Regions surrounding vessel cavities, in addition to tissue surfaces, had significantly lower peak downstream electrotonic currents than well coupled myocardium ( Recent advances in clinical electrophysiological mapping procedures, experimental optical mapping as well as computational modelling, have provided the ability to record and understand the nature of the electrical reentrant circuits which drive many types of cardiac arrhythmias. Such knowledge has highlighted the importance of structural anatomy in providing an essential substrate which stabilises reentrant circuits, particularly relevant in incessant ventricular tachycardia Conduction block within slow conducting pathways or isthmuses of surviving tissue through ventricular scar has been shown to trigger reentry Electrotonic loading effects are known to influence many different aspects of cardiac electrophysiology Structural anatomical heterogeneity within the myocardium, due to the presence of intramural blood vessels, endocardial structures such as trabeculae and papillary muscles, and extracellular cleft spaces, introduce the presence of boundaries and complex changes in cardiac fibre architecture that significantly affect local electrotonic loading. How the consequential effects of alterations in electrotonic loading in the vicinity of such fine-scale structural heterogeneities combine together to affect local refractoriness, excitability and the ability to successfully capture a focal triggered stimulus, is currently not well-understood. Furthermore, should a focal ectopic beat actually elicit propagation, it is also not clear the conditions under which the induced wavefront will become reentrant, requiring some form of conduction block or wavebreak to also occur, which will also be strongly dependent upon local structural anatomy and propagation pathways.In this study, we aim to develop an in-depth, quantitative understanding of the biophysical processes by which fine-scale structural heterogeneities affect local electrotonic loading and the resulting direct effects on functional electrophysiological tissue properties and consequent arrhythmogenic implications. By using a combination of idealised and anatomically-detailed computational ventricular wedge models, we directly quantify fundamental relationships between electrotonic loading, refractoriness and successful capture of focal ectopics, and the dependence upon immediate structural heterogeneity, focussing on large sub-epicardial blood vessels, and also trabeculations. Our findings elucidate the important role played by physiological anatomical heterogeneity in arrhythmia initiation by ectopic mechanisms, which may have important implications in the setting of more significant pathological structural heterogeneity.Two separate unstructured, tetrahedral finite element meshes were used, each representing left-ventricular (LV) wedge preparations, but differing in their level of detail and complexity. The first model constituted a simplified, idealised representation of an LV wedge of cuboid dimensions Transversely-rotational fibre orientation was assigned to the models, rotating ±60° between epi- and endocardial surfaces. A previously described algorithm based-on a Laplace-Dirichlet approach l) and cross-fibre (t) directions within the intracellular  All protocols described below were applied to the idealised wedge model, with selected protocols also applied to the anatomically-detailed model.Two separate wavefront propagation directions were considered: circumferential  and transmural . A basic cycle length (BCL) of 400 ms was used for 10 paced beats to achieve steady-state. Electrophysiological metrics were then quantified on the 11th beat. APDs were defined as the time-point between depolarisation (positive crossing of 0 mV threshold) and 90% repolarisation and were calculated at all node points within the models. Maximum AP upstroke velocity was also calculated for all nodes as the maximum positive rate of rise during depolarisation with an output time discretisation of 0.1 ms.µA/cm2. The calculated electrotonic current is equivalent to the total transmembrane current, Total electrotonic current at each node was calculated directly from the diffusion term of the bidomain equation Finally, the effects of restitution on the above electrophysiological metrics was performed by progressively decreasing BCL 400 ms to 170 ms, until loss of capture, with 10 paced beats performed at each BCL. Progressively decreasing increments were used: 50 ms , we realise that sites with a high electrotonic loading ‘share’ a large proportion of the current used to stimulate them with surrounding tissue. Thus, during focal stimulus, maximum changes in 2, duration 2 ms); a strength of less than this would not compute an ERP as propagation would never be inducible within parts of the domain. The initial state of the tissue for ERP calculation in both models was as following a transmural propagation stimulus at a BCL of 170 ms. In all cases, ERP was defined relative to the prior activation time at each individual focal region following activation by the preceding S1 transmural wavefront propagation.Effective Refractory Period (ERP) is defined as the minimum time  after being fully activated (eliciting an AP) that a region of tissue may once again capture a focal stimulus and initiate another propagating wave of activity. Successive stimuli were applied to small regions of tissue within the models  was performed using a previously described approach Pacing protocols were applied to both the idealised and anatomically-detailed models, as described in Section. 