction error induced by sensory input and implementing changes in (expected) precision. Over the past few years, there have been further simulations of the mismatch negativity under predictive coding e.g.,  and ther55.1A receptors in the inferior colliculus , as well as classical neuromodulators such as acetylcholine (ACh), dopamine and serotonin are natural candidates to implement precision or gain control in prediction error signalling . This learning implicates short-term synaptic plasticity and neuromodulatory effects of the sort mediated by NMDA-type glutamate  and ACh receptors (for mediating attention through expected precision). Prediction errors, on the other hand, are conveyed by ascending (driving) connections that use fast AMPA-type glutamate receptors. This asymmetry is not only supported by empirical differences between ascending and descending projections but can be deduced from the form of Eqs. nicotine  was admiSimilarly, a DCM study investigated the effects of galantamine  on mismatch negativity . An analAnother DCM study investigated the mechanisms underlying the effects of NMDA-receptor antagonist ketamine, administered in a roving oddball paradigm . BesidesBeyond pharmacological manipulations, sensory precision can be modulated by various exogenous and endogenous factors. Decreasing stimulus visibility, for instance, has been shown to abolish repetition suppression effects , consistAttention has often been operationalised as the relevance of a particular stimulus for the task at hand . A direcIn contrast to attention, which has been be linked to low-level (sensory) precision, manipulating precision at higher contextual levels has been addressed by manipulating repetition probability – building upon a distinction between repetition suppression and expectation suppression. The expectations here rely upon context , and thus are presumably located at high hierarchical levels that can represent contextual factors. According to predictive coding, descending predictions of incoming repeated stimulus should suppress the prediction error resulting from the presentation of this stimulus. However, if the stimulus sequence is constructed such that stimulus alternation is more likely than repetition, presenting an identical stimulus twice should result in a prediction error, while valid expectations of a different stimulus should suppress prediction error. The initial finding that stimulus repetition probability affects repetition suppression  was baseInterestingly, the effects of repetition suppression and expectation suppression have been shown to be dissociated in time using MEG . While rAs discussed above, repetition suppression is subject to attentional modulation. Similarly, the interaction of expectation and repetition has been shown to depend on top-down attention . When paThe results of studies investigating the interactions between repetition and attention are in line with the nonlinear, modulatory nature of precision or gain control in predictive coding. Due to this nonlinearity, independent manipulations of precision at different hierarchical levels might even have antagonistic effects on neural responses in sensory areas . Therefo5.3Besides repetition suppression effects, several studies have reported repetition enhancement effects, or increased neuronal responses to repeated stimuli , where the amplitude of BOLD repetition suppression – as well as behavioural performance  – was shown to depend on the exposure duration of the first stimulus in a non-linear fashion. At the shortest exposure duration examined (40 msec), BOLD response to subsequently repeated stimuli was not significantly suppressed, and in some areas was nominally enhanced  neural response to repeated stimuli.The connections between cortical areas are subject to modulation of post-synaptic gain to specific inputs. Synchronisation of pre-synaptic inputs has been proposed as a likely candidate for controlling post-synaptic gain . As discSimilarly, several competing hypotheses had previously been proposed to account for a phenomenon closely related to repetition suppression, namely the mismatch negativity. According to the model-adjustment hypothesis , the MMNCrucially, beyond accommodating previous, more particular accounts of repetition suppression, predictive coding – by postulating a central role for descending connections in perceptual inference and learning in conveying predictions of sensory inputs and their precision – readily explains contextual effects of attention, expectancy, and confidence on neural responses to repeated stimuli. At the same time, it generates testable hypotheses in terms of laminar specificity of prediction and prediction error propagation, as well as the key involved neurotransmitters and neuromodulators, whose interactions will be reflected in the amplitude and dynamics of observable repetition effects.7The predictive coding framework offers a mechanistic explanation of repetition suppression in terms of optimised predictions about the content and precision of sensory inputs. This dual role of predictions, mediated by descending connections between levels of the cortical hierarchy, explains how contextual factors such as attention, contextual expectancy, and prior experience might modulate the effect of stimulus repetition – by simultaneously minimising prediction errors in perceptual inference, and increasing their precision due to perceptual learning. The precision-modulation of the underlying prediction error suppression is likely due to NMDA-receptor dependent plasticity and classical neuromodulators such as ACh. The ensuing modulation of inhibitory populations in sensory regions will influence neural activity in superficial layers that are populated by neurons encoding prediction errors. By bridging the neurophysiological and computational levels of description, the predictive coding framework reconciles several accounts of repetition suppression and offers insights into the most general principles of neuronal message passing.


Critical Care, Tagami et al. [In the March 2017 issue of During resuscitation, the main objective of cardiopulmonary resuscitation is to maintain blood flow pending etiological treatment, and to try and restore sinus rhythm . AntiarrPotassium chloride (KCl) is the major component of cardioplegia solutions used to arrest the heart during cardiac surgery under extracorporeal life support (ECLS). This effect is fully reversible, enabling return of spontaneous circulation after its administration. Twenty-five years ago, Beyersdorf et al.  observedIn cardiac arrest due to refractory VF, the proposition of direct intravenous KCl injection can primarily be surprising, because there is a suspected ischemia–reperfusion syndrome with metabolic acidosis and thus hyperkalemia, underlying the use of the pure potassium blocker nifekalant . ConversInvestigating the efficacy of direct intravenous KCl injection for OHCA due to irreversible VF seems therefore to be a relevant research pathway, to define the potential benefit of this treatment in the therapeutic strategy as compared with conventional antiarrhythmic drugs.


Dear Editor,Epidemiology and Health in March 2017. The aim of the study was to assess postpartum modern contraceptive use and associated factors among postpartum women in northern Ethiopia. A multivariable logistic regression model showed that the following independent variables were the most important determinants of postpartum modern conception use in the town of Aksum: maternal educational level (secondary and tertiary education level), family planning counseling during pregnancy and during prenatal and postnatal care, having postnatal care, resuming sexual activity, menses returning after birth, and experiencing problems with previous contraceptive use [We read the paper entitled ‘Postpartum modern contraceptive use in northern Ethiopia: prevalence and associated factors,’ written by Abraha et al. , which wRegardless of the results obtained from the model, it should be explained that accurate predictors or determinants of a dependent variable cannot be reliably identified by a cross-sectional study because predictors must be identified based on cohort studies ,3. In ot


Intranasal delivery provides a practical, noninvasive method of bypassing the blood-brain barrier (BBB) in order to deliver therapeutic agents to the brain. This method allows drugs that do not cross the BBB to be delivered to the central nervous system in a few minutes. With this technology, it will be possible to eliminate systemic administration and its potential side effects. Using the intranasal delivery system, researchers have demonstrated neuroprotective effects in different animal models of stroke using erythropoietin (EPO) as a neuroprotector or other different types of EPO without erythropoiesis-stimulating activity. These new molecules retain their ability to protect neural tissue against injury and they include Asialoerythropoietin  carbamylated EPO (CEPO), and rHu-EPO with low sialic acid content (Neuro-EPO). Contrary to the other EPO variants, Neuro-EPO is not chemically modified, making it biologically similar to endogenous EPO, with the advantage of less adverse reactions when this molecule is applied chronically. This constitutes a potential benefit of Neuro-EPO over other variants of EPO for the chronic treatment of neurodegenerative illnesses. Nasal administration of EPO is a potential, novel, neurotherapeutic approach. However, it will be necessary to initiate clinical trials in stroke patients using intranasal delivery in order to obtain the clinical evidence of its neuroprotectant capacity in the treatment of patients with acute stroke and other neurodegenerative disorders. This new therapeutic approach could revolutionize the treatment of neurodegenerative disorders in the 21 century.


Gastroparesis is defined as a combination of chronic dyspeptic symptoms and delayed emptying of a solid test meal. It remains a difficult-to-treat disorder with a significant impact on quality of life. Although gastroparesis is defined by delayed emptying, several important studies did not find a correlation between this biomarker and symptom severity or treatment success. Thus, some of the more recent trials explored strategies that ranged from antiemetics to antidepressants. Although dietary management showed benefit, most of the other interventions were barely superior to placebo or were not superior at all. Placebo responses were often quite high and this complicates the assessment of active agents. While it complicates the design and interpretation of clinical trials, high response rates for active and sham interventions indicate that we can achieve symptom relief in many patients and thus give them some reassurance. If indeed most therapies are only marginally better than placebo, the differences in adverse effects should be weighed more strongly, a point that is especially important in view of the controversy surrounding metoclopramide. Mechanistic studies introduced the network of macrophages as another potentially important player in the development of gastroparesis. Results are too preliminary and are largely based on preclinical data but show up- and downregulation of cellular elements controlling gastric function. Thus, future developments may teach us how they interfere with some of these mechanisms in clinical settings, potentially making gastroparesis a reversible process. About 60 years ago, Kassander coined the term gastroparesis, describing it as a consequence of vagal neuropathy in diabetics with secondary complications2. We have since gained significant insight into the normal and abnormal function of the stomach, developed and standardized a disease definition and diagnostic approaches, and employed a wide range of treatments from diet to medications and endoscopic and surgical therapies. Current consensus statements highlight the importance of delayed gastric emptying, typically documented by measuring the exit of a solid test meal from the stomach, and the chronic presence of dyspeptic symptoms, such as nausea, vomiting, post-prandial fullness, early satiety, anorexia, and abdominal discomfort3. Yet, given the repeatedly disappointing results of interventions that target disease-defining mechanisms, such as motilin or ghrelin agonists8, are we still facing a “conundrum”, as Longstreth lamented more than a decade ago9? Since then, several investigations have shifted our understanding of the start of gastroparesis and this has affected clinical practice.Eating not only ensures survival but also is part of the social fabric. Meals often function as times for social interaction, as symbols of hospitality, or as a central aspect of celebrations. Thus, illnesses that limit the ability to partake in this important daily activity have a disproportionately high impact on quality of life6. Yet gastric emptying does not consistently correlate with symptom severity, whether assessed with a labeled meal or a large undigestible particle, such as the wireless motility capsule11. This limited correlation could be due in part to a focus on the wrong endpoint, such as half-emptying time, rather than the fraction of a test meal retained after a predefined time12. The relationship between gastric emptying and symptoms improved when acute, meal-induced rather than recall-based severity ratings covering a longer period were used15. These chronic complaints and acute, experimentally evoked symptoms are conceptually related; however, trial endpoints and drug approval criteria target the chronic symptoms as clinically validated and relevant endpoints. Interestingly, a recent cohort study described similar symptoms and symptom severity between type 1 and type 2 diabetics, even though gastric emptying was slower in type 1 diabetes, thus similarly pointing away from this variable as the sole or even primary determinant of disease severity16. Conversely, the perceived benefit of treatments does not correlate with changes in gastric emptying17. Lastly, gastric emptying does not reliably do what it is supposed to do as a biomarker, namely differentiate patients with overlapping symptoms but different disorders. Close to 90% of patients with gastroparesis meet the consensus definition of functional dyspepsia and about 20% of patients with functional dyspepsia have impaired emptying independent of their symptom pattern18. Similarly, a recent study characterized patients with chronic unexplained nausea, which phenotypically overlapped with gastroparesis except for the normal results of gastric-emptying studies19. The overlap goes beyond symptoms and changes in emptying as detailed physiological studies showed coexisting changes in accommodation and sensory mechanisms in gastroparesis20. If we look at mechanistic studies, we still see more parallels than differences as patients with otherwise-unexplained nausea and vomiting share some of the microscopic abnormalities on full-thickness gastric biopsies that otherwise have been associated with gastroparesis21. Looking for alternative explanations related to gastric motor function, a small study suggested a potential correlation between symptoms and integrated motor activity22, a finding that subsequent investigations did not confirm in a substantially larger cohort with 209 participants11. Thus, we may deal with a spectrum of abnormalities that overlap in symptoms, test results, and underlying pathophysiology. Instead of making gastric emptying a key criterion for diagnosis and treatment, we may reconceptualize dyspeptic syndromes as a potential consequence of several coexisting mechanisms that contribute to a problem. Delayed emptying can clearly be one of these changes and may contribute to symptoms or symptom severity, especially the severity of nausea and vomiting18 or post-prandial symptom exacerbations15. Some recent data also point to the potential prognostic value of gastric-emptying data with apparently counterintuitive results, as slower emptying at baseline was associated with a better prognosis after close to one year of follow-up23.As mentioned above, gastroparesis is defined by a delay in gastric emptying, which differentiates it from functional dyspepsia or chronic unexplained nausea. The impaired emptying has traditionally been the target of treatments and often functioned as a key endpoint in clinical trials24. Conversely, interleukin-10 activates M2 macrophages, which protect the ICC population26. With animal models showing up- or downregulation of ICCs through such mechanisms and associated changes in gastric emptying7, do we need to look at this motility disorder as a problem at the interface between the immune system and neuromuscular transmission? Bharucha and colleagues recently explored this question by trying to induce heme oxygenase-1, which is expressed in M2 macrophages and plays an important anti-inflammatory role8. Their pilot data show a potent induction of the enzyme but no influence on symptoms or emptying28. Even if macrophages or specific enzymes have not yet found their way into successful treatment strategies, these findings illustrate how preclinical data are translated into novel treatment strategies. In addition, the underlying preclinical data may function like a ray of hope as they suggest that cellular abnormalities linked to the development of gastroparesis are potentially reversible.Animal models and human data have identified a network of macrophages in the muscular layer of the stomach, which may link the immune system to changes in gastric motor function. Activation of M1 macrophages triggers the release of tumor necrosis factor alpha, which, in turn, leads to a decrease in interstitial cells of Cajal (ICCs), the electrical pacemaker cells of the gut30, the potential role of pyloric dysfunction in gastroparesis has surfaced again in two forms. On the diagnostic side, the EndoFLIP  device allows detailed assessments of the pylorus’s active and passive mechanical properties, which seem to be altered in gastroparesis and appear to correlate with symptoms and emptying32. If this test is confirmed, we need to examine whether it enables us to identify a subgroup that could indeed benefit from interventions targeting this muscle structure. On the therapeutic side, per-oral endoscopic myotomy not only is feasible but also comes with impressive preliminary findings in early open-label trials33. In light of the similar early enthusiasm with botulinum toxin therapy, followed by disappointing results in randomized controlled studies37, it is essential to integrate more detailed functional assessments of this muscle  and to assess the true impact of this intervention with controlled trials.One conceptual model envisions impaired opening of the pyloric channel as a cause for gastric retention, providing a rationale for treatments targeting this muscle. After two negative trials of botulinum toxin injection into the pylorus23. As already indicated, the observational design of this cohort study does not allow conclusions about specific steps. Yet it is important to contrast this message with the results of randomized controlled trials completed recently. We compiled data from double-blinded placebo-controlled trials published within the last decade and examined response rates for dichotomized endpoints  becomes more important. While the effects of antiemetics and other agents commonly used in gastroparesis have been less systematically studied, the controversies surrounding metoclopramide clearly highlight the relevance of this criterion in gastroparesis48.Several other observations about treatment options and impact have emerged during the last decade. First, a consortium of tertiary referral centers completed longitudinal studies that tracked symptoms and findings in patients who received treatment at the various sites for gastroparesis. Though not standardized and thus limiting our ability to assess specific treatment approaches, management was largely driven by the very