2 (67% reduction) for circumferential propagation and 2 vs 2 (2) than the well-coupled node during circumferential propagation, but a lower value (2) during transmural propagation. The peak positive electrotnic current is almost 3-fold larger at the proximal node relative to the distal node for circumferential propagation (2 vs 2), whereas this difference is only 45% for transmural propagation (2 vs 2). Comparing with the well-coupled node, the peak positive electrotonic current is 45% larger (2) and 26% (2) for circumferential and transmural propagation directions, respectively.A similar pacing protocol was conducted on the anatomically-detailed wedge model. Restitution of the changes in electrophysiological properties noted above were then examined in the simple model. The stimulation protocol described in Section was applied to both idealised and anatomically-detailed wedge models to quantify focal electrotonic loading, focussing on small regions in the neighbourhood of high structural heterogeneity in each case. Finally, ERP was computed using the algorithm described in Section at a number of different spatial locations surrounding the blood vessel cavity in the idealised model, the results of which are plotted in The spatial distribution of ERPs close to the endocardium shows a slightly lower range of values compared to that seen at the epicardium, as there are no large vessels to create narrow channels of tissue. As expected, ERP is low close to the surface boundaries, both the endocardium itself and also the surface of the trabecula However, ERP is seen to increase as these surfaces join together at the narrow strand of tissue which joins the trabecula to the endocardium. Not only is ERP then significantly higher within the mid-myocardium, but it is also seen to increase to similar levels within the bulk of the trabecula itself, which represents a relatively thick structure approximately>1 mm in diameter. Consequently, this results in significant heterogeneity in ERP within this relatively small spatial region.Simulations were also conducted in which tissue was stimulated at the same timing as the above case in which reentry was initiated (163 ms), but in locations where ERP was relatively uniformly high  and low . The former case resulted in the stimulus failing to capture at all, as all tissue near the stimulus site was not yet recovered, whilst the latter case lead to the stimulus capturing relatively uniformly and eliciting wavefront propagation that did not initiate reentry.Although much knowledge exists regarding how anatomical substrates stabilise and support reentrant activity, little is currently known about the role structural anatomy plays in the genesis of the initial trigger that establishes the first reentrant beat. Particularly, it is not well understood how the small isolated regions of tissue which provide the trigger may successfully over-come intrinsic current source-sink mismatches to elicit wavefront propagation into the surrounding tissue, and subsequently setup a reentrant circuit. In this study, we have used anatomically-detailed computational models to demonstrate how physiological structural heterogeneity significantly reduces local tissue electrotonic loading, directly affecting tissue excitability, providing an important substrate to facilitate the capture of focal activity and initiation of reentry.The detailed modelling performed in this study has allowed us to quantify and understand the biophysical processes by which electrotonic loading is reduced by the presence of fine-scale structural features - such as intramural blood vessels and endocardial trabeculations - due to an increase in intracellular boundaries and a corresponding localized change in the ratio between boundary and myocardial volume. During wavefront propagation, the presence of structural heterogeneity was seen to have little affect on APD, with the expected dispersion of APD along the pacing direction dominating Differences were seen in the absolute magnitude  and the focal electrotonic loading, a quantity which depends upon tissue conductivities, fibre orientation and presence of boundaries within a localised three-dimensional volume surrounding the focal source. Due to the highly complex nature of the models used here, we sought to develop and apply an elegant measure of focal loading as the change in Simulation of paced wavefronts allow active quantities such as upstroke durations and electrotonic currents to be explicitly quantified and their spatial distribution with respect to structural heterogeneity examined. In this context, Of key relevance to the interpretation of the above findings lies in linking this reduction in electrotonic loading, driven by structural heterogeneity, to its functional consequences in terms of capture of focal stimuli. A highly-useful clinical measure of the susceptibility of a region for capture of a focal stimulus is in the determination of the ERP for point stimulation. In this study, for the first time, we computed the spatial variation of ERPs around key areas of high structural heterogeneity within highly anatomically-detailed models, made possible through the development of a computationally-efficient ERP protocol for use at the tissue level. Despite seeing very little variation in APD and repolarisation times in the vicinity of fine-scale structural features during wavefront propagation, significant heterogeneity was witnessed in ERP. The large reduction in ERP around structural features and tissue boundaries is, we believe, primarily explained by the changes in the excitability of the tissue in these regions, introduced by the close proximity of intracellular discontinuities. The corresponding reduction in electrotonic loading in these areas means that, upon stimulation with a given strength of current, less of the stimulating current diffuses away to neighbouring tissue, meaning that more is able to be used by the stimulated region itself to help bring it to threshold and depolarise. The tissue in these regions is therefore more excitable, with a corresponding lower ERP.The ERP protocol used in this study used a transmural stimulus as its S1 pre-conditioning pulse. As noted above, although there was no noticeable heterogeneity in APD surrounding the vessel cavity , there was a minor gradient in APD (changing by just a few ms) along the direction of wavefront propagation . In the Due to the high anatomical complexity of the network of coronary vessels and endocardial structures represented within the models, very high gradients in ERP occurred within close proximity to one-another. Specifically, differences of approximately 40 ms in ERP (approximately 25% of APD) were seen within spatial regions of approximately 1 mm, as witnessed in It should be noted here that ERP also depends upon the specific nature of the protocol used in its calculation. For example, stimulating with a stronger stimulus or over a larger tissue volume would have caused a reduction in ERP values. Here, we carefully selected the combination of smallest feasible tissue volume and lowest  stimulus strength to just elicit propagation in the regions of well-coupled myocardium, thus most closely resembling a physiological scenario of stimulus due to a mechanism such as a spontaneous earlyafter depolarisation. Finally, it should also be noted that the previously mentioned effects of complex fibre orientation also appears to have an impact on ERP. This is thought to be responsible for the slight heterogeneous variation in ERP in We have identified that the stark gradients in ERP witnessed around fine-scale anatomical features within the myocardium provide the necessary substrate for arrhythmogenesis upon delivery of a focal stimulus. A correctly-timed stimulus within a high gradient of ERP is expected to cause unidirectional conduction block; tissue will capture and initiate propagation into the direction of low ERP, whilst propagation will not be possible into the direction of higher ERP as the tissue has not fully recovered and cannot be excited. However, unidirectional block does not necessarily directly cause reentry; it is the fact that these regions of high ERP gradient are located in close proximity to structural heterogeneity , that provide this additional key mechanism. As shown in Following myocardial infarction, significant structural remodelling may occur leaving highly heterogeneous regions of tissue. As well as consisting of regions of dense fibrosis, infarct scars often contain surviving myocyte bundles which form channels of healthy, excitable tissue through the scar, along with border-zone regions representing the area between viable healthy tissue and non-viable scar that contain an admixture of surviving myocytes and fibrotic scar tissue Recent studies The initial focal triggered stimulus, represented here by a ‘forced’ stimulation, may result from a number of physiological processes such as early- or delayed-afterdepolarisations or abnormal automaticity Although a range of electrophysiological metrics were analysed during this work, localised spatial variations in conduction velocity and its restitution were not investigated. ERP is associated with excitability at high pacing rates, and therefore plays an important role in both arrhythmogenesis and the stability of reentrant waves. Thus, restitution effects of conduction velocity and particularly localised differences with respect to structural heterogeneities may prove to provide an important mechanism of arrhythmogenesis and stability due to non-uniform conduction during rapid pacing. In this current study, we have focussed our attention more on arrhythmia initiation due to focal stimulus, and thus we leave the effects of large-scale heterogeneities upon localised conduction velocity in arrhythmias as an interesting avenue of future investigation.The results presented in this study use a recent rabbit ventricular cell model. However, the extent of electrotonic APD modulation is known to depend upon the membrane resistivity during repolarisation, with different degrees of APD modulation being noted for different species with inherent different membrane electrophysiological properties The presence of physiological structural heterogeneity, in the form of intramural vessel cavities and complex endocardial trabeculations, significantly reduces local electrotonic loading during wavefront propagation and focal stimuli. This reduced loading directly affects tissue excitability, increasing upstroke velocity and significantly reducing ERP around fine-scale structures, despite no corresponding significant spatial variation in APD during normal wavefront propagation in the very same region. ERP gradients that exist in the vicinity of such structures provide a mechanism of arrhythmogenesis upon focal stimuli by establishing unidirectional conduction block and combining with the structures themselves to initiate reentry. These fundamental findings may have increased significance under conditions of fibrosis in the presence of extensive tissue and electrophysiological remodelling.