experts who set the tone in discussions about the diagnosis and treatment of this illness. The data seem to be disappointing as even in expert hands, symptoms largely remain stable with less than one third of the patients improving during follow-updpoints  or changdpoints . Virtual49. Impaired motility clearly contributes to the pathophysiology of these common problems but includes more than just a delay in emptying. Thus, we need to assess whether other non-invasive tests, such as magnetic resonance imaging or single-photon emission tomography51, can provide a more comprehensive assessment of gastric motor function from accommodation to emptying and whether more detailed information allows more targeted and more effective therapy. If indeed emptying poorly correlates with symptoms and treatment effects, what can we offer beyond dietary advice? Recent studies give us some sense of direction, as anxiety and depression are associated with symptom severity10 and as antidepressant use functioned as a positive outcome predictor23. These results correspond to detailed mechanistic studies in functional dyspepsia that highlight the importance of affect53. Yet we still have to resolve some apparent contradictions, as well-designed trials led to inconsistent results with tricyclics not being superior to placebo in gastroparesis, while there was a benefit in a large cohort of patients with functional dyspepsia which included persons with impaired gastric emptying55. Going back to our call for a better and more comprehensive phenotypic characterization of patients, we may be able to identify subgroups with markers other than gastric emptying alone that may allow more targeted and more successful therapy. Concerns about metoclopramide led to a decreasing use of prokinetics with an apparent increase in antiemetics. Given the importance of nausea and vomiting as hallmark symptoms of gastroparesis, this development seems intuitively reasonable. However, the marginal benefit seen with aprepitant56 demonstrates the need for evidence rather than intuition. Most of our insight into the efficacy of antiemetics comes from the treatment of chemotherapy-induced nausea, which is typically an acute and only transient problem. Although the clinical manifestation may look identical, the underlying mechanisms will almost certainly differ, forcing us to better define the true benefit of these agents in gastroparesis. Perhaps most importantly, we need to develop approaches that will enable us to provide better prognostic information and treatment for patients where they are most commonly seen, outside the tertiary referral centers.With at least some of the novel treatments not meeting expectations, gastroparesis remains a difficult-to-treat disorder. However, there have been clear advances in our understanding of this and perhaps some of the other closely related diseases. In our short review, we have tried to point to potential areas for future studies. One key question, is whether we should see gastroparesis as a distinct disorder or whether it belongs in the spectrum of syndromes labeled as functional dyspepsia, as has been suggested


Finding a treatment for spinal cord injury (SCI) focuses on reconnecting the spinal cord by promoting regeneration across the lesion site. However, while regeneration is necessary for recovery, on its own it may not be sufficient. This presumably reflects the requirement for regenerated inputs to interact appropriately with the spinal cord, making sub-lesion network properties an additional influence on recovery. This review summarizes work we have done in the lamprey, a model system for SCI research. We have compared locomotor behavior (swimming) and the properties of descending inputs, locomotor networks, and sensory inputs in unlesioned animals and animals that have received complete spinal cord lesions. In the majority (∼90%) of animals swimming parameters after lesioning recovered to match those in unlesioned animals. Synaptic inputs from individual regenerated axons also matched the properties in unlesioned animals, although this was associated with changes in release parameters. This suggests against any compensation at these synapses for the reduced descending drive that will occur given that regeneration is always incomplete. Compensation instead seems to occur through diverse changes in cellular and synaptic properties in locomotor networks and proprioceptive systems below, but also above, the lesion site. Recovery of locomotor performance is thus not simply the reconnection of the two sides of the spinal cord, but reflects a distributed and varied range of spinal cord changes. While locomotor network changes are insufficient on their own for recovery, they may facilitate locomotor outputs by compensating for the reduction in descending drive. Potentiated sensory feedback may in turn be a necessary adaptation that monitors and adjusts the output from the “new” locomotor network. Rather than a single aspect, changes in different components of the motor system and their interactions may be needed after SCI. If these are general features, and where comparisons with mammalian systems can be made effects seem to be conserved, improving functional recovery in higher vertebrates will require interventions that generate the optimal spinal cord conditions conducive to recovery. The analyses needed to identify these conditions are difficult in the mammalian spinal cord, but lower vertebrate systems should help to identify the principles of the optimal spinal cord response to injury. Recovery from a spinal cord injury (SCI) in mammals, including humans, is minimal. Despite hopes, and claims, for effective therapies, the outlook for targeted improvement remains poor. Injury is associated with the loss of motor, autonomic, and sensory function, as well as the development of deleterious effects like spasticity and neuropathic pain . While sTreatments for SCI have been categorized as those that rescue, reactivate, and rewire . Rescue Issues of replication in studies that attempt to promote regeneration or alter the effectiveness of spared axons may reflect the fact that the descending input to the spinal cord is only one of several factors needed to generate effective outputs. The most obvious is that while promoting regeneration or manipulating the properties of spared axons seems necessary and reasonable, these approaches can only be beneficial if they lead to functionally appropriate interactions below the lesion site.Locomotor networks below lesion sites remain after injury, and can control complex movements if they receive appropriate inputs. This has been examined with epidural stimulation of the spinal cord in experimental and clinical studies , while stage 1 animals fail to show any recovery . In larval and juvenile adult lampreys that recovered well , swimming parameters  assessed from electromyograms did not differ significantly to unlesioned animals . Recovery thus restores normal locomotor function.To assess locomotor activity after recovery from lesioning we scored swimming behavior on a six-point scale based on Figure 3Ai). While stage 5/6 activity can occur without any regeneration . This, together with the potential for recovery in some cases in the absence of regeneration, suggests the involvement of other factors in recovery.Stage 1/2 animals typically lacked regeneration, shown by the absence of activity in the spinal cord below the lesion site when stimulating above the lesion site ; poor co-ordination across the lesion site associated with differences in the frequency or pattern of activity; and defects in the reciprocal coupling between the left and right sides of the spinal cord above and below the lesion site . In an analysis of 16 stage 1/2 animals, none were able to generate activity below the lesion site, and 7 were also incapable of generating reliable alternating activity above the lesion site. Even when there was alternation above the lesion site, alternating activity was absent 20–80% of the time . These defects need to be characterized as they could suggest influences on the success or failure of recovery. Their potential range and variability of defects means that this will require a large sample size.While analyses typically focus on successful recovery, analyses of the small proportion of animals that fail to recover could be more informative. Poor recovery is associated with several behavioral defects see , includiin vitro preparations by In addition to monitoring swimming in intact animals, spinal cord function can be examined in the isolated spinal cord using fictive locomotion evoked by glutamate-receptor agonists e.g., . This alWhile fictive locomotion offers experimental advantages, there are issues surrounding its use in SCI given the hypothesis that spinal cord perturbation evokes compensatory changes. Fictive activity varies between experiments for a given drug concentration, and shows a prolonged and variable development that requires preparations to be left for some hours before activity is examined see . HemisecThe morphology of regenerated axons have been studied extensively in lamprey. Analyses typically focus on the larger Muller reticulospinal axons, as these can be uniquely identified . These an = 42) and above (n = 22) and below a lesion site (n = 14) were examined in response to presynaptic stimulation at 20 Hz to characterize the initial and activity-dependent properties of the connections , rise-time and half-width, did not differ significantly in lesioned and unlesioned animals. This suggests against any compensation at individual synapses for the reduced innervation of the sub-lesion spinal cord that will occur given that regeneration is never complete , suggesting increased variability after lesioning. In unlesioned animals most connections depressed over spike trains . The proportion of connections showing different forms of plasticity was the same below the lesion site as in unlesioned animals, but above the lesion site connections usually facilitated . While this analysis is on lateral tract axons, these features were also seen for putative Muller axons : the initial slope represents Q , the width represents N (the number of release sites), and the degree of curvature represents Pr (the transmitter release probability). These analyses were performed in the larger medial column Muller axons, firstly to relate functional properties to the ultrastructural features in Regenerated Muller axons in the ventromedial region of the spinal cord make fewer synapses below the lesion site, contain fewer vesicles, and have smaller active zones than comparable synapses from unlesioned animals . These uIn regenerated axons below the lesion site N was generally reduced, consistent with the sparser anatomical connectivity ; Pr was While these analyses are not definitive, they suggest lesion-induced changes of reticulospinal synapses that differ above and below the lesion site. The increased Q below the lesion site could scale regenerated inputs so that they match unlesioned synapses despite the sparser connectivity. The postsynaptic increase in Q but reduced Pr above the lesion site allows connections to facilitate without the reduction of the initial input that would occur with a reduction of Pr alone, resulting in a net increase in connection strength above the lesion site see . A reducn = 4) making the data very preliminary. These connections had initial inputs that were markedly smaller than unlesioned connections (<0.3 mV) and tended to depress strongly, to the extent that the connections failed . This suggests a potential failure of the postsynaptic scaling of the connection (increase in Q), while compromised function over repetitive stimulation could reflect a failure of activity-dependent replenishment mechanisms . Averaging these responses to give mean values does not seem appropriate. Synapses vary in unlesioned animals . Effects differed in poorly recovered animals, depending on whether regeneration occurred: in poor recovery with regeneration excitability was not increased locally only distally below the lesion site , but without regeneration excitability was increased immediately below the lesion site but distal effects were reduced . This suggests several aspects: firstly, increased excitability locally (1–5 segments) below the lesion site is needed for good recovery; secondly, excitability changes occur diffusely below the lesion site,  in response to spinal cord stimulation at different supra- and sub-lesion sites  or other divisions  that may better reveal a link between the excitability changes and degree of regeneration. Alternatively, rather than being “noise” associated with these effects, variability may be a signal that allows the spinal cord to sample a range of options to optimize recovery , or it rFigure 3Av), and a significant reduction of activity when stimulating across the lesion site, indicative of a lack of functional regeneration. In animals that showed good recovery, regeneration had occurred and there was a significant increase in excitability ipsilateral to stimulation above the lesion site . However, below the lesion site excitability did not differ significantly to unlesioned animals. This suggests that in adults as regeneration occurs sub-lesion excitability falls back to match that in unlesioned animals. However, this restoration was associated with changes in functional properties and spinal cord organization (see below).Juvenile adults also showed changes in excitability. In contrast to larvae these were significantly related to different degrees of recovery. Animals that failed to recover had significantly increased excitability ipsilateral to the stimulation site up to 10 segments below the lesion  and the magnitude of the excitability changes below the lesion site . However, animals that failed to recover showed negative correlations between the degree of regeneration and excitability . As a result, the synaptic excitation:inhibition ratio was increased in animals that recovered well compared to unlesioned animals , but was reduced in animals that failed to recover  were all significantly increased in motor neurons below the lesion site compared to unlesioned animals . These effects could reflect presynaptic and postsynaptic changes. An ultrastructural analysis of synaptic terminals from putative network interneurons showed that in lesioned animals the total and docked vesicle numbers were significantly greater  and the vesicle diameter and synaptic gap significantly reduced at asymmetric (putative excitatory) synapses. However, there was a reduction in the post-synaptic density length at symmetric (putative inhibitory) synapses (Synaptic effects below the lesion site were examined in more detail using TTX-resistant miniature EPSPs (mEPSPs) to remove the influence of changes in cellular excitability. The amplitude, frequency, and half-width of mEPSPs were all increased, and there was an increase in summated or “double” mEPSPs . This was significantly larger in animals that recovered well than those that recovered poorly where it was small or absent, suggesting another potential factor in good recovery. The mechanism underlying this effect is unknown. It does not seem to reflect an L-type calcium, persistent sodium, or NMDA conductance , which could be facilitated by the larger glutamatergic vesicle pool. We tried to mimic the effect in unlesioned animals using strontium to increase spontaneous release and N-ethylmaleimide to increase the number of docked vesicles . Unlike larvae, where increased inhibition was associated with poor recovery, in juvenile animals increased inhibition also occurred in good recovery , excitability; Figure 1Diii) and changes in excitability  suggest that there are changes above the lesion site. This may reflect the degeneration of damaged neurons above the lesion site. In addition, there are ascending propriospinal inputs to the spinal cord and brainstem that will be affected by a lesion  may also be programmed directly to increase propriospinal signals across the lesion site to compensate for the reduction of descending brainstem inputs . The frequency of spontaneous excitatory and inhibitory synaptic inputs was increased above and below the lesion site compared to unlesioned animals, but spontaneous inhibitory inputs were greater below and excitatory inputs greater above the lesion . The increased excitatory input above the lesion was lacking in animals that failed to recover , again suggesting a potential role in good recovery, but the increased inhibition below the lesion site occurred irrespective of the degree of recovery. The changes in supra-lesion excitation were supported by the increased feedforward excitatory interactions above the lesion site seen in paired recordings from EINs and motor neurons . However, feedforward inhibition was increased below the lesion . These effects suggest a potential difference in the reorganization of sub and supra-lesion locomotor networks  and the slow synaptic depolarization did not differ significantly above and below the lesion site to the properties in unlesioned animals . However of <0.1  to a pronetworks ,n = 2), suggesting that regeneration was necessary. Interestingly, recovery was faster after the second transection . This may be “noise”  that are identified by their ability to make monosynaptic connections onto motor neurons  and the Figure 6A; Proprioceptive inputs can be examined simply in the lamprey as the proprioceptive edge cells are located on the lateral margin of the spinal cord . They seFigures 6Bi,Bii; Figure 6Ci), effects associated with an increase in the excitability in response to current injection . There also seems to be an interaction of sensory systems with the locomotor network, as after lesioning there was an increase in spontaneous synaptic inputs in the edge cells . But in lesioned animals, where proprioceptive activity was potentiated (see above), GABA was less effective .Bicuculline could cause long-lasting sensory discharges, suggesting that unregulated proprioceptive feedback would disrupt locomotion. Bicuculline can improve locomotor function after SCI when the lesion was made in adult cats and resulting recovery was poor; but disrupted activity when the lesion was made in kittens that subsequently recovered good locomotor function, suggesting that increased GABAergic inhibition was needed for recovery ,b. This Somatostatin, which co-localizes with GABA , did not5-HT is arguably the dominant transmitter system studied after SCI. 5-HT has significant effects on motor outputs and sensory processing in the unlesioned spinal cord , and theFigure 8A). This may negatively affect recovery by reducing the excitation:inhibition ratio from an optimal level (see above). 5-HT reduced the post-action potential sAHP and increased excitability in control animals and animals that recovered well (above and below the lesion site), but had no effect in animals that didn’t recover , again suggesting a potential role in good recovery. A striking effect was that the 5-HT-mediated reduction of glutamatergic inputs, a highly consistent modulatory effect in unlesioned animals , suggesting that the synaptic effects of 5-HT are reduced after SCI.The effects of 5-HT differed in unlesioned and lesioned animals that recovered well or poorly. 5-HT consistently evoked a hyperpolarization of the membrane potential in unlesioned animals . However, 5-HT seems to be essential during recovery. Depleting 5-HT with p-chloro-phenylalanine (PCPA), a 5-HT synthesis inhibitor, markedly disrupted activity in unlesioned animals , suggesting a necessary role for endogenous 5-HT in normal locomotor activity. Animals usually recovered normal swimming after removal of PCPA for 5 days . However, PCPA was without affect when it was applied after locomotor function had recovered , suggesting that endogenous 5-HT becomes less important once recovery had occurred. These results are based on small numbers of animals. Given the variability of effects, especially after lesioning, further experiments are needed.We have also examined the effects of 5-HT on locomotor behavior . 5-HT tyr 5 days . When leThe simple conclusion from these analyses is that modulatory effects differ in lesioned animals. This seems to be a conserved effect (see (1)Regeneration is usually necessary for recovery, but as recovery can occur in its absence it may not be an absolute necessity. This does not negate the importance of promoting regeneration, but means that other approaches, for example electrical or pharmacological stimulation, could substitute for regeneration given optimal conditions.(2)Regeneration is not restoration: regeneration is never complete, regenerated axons project ectopically, and regenerated synapses have a different ultrastructure. Even though pre-lesion functional properties are restored at regenerated synapses, this reflects changes in synaptic function.(3)As inputs from individual regenerated axons match those in unlesioned animals, there is no compensation at individual synapses for the reduced descending drive caused by incomplete regeneration cf. . There m(4)Compensation seems to occur in locomotor networks through wide-ranging changes in cellular and synaptic properties and synaptic reorganization. The net effect of the cellular and synaptic changes seems to be an increase in spinal cord excitability, although this requires balancing by a parallel increase in inhibition.(5)Even though regeneration is usually necessary for recovery, it is not sufficient. Regeneration does not equal recovery: recovery instead depends on how altered regenerated inputs interact with altered sub-lesion networks.(6)Proprioceptive inputs are potentiated after SCI. The lack of correlation between the sensory changes and recovery could suggest that sensory inputs have no influence on recovery, which seems unlikely given the powerful sensory entrainment/reflex effects , or thatFigure 10): the same behavioral output is generated by an anatomically and functionally different spinal cord. Recovery is thus more than reconnecting the two sides of the spinal cord. Sub-lesion changes may adapt the spinal cord to the reduced descending excitatory drive, while supra-lesion changes may generate stronger proprioceptive or mechanical responses to relay activity across the lesion site. Potentiated sensory feedback may in turn be needed to regulate the activity in these altered networks. While these are plausible responses to injury, we lack insight into their direct roles. Understanding their influence and underlying mechanisms will allow them to be targeted to improve functional recovery. Even if not targeted directly, as they will influence any regenerative, prosthetic, training or pharmacological input, understanding these changes is necessary to any rational intervention after SCI.The restoration of normal locomotion after SCI thus reflects regeneration and functional changes in locomotor networks and sensory inputs We need to understand the properties of regenerated synapses. While synaptic properties will ultimately determine what regenerated axons will do, this aspect has received very little attention compared to axonal regeneration across lesion sites. The changes in the sub-lesion spinal cord mean that restoring a pre-lesion descending input will be unnecessary, unsuccessful, or even undesirable. We instead need to ensure that regenerated inputs make appropriate connections with sub and supra-lesion networks. Anatomical and functional connectivity arises in an orchestrated developmental process that may be difficult to recapitulate in adults , and can identify features associated with successful synaptic re-integration that can be applied to mammalian analyses.ults see , but und(2)A core requirement is the need to determine whether the changes in spinal cord networks are beneficial or deleterious to recovery. Sub-lesion changes in locomotor networks and sensory systems are routine across systems. However, their roles are generally complex and unclear: even deleterious effects  can be beneficial under some conditions . The rouThe impact of any effect will depend on its intrinsic properties and how it interacts with the locomotor network. Identifying these changes and their impact on recovery is crucial, but can be difficult in mammals . Lower v(3)We need to understand the interventions needed after SCI. Self-organization through compensatory changes may underlie spontaneous recovery in lower vertebrates or neonatal mammals, but the limited recovery of adult mammals suggests that these mechanisms are insufficient, absent or, like the regenerative capacity of the spinal cord, actively suppressed. We need to understand how to intervene to trigger effects conducive to recovery, and when to intervene given that effects can be beneficial or deleterious depending on timing We need to understand the relevance of variability after SCI. Parameters vary in spinal cord circuits (e.g., Analyses of variability need the characterization of identified parameters pre and post-lesion. This is possible in mammalian systems (but is easier in lower vertebrate systems (e.g., of excitatory network interneurons in lamprey and zebrafish; Testing the role of variability requires that it is manipulated without changing mean values. This can be difficult to do experimentally in any system, but the characterization of variability after SCI will allow computational analyses.ts e.g., , and thits e.g., , the appts e.g., . Analysets e.g., . While ets e.g., , suggestts e.g., . This mats e.g., . While nts e.g., . Analyse systems , but is 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.


Genomic sequencing in cancer (both tumour and germline), and development of therapies targeted to tumour genetic status, hold great promise for improvement of patient outcomes. However, the imminent introduction of genomics into clinical practice calls for better understanding of how patients value, experience, and cope with this novel technology and its often complex results. Here we describe a protocol for a novel mixed-methods, prospective study (PiGeOn) that aims to examine patients’ psychosocial, cognitive, affective and behavioural responses to tumour genomic profiling and to integrate a parallel critical ethical analysis of returning results.This is a cohort sub-study of a parent tumour genomic profiling programme enrolling patients with advanced cancer. One thousand patients will be recruited for the parent study in Sydney, Australia from 2016 to 2019. They will be asked to complete surveys at baseline, three, and five months. Primary outcomes are: knowledge, preferences, attitudes and values. A purposively sampled subset of patients will be asked to participate in three semi-structured interviews (at each time point) to provide deeper data interpretation. Relevant ethical themes will be critically analysed to iteratively develop or refine normative ethical concepts or frameworks currently used in the return of genetic information.This will be the first Australian study to collect longitudinal data on cancer patients’ experience of tumour genomic profiling. Findings will be used to inform ongoing ethical debates on issues such as how to effectively obtain informed consent for genomic profiling return results, distinguish between research and clinical practice and manage patient expectations. The combination of quantitative and qualitative methods will provide comprehensive and critical data on how patients cope with ‘actionable’ and ‘non-actionable’ results. This information is needed to ensure that when tumour genomic profiling becomes part of routine clinical care, ethical considerations are embedded, and patients are adequately prepared and supported during and after receiving results.ACTRN12616000908437.Not required for this sub-study, parent trial registration  Despite treatment advances in recent years, survival rates in some cancers have only minimally improved.  As such,There are generally two types of genomic sequencing in the cancer context: TGP and germline genomic sequencing (GGS). TGP involves laboratory analysis of somatic tissue to identify somatic driver mutations in tumours, whereas GGS involves examination of the entire genome through a blood sample seeking germline variants associated with cancer predisposition syndromes. This study focuses on TGP. A second study examining the psychosocial, behavioural and ethical issues and outcomes of GGS on cancer probands and their blood relatives is also underway and its protocol will be published separately. The two sub-studies together represent The P  i(n) Ge (genomics) On (oncology) PiGeOn project.TGP involves panel or whole genome testing of DNA derived from tumours. This may identify gene variants that can inform choice of treatments targeting the specific gene variants present in the tumour. TGP can identify somatic variants that: i) affect treatment ; ii) do not affect treatment (non-actionable); or iii) are of uncertain significance. TGP can be followed by further confirmatory testing if it is suspected that gene variants may have a germline origin .The promise of genomic medicine will only be realised if patients understand and benefit from identification of gene variants; that is, they need to understand the chances of finding actionable variants, and any potential implications of results. While uncertainty in terms of potential information available pervades TGP less than GGS due to the smaller scope of testing, there remains an element of uncertainty due to the poor prognosis of most patients and the uncertainty of the results being clinically actionable, and also the chance of finding a germline variant with the subsequent implications of testing for the recipient’s blood relatives. Practitioners who obtain consent for TGP face the challenges of conveying these uncertainties to both ensure informed choice regarding testing and to mitigate any unrealistic expectations.Further, although uncertainty may be shared among stakeholders, such as during education sessions between physician and patient, the extent of intricacy within genomic testing can lead to both parties remaining unaware of their areas of ignorance.  The expeWe have little understanding of the ethical, psychosocial and behavioural implications of providing cancer patients with results from TGP, and whether it will be experienced differently to other medical tests. The published evidence has been gathered primarily from research participants who may or may not have cancer, where the potential benefits of a gene variant being present (such as access to novel therapeutic options) differ. Several North American studies –7 presenOverall, very little is known about the knowledge, preferences, attitudes and values of cancer patients who have actually undergone TGP, their experience of uncertainty, their behaviour on receipt of TGP results, or the psychological effects these results may have. Only a handful of studies have explored responses in cancer patients who have actually been offered TGP. These have primarily noted patient report of information overload and misunderstanding, leading to unrealistic expectations, anxiety and uncertainty –10. PatiOncologists were among the first clinicians to incorporate TGP into routine management to guide treatment choice. Unifocal profiling goes back to estrogen receptor status testing in breast cancer and then EGFR mutation in lung cancer, with recent testing of the KRAS gene in colorectal cancer and the BRAF gene in melanoma. , 12 It iThis paper outlines the protocol of the first Australian study collecting longitudinal data on cancer patients’ experiences of TGP over time..The design and measures for the study are guided by Protection Motivation Theory  and DiffIn this setting, protection motivation will be the result of perceived susceptibility to disease progression and death, and fear of cancer progression, as well as participant knowledge of, attitudes to, and value given to, TGP (as a strategy to guide more effective treatment and thus reduce the threat of disease progression). Patients who perceive TGP to be valuable will be more likely to remain satisfied with their decision to undertake TGP, regardless of results, and to act on positive results by joining appropriate clinical trials of tailored treatments. Intolerance of uncertainty may make TGP less attractive and increase the likelihood of regret, doubt and poor psychological outcomes.Before and after receipt of results: evaluate knowledge of TGP, preferences for genetic information, attitudes (positive and negative) and the value placed on TGP;After receipt of results: identify behavioural, decisional and psychological outcomes from TGP along with their respective predictors; andContextualize the findings via a critical bioethics analysis.The aim of this study in adults with advanced cancer undergoing TGP is to:At baseline (after giving informed consent to undergo TGP), advanced cancer patients will have moderate to poor knowledge about TGP .At baseline, advanced cancer patients will have primarily positive attitudes to, and highly value, TGP.who receive an actionable result, most will:Pursue personalised treatment in a clinical trial setting .Cope well with results, and experience low decisional conflict and regret and high decisional satisfaction .Have higher hope, less cancer-related anxiety, lower fear of cancer progression and lower general anxiety and depression than those who do not receive actionable results .Of advanced cancer patients no actionable variants identified will:Those advanced cancer patients who have Struggle to cope with non-results .Have levels of hope, cancer-related anxiety, fear of cancer progression and general anxiety and depression similar to published results for advanced cancer patients who have not undergone TGP, within 1 month of receiving results and 5 months later .5.Age, education level, ethnicity, and gender;Tolerance of uncertainty and baseline knowledge; andPreferences and attitudes to TGP.Predictors of behavioural, decisional and psychological outcomes within 1 month of receiving results and 5 months later, will include:Specific hypotheses are that:Methodology in critical bioethics analysis is not hypothesis-driven and to this end there are no hypotheses relating to this part of the analysis.The Genomic Cancer Medicine Program includes a large cohort study funded by the NSW Ministry of Health, Australia, the Molecular Screening and Therapeutics (MoST) Program. MoST is recruiting 1000 adult patients with pathologically confirmed advanced or metastatic solid cancer with a particular focus on rare or neglected cancers. Participants undergo TGP, and receive results at approximately 11 weeks. Their treating oncologist is informed of results and discusses results and treatment options with the patient. Possible treatment options identified through TGP include a clinical trial offered as part of the MoST program, another clinical trial available in Australia, or off-label treatment through the treating clinician. This cohort offers the opportunity to explore short- and long-term impacts on behaviour and psychological morbidity of receiving TGP results. The PiGeOn project constitutes a psycho-social/ethical sub-study in this cohort, employing mixed methods to gather patient-reported outcomes and qualitative data.This is a mixed method, prospective, cohort sub-study of a TGP programme enrolling patients with advanced cancer.Participants will be recruited by the parent programme, from incident and prevalent cases at three large tertiary oncology units in Sydney hospitals. All suitable participants will provide informed consent using established procedures for genetic research, validated by large genetic studies. , 14 The The target population includes patients with pathologically confirmed advanced or metastatic solid cancer of any histologic type. Patients with rare or neglected cancers  will be given priority.Inclusion criteria include: age 18 years or older; pathologically confirmed advanced and/or metastatic solid cancer or an earlier diagnosis of a poor prognosis cancer; sufficient and accessible tissue for TGP; failed all standard anticancer therapy or receiving last line of standard therapy at time or enrolment; Eastern Cooperative Oncology Group Performance Status Measure (ECOG) performance status 0, 1 or 2; willing and able to comply with all study requirements, including timing and/or nature of required assessments; and written informed consent.Exclusion criteria include: suitability for standard therapy, if the patient has not been previously treated; specific co-morbidities or conditions which may contraindicate participation or compromise assessment of key outcomes; history of another malignancy within 2 years prior to registration: pregnancy, lactation or inadequate contraception.Patients will provide consent to MoST staff for the psychosocial (PiGeOn) sub-study at the same time as they give consent to the parent TPG program. Thus at baseline they will have already considered and given consent to TGP, but not yet had TGP. Participants will be asked to complete a questionnaire  at baseline (T0), 1 to 4 weeks after receiving TGP results , and at 2 months (T2) after T1. These timeframes have been chosen to allow impacts of the test results to be captured in both the short-term (when a treatment decision is being made) and longer-term . A small subset (around 20–40 participants) will be invited to participate in semi-structured interviews at the same assessment time points to explore in more detail attitudes towards TPG and its associated psycho-social, behavioural and ethical aspects. See Fig. The following patient-reported measures will be collected  outcome measures will be administered at baseline only:This 2-item measure adapted from Hay et al.  assessesA 9-item, multiple choice, study-developed questionnaire assessing knowledge of the purpose of TGP, likely frequency of informative results, cancers in which informative results are more likely to be found, availability of tailored treatment options, and source of genetic knowledge. Scores are summed, with high scores indicating greater knowledge.Four items adapted from Rosenberg et al.,  assessinA hypothetical time trade-off scenario based on those used in three previous studies. –19 Six iFour Likert-scale items adapted from Tabor et al.,  assessinEight Likert-scale items from Kasparian et al.,  measurinNote that to reduce participant burden at baseline, measures of psychological morbidity  were not included at T0.The following outcome measures will be administered at the first follow-up only:The 25-item Multidimensional Impact of Cancer Risk Assessment  assessinTen items from the Decisional Conflict Scale  compriseThe following outcome measures will be administered at the first and second follow-ups:The 15-item Impact of Events Scale  assessesThe 14-item Hospital Anxiety and Depression Scale  compriseThe 12-item Herth Hope Index  measuresThe following outcome measure will be administered at the second follow-up:The 5-item Decisional Regret Scale  measuresThe following outcome measures will be administered at baseline and first follow-up:Two open ended questions adapted from an earlier study  assessinThe 6-item Satisfaction with Decision scale  measuresThe following outcome measures will be administered at baseline and all follow-ups:Three items from the Concerns about Recurrence Questionnaire , adaptedParticipants indicate perceived likelihood of having a gene fault that increases risk of cancer progression on a visual analogue scale (0–100%) adapted from Kasparian et al. A subset of participants will be invited to participate in three semi-structured interviews (at baseline and each follow-up). Recruitment will continue until saturation is reached.  As the mThe MoST study plans to recruit 1000 patients over three years. Thus, with a conservative retention and survival estimate of 75%, this is reduced to at fewest 469 patients. With this sample size estimate, assuming 15% of patients receive an actionable result and using a significance level of 0.05, this project: has 90% power to detect a mean change of 5.8 and 2.6 points on the Impact of Events scale for patients with actionable and non-actionable results respectively   and a meInterviews will be recorded and transcribed verbatim and manually coded line-by-line. Transcripts will be subjected to Framework Analysis,  allowingEthical concepts and analysis will be relevant throughout this project, and will be iteratively and critically reflected upon as the study progresses. Quantitative survey items allowing free-text responses regarding benefits and drawbacks of TGP will allow the study team to see whether any responses assist in identifying relevant ethical themes; such as how participants view autonomous decision-making or cost considerations. Data from qualitative interviews will be critically compared with bioethics literature regarding concepts such as family communication, duties to disclose, what constitutes autonomous decision-making, approaching uncertainty in TGP, the distinction between research and clinical practice, obligations at the conclusion of a research project and determining when it is appropriate to offer testing; and to whom. The result will be a series of normative positions, defensible using ethical reasoning and informed by empirical data.Mean differences in outcomes will be compared using t-test (continuous) or chi-squared tests (dichotomous). Non-parametric tests will also be used where appropriate. Temporal changes in scales will be investigated by calculating the difference between time-points. Multiple (continuous outcomes) or logistic (dichotomous outcomes) regression will be used to adjust for the effect of confounders and identify predictors of outcome. Linear mixed models and logistic mixed models will be carried out in R: A language and environment for statistical computing using package nlme for the linear mixed model and lme4 for the logistic mixed model. Assumptions of normality of residuals and homogeneity of variance will be checked visually though diagnostic residual plots. Multivariable models will be constructed with the inclusion of all potential confounders, and those predictors that show weak evidence for an association with the outcome in univariate analysis. Backwards elimination followed by forwards addition will be used to select predicting variables in the final model. Known and identified confounders will be included regardless of their statistical significance. Collinear independent variables will be identified and removed.The PiGeOn study advances the field by identifying the knowledge, values, attitudes and coping strategies of patients with regards to TGP, determining how these, and other factors, predict subsequent cancer-related behaviour and psychosocial outcomes, and aligning these with current ethical norms. It will be the first Australian study to collect longitudinal data on cancer patients’ experience of TGP over time.The trial experience of the first 100 patients revealed the unexpected finding that a significant proportion of the cohort was eligible for both this sub-study, and the twin psychosocial sub-study, which involved participants undergoing GGS. This group will be treated as a separate cohort, as they will be facing the combined challenges of uncertainty related to prognosis/treatment related outcomes of the MoST component, as well as the germline implications for identifying increased cancer risk for blood relatives. Previous research using hypothetical scenarios unsurprisingly suggests that patients with cancer have greater concerns about GGS than TGP.  This stuMethodological issues considered in the development of this study included the timing of the psychosocial assessments, including questionnaires. Timing was crucial in order to capture participant experiences and this was modified early based on data collected in the initial phase of the programme. Given it is expected that 5–10% of the cohort will have actionable somatic mutations, sampling for the qualitative component will be manipulated between baseline and follow-up 1 to allow for exploration of the reaction to both return and non-return of actionable results. The follow-up 1 cohort will be reassessed qualitatively at the subsequent follow-ups. It is recognized that there may be challenges in maintaining the follow-up of patients not receiving actionable results, although cancer populations are known to be highly motivated to contribute to research.  It will Given the information needs and preferences are expected to vary, the qualitative and ethical components have been included to capture and critically reflect on the complexity of and nuances within patient expectations, their understanding of the implications of TGP, the likelihood of receiving an actionable somatic result and their attitudes towards sharing results with potential familial repercussions even if they did not receive such a result. Qualitative research has value in understanding these processes. Genomic sequencing (tumour and germline) is likely to become pervasive across healthcare, and to influence cancer diagnosis, treatment and prognostication. It gives rise to testing methods that are both diagnostic and predictive on a scale not previously seen in healthcare. The PiGeOn study results will inform ongoing ethical debate on issues relevant to the large-scale introduction of genomic testing into the clinical space, such as guidelines for truly obtaining informed consent for genomic testing as well as assenting to unknown future research. In addition, although somatic testing rarely identifies germline mutations, relevant to family members, it has already been reported in our study and others.  The PiGeGiven the dearth of information on participant responses to actual TGP for cancer, this study is an important step in filling the gap in knowledge about how best to introduce this form of genomic testing into clinical practice.


Science, Klaeger et al. analysed cellular targets of 243 drug candidates providing a large repository of data for drug repurposing.Protein kinases are major drug targets for oncology. The large size of the kinome, active site conservation and the influence of activation states on drug binding complicates the analysis of their cellular mode of action. In a recent article in  Also in 2017, four additional kinase drugs have been approved by the FDA (http://www.brimr.org/PKI/PKIs.htm)2.Deregulation of signalling pathways is a hallmark of cancer. It is therefore no surprise that protein kinases have emerged as one of the most successful target classes for the development of cancer treatments. Currently 37 small molecule kinase inhibitors are approved by the FDA; almost all of them for applications in oncology. Based on the large number of kinase inhibitors that are in clinical trials at present it is highly likely that we will see many more approvals soon. This prediction is supported by the large number of FDA and EMA approvals during the past 4 years that have contributed half of our clinical arsenal of kinase-targeted drugsScience, Klaeger et al.3 analysed the cellular targets of 243 clinically studied kinase drug candidates using chemical proteomics. This comprehensive study provides a large repository of data for drug repurposing, target identification, and the rational design of novel kinase inhibitors. Surprisingly, a number of non-kinase off-targets have also been identified that may lead to side effects in the clinic.The mode of action of many kinase inhibitors is, however, difficult to understand. Although most kinase inhibitors have been developed against a specific kinase target, many are promiscuous, often inhibiting multiple kinases in key signalling pathways, but how efficiently these pathways are inhibited on a cellular level is an unresolved question. Polypharmacology may be required for treatment efficacy in diverse cancer types that display a complex landscape of genetic lesions, but the lack of understanding of the mechanisms that result in treatment benefits limits further clinical development and the identification of new disease-modulating kinase targets. The characterisation of kinase drug selectivity is an important prerequisite for our understanding of the cellular mechanisms that lead to drug response. In a recent issue of 7. The data from these profiling studies have had a major impact on kinase drug discovery by highlighting off-target liabilities and unexpected activity on kinase targets that led to repurposing, in some cases, and casted doubt on validation studies that used these promiscuous inhibitors as tool compounds. However, these selectivity profiles were generated using purified isolated catalytic domains, neglecting the effect of regulatory domains in the native full-length kinase, posttranslational modifications, and the influence of the cellular environment that may modulate inhibitor efficacy by cofactor competition, the binding of protein interaction partners, and cellular location.Kinome-wide in vitro screening technologies have previously revealed the complexity of the target space inhibited by commonly used kinase tool compounds, as well as inhibitors that were studied clinically during that time8 used an array of fluorescent-based assays and highlighted the importance of the cellular environment, such as cellular location, ATP, or metabolite concentration and the use of full-length kinases, but this approach used ectopically expressed kinases and cannot currently validate selectivity on a kinome-wide scale. In contrast, the kinobeads technology used in the Klaeger3 study makes use of cellular extracts containing endogenous full-length proteins in complex with regulatory proteins and harbouring posttranslational modifications, and considers the presence of metabolites and cofactors9. This assay is run in the format of a competition binding assay, where a number of promiscuous kinase inhibitors are immobilised on a bead to capture the cellular kinases. In the inhibitor-treated lysates, the kinase inhibitor will compete to bind the kinase; binding can be quantified in pulldown assays with the help of mass spectrometry. Measuring this competition in an inhibitor-concentration-dependent way, thousands of proteins can be assayed in parallel resulting in dissociation constants (KD) for every drug interaction with cellular proteins. However, the assay may miss interactions with kinases that are absent or weakly expressed in the cell lines used, have intrinsic and inhibitor independent affinity for the beads, or that are not captured by the immobilised inhibitors. Despite these limitations, validation studies showed that kinobeads capture a comprehensive set of kinases and the use of several diverse cell lines mitigates the risk that an off-target is not expressed in a certain cell type9.Assays have now been developed to allow broad cellular profiling. First, Vasta et al.7 and bromodomains11, as well as other nucleotide-binding proteins. The non-protein kinase targets of clinical inhibitors identified by the Klaeger et al.3 study comprise metabolic kinases, such as pyridoxal kinase and other nucleotide binders, proteins that bind flavin adenine dinucleotide, and the haem-binding enzyme ferrochelatase. GPCRs and bromodomains cannot be detected using kinobeads as the immobilised inhibitors do not bind any of these targets. Using four diverse cell lines , and COLO 205) a total of 253 kinases were identified binding to kinobeads. This assay format revealed that the chosen clinical kinase inhibitor set targeted 220 kinases with high affinity (in the nanomolar KD) providing an interaction map of the currently druggable kinome with clinical inhibitors.The kinobead assay has the further advantage that some non-kinase proteins can also be identified as targets of clinical kinase inhibitors. A number of non-kinase targets have been described to be potently inhibited, including proteins that are structurally very distinct from kinases, such as G-protein-coupled receptors (GPCRs)12. The recent approval of several irreversible inhibitors targeting EGFR and Bruton’s tyrosine kinase spurred interest in these types of inhibitors; however, Klaeger et al.’s3 kinome bead assays showed that the covalent binding mode alone is not sufficient to confer high selectivity for the intended target.The selectivity of clinical inhibitors ranged from largely promiscuous compounds targeting more than 100 kinases to inhibitors with exquisite selectivity for just one target, suggesting that clinical efficacy may not always require simultaneous inhibition of several kinases. Examples of highly selective compounds were the MET receptor tyrosine kinase inhibitor capmatinib, the epidermal growth factor receptor (EGFR) inhibitor lapatinib, and the checkpoint kinase 1 inhibitor rabusertib. Irreversible inhibitors are often thought to be more selective due to bond formation with cysteine residues that are only present in a few kinases13. The study by Klaeger et al.3 showed that cabozantinib is also a potent inhibitor of the tyrosine kinase fusion product FLT3-ITD, suggesting potential application in FLT3-ITD positive acute myeloid leukaemia (AML). Indeed, cell lines bearing the FLT3-ITD rearrangement but not wild type AML cell lines were sensitive to cabozantinib treatment, which potently inhibited phosphorylation of the FLT3 downstream target STAT5 and showed efficacy in a xenograft model. The data will be available in a database and it is likely that, based on the provided selectivity profiles, more repurposing opportunities will emerge.The authors provide several examples how this large array of profiling data can be used for translational research. An obvious way is the repurposing of approved drugs. The MET/VEGFR inhibitor cabozantinib is currently approved for medullary thyroid cancer and advanced renal cell carcinoma14. The only clinical kinase inhibitor developed for MELK (OTS-167) had a very broad kinase activity making it not suitable as an inhibitor to study MELK function in squamous cell carcinoma, but kinobead profiling identified an additional 16 inhibitors, including the approved drug nintedanib that may be more suitable for mechanistic studies, or that may serve as a starting point for the development of selective MELK inhibitors using a pharmacological validated inhibitor scaffold.Apart from drug repurposing, the data from this assay can also be used for the identification of novel kinase targets. For example, expression profiling using kinobeads and 15 non-small cell lung cancer (NSCLC) tumours identified the known cancer targets EGFR and MAP2K1 (MEK1), as well as the kinases DDR1 (discoidin domain receptor tyrosine kinase 1) and MELK . Tissue arrays of a cohort of 375 NSCLC, squamous cell carcinoma, and adenocarcinoma patients showed no significant correlation with EGFR and DDR1 expression in a retrospective survival analysis, but these data revealed a moderate overall correlation with MELK expression levels. Separate analysis suggested poor survival of patients with high MELK levels in squamous cell carcinoma but not adenocarcinoma patients, in support of earlier studies3 provides an exciting data source describing the target landscape of current kinase inhibitors. This large repository of selectivity data will be highly informative for basic research scientists, as well as drug discovery and clinical repurposing of approved kinase drugs. Our repository of kinase drugs and drug candidates is growing rapidly, and it is therefore highly likely that comprehensive profiling data as presented in this study will be the one of the main sources for future target identification and the development of new therapeutic concepts.The study by Klaeger et al.


Treating preschool age children (PSAC) with schistosomiasis remains a challenge. Without a pediatric praziquantel (PZQ) formulation, the inclusion of this age group in control programs is limited, and general access to treatment in routine care settings is severely bottlenecked. There are, however, current platforms that target PSAC in primary health care such as the integrated management of childhood illnesses (IMCI), which could integrate PZQ in their portfolio and deliver a pediatric PZQ formulation when available. In addition, other age groups such as school-aged children (SAC) could also benefit from the IMCI’s successful strategy and be treated in health centers using a similar approach. This Viewpoint article reports a summary of a symposium held at the American Society of Tropical Medicine and Hygiene national meeting in 2014 that brought together six experts in different areas in the field of pediatric schistosomiasis to form a working group that could provide recommendations for the inclusion of PSAC in the IMCI and other existing preschool outreach programs. This was to develop and adapt methodologies to fill existing gaps left by current mass drug administration (MDA) programs and synergize efforts for schistosomiasis control more broadly. Foremost, this includes a better definition of subclinical disease in young children to integrate into ICMI guidelines and further demonstration of the benefit of expanded access of treatment to children of all ages by encouraging universal access.Over 123 million children suffer from schistosomiasis worldwide, but only SAC are presently targeted for MDA with PZQ treatment in WHO-led preventive chemotherapy (PC) efforts . NeverthThe IMCI is one of WHO/UNICEF’s most successful strategies in reducing mortality in children under five years of age . Since iIn most endemic areas, individual case management for clinical schistosomiasis is not happening, because the disease is poorly recognized in younger children . This isNutritional status is considered a key “vital sign” in the assessment of the pediatric patient, integrating both long- and short-term insults to health. Schistosomiasis is well known to cause deficits in linear growth, leading to chronic undernutrition (stunting) as well as acute undernutrition (wasting) . As pracProvision of PZQ as part of IMCI would also provide an opportunity to treat well-documented, schistosomiasis-associated anemia . ImproveFemales with schistosomiasis carry an additional burden of reproductive tract disease that has been neglected in clinical management since its discovery more than 120 years ago. Female Genital Schistosomiasis (FGS) causes bleeding and three distinct lesions: grainy, sandy patches, rubbery papules, and homogenous yellow patches. Studies in Tanzania and Zimbabwe found that women with FGS have a 3- to 4-fold increased risk of having HIV; they also seem to have increased secondary infertility, contact bleeding, pain, and stress incontinence . Girls aThe perfect timing of treatment for the prevention of lesions (and HIV) should therefore also be explored, but it is within reason to assume that preschool treatment could have a tangible impact on preventing genital lesions .Schistosoma-endemic regions may be beneficial for preventing downstream educational loss and cognitive impairment.The evidence for educational or cognitive benefit of deworming of any kind is currently debated ,7. The eEpidemiological studies with global positioning system data loggers have revealed that PSAC can have surprisingly high levels of daily and cumulative water contact within the first three years of life . With moSchistosoma species [The safety of PZQ in PSAC has been widely established across  species ,19,20, b species . However species . Their t species .We have identified important treatment gaps in the ongoing MDA strategy for schistosomiasis that leave infected children untreated. A synergistic approach to schistosomiasis control could integrate PZQ delivery through ongoing primary health care strategies (IMCI) both for PSAC as well as SAC, with ongoing MDA efforts that currently exclude PSAC. When the PZQ pediatric formulation becomes available, a solid clinical care pathway with an optimized dosing strategy needs to be in place to allow an efficient delivery into existing programs.Identification of schistosomiasis individual cases using a detailed clinical classification that could be taught to health care workers so they could identify children with suspected disease, and in areas where prevalence is known to be high, provision of treatment could be the first standard of care  Integration of PZQ within the IMCI when the PZQ pediatric formulation becomes available. PC to PSAC can be synergized with other preventive measures  when a pediatric formulation is available. Meanwhile, while waiting for a pediatric formulation, the use of crushed tablets should be continued on a case-by-case basis, which is common practice when very young children need other essential drugs [We propose a step-wise approach to identify and treat children of any age with schistosomiasis through (1)  of care . This isproaches . (2) Accal drugs  and whical drugs . It is t