Bistable stimuli are one of the most popular approaches to studying the neural mechanism of conscious visual perception. Such stimuli contain conflicting information, which the visual system cannot integrate into a unified percept. This causes the perceptual state of the observer to change every few seconds between the two interpretations while the physical stimulus remains the same. Binocular rivalry is an example of such perceptual phenomena with ambiguity achieved by presenting one image to one eye and a different image to the other eye. Perceptual changes during binocular rivalry are particularly vivid, and closely resemble a physical image exchange.The study of neural mechanisms of bistable perception and binocular rivalry revealed the involvement of multiple areas across different levels of the visual hierarchy . Under certain conditions, however, perceptual states can be inferred objectively, without self-report. An example recent study by Frässle et al.  took advThe authors used fMRI to monitor brain activity when observers experienced either genuine binocular rivalry or a “replay” . These conditions were performed while subjects either actively reported their perceptual state or just passively viewed the stimuli.per se.Such experimental design allowed Frässle et al. to make an important observation. Comparison of rivalry with replay in the passive viewing condition showed less activation in the frontal cortex compared to active report condition. The most prominent decrease was observed in the left superior and bilateral middle frontal gyri. According to the authors, decrease of frontal activity implies that frontal lobe areas play a role in introspection and motor response generation, but not in perceptual changes Frontal lobe takes about one third of the cortical volume, and is comprised of multiple architectonically and functionally distinct areas Stuss, . Given tPrevious studies comparing perceptual changes in rivalry with replay frequently reported activation in the inferior frontal cortex (IFC), middle frontal gyrus (MFG) and superior frontal gyrus (SFG) (Lumer et al., Besides, passive viewing involves not only less self-monitoring, but also less attention. Attention, in turn, is linked to increased activity in frontal, but also parietal and extrastriate areas. The reduction of activity at multiple non-frontal brain locations (Table 2 in Frässle et al., Besides singling out the effects of self-monitoring from those of attention during passive viewing, it is important to dissociate the effects of self-monitoring and those of motor response. The ability to report one's conscious experience is not a prerequisite for conscious perception, but this may not hold for self-monitoring and introspection. Can we be sure that we are aware of something without reflecting about it? In fact, several approaches to consciousness define self-awareness as its essential component (Van Gulick, Almost two decades ago Crick and Koch  proposedThe 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.


However, many studies lack the use of endothelial cells in their approaches leaving out an important factor to enhance vessel ingrowth and anastomosis formation upon implantation. In addition, the adequate endothelial cell type needs to be considered to make these approaches bridge the gap to in vivo applications.The use of cell-laden hydrogels to engineer soft tissue has been emerging within the past years. Despite, several newly developed and sophisticated techniques to encapsulate different cell types the importance of vascularization of the engineered constructs is often underestimated. As a result, cell death within a construct leads to impaired function and inclusion of the implant. Here, we discuss the fabrication of hollow channels within hydrogels as a promising strategy to facilitate vascularization. Furthermore, we present an overview on the feasible use of removable spacers, 3D laser-, and planar processing strategies to create channels within hydrogels. The implementation of these structures promotes control over cell distribution and increases oxygen transport and nutrient supply  The blood and lymphatic vascular systems provide most tissues in our body with a supply of nutrients and oxygen and ensure proper waste removal. When cells grow beyond the diffusion range of oxygen, hypoxia initiates a sequence of events to release signals to attract new blood vessels. The same events are triggered when a cell-laden construct is implanted into a host. However, blood vessels require several weeks to invade a millimeter-sized implant for complete vascularization. As a result improper nutrient supply following cell death in the core of such an implant can cause problems in engineered tissue integration  has been encaged by rapid processing of the surrounding hydrogel (Torgersen et al., A comparison of techniques presented is summarized in Table For every approach that aims at promoting vascularization, it is inevitable to consider endothelial cell biology. While a detailed discussion of the adequate cell source would go beyond the scope of this review, a few key points are highlighted in this section. The endothelium is a heterogenous organ where cells have major phenotypic differences dependent on the organ, location, vessel, and vessel size Aird, . Umbilicin vitro constructs. Therefore, successful hydrogel integration and cell survival can be achieved using accurate and feasible engineering techniques with equal consideration of vascular biology.In most soft tissue engineering applications, implantation of inadequately vascularized hydrogels will result in hypoxia or even anoxia and cell death. To address this problem, fast ingrowth of host blood vessels into a scaffold needs to be considered in the design of tissue-engineered constructs. Endothelialized channels and interconnected structures aim to resemble native microvasculature thus promoting survival and function of cells embedded in an implant through formation of anastomoses with host tissue. A variety of reproducible and accurate techniques exists to generate channeled hydrogels. Despite all efforts to develop feasible methods to alter microstructures, only a few studies incorporate endothelial cells in their hydrogels. Unfortunately, the majority of publications show a proof-of-principle rather than demonstrating its applicability for vascularization using an adequate cell source. Microvascular endothelial cells would be ideal to be embedded in engineered microvessels while macrovascular endothelial cells such as HUVEC have their limitations but are suitable for preliminary tests. Additionally, EPCs from peripheral or umbilical vein blood offer a clinically relevant cell source as they can be harvested minimally invasively and used autologously. Despite all these intriguing advantages, this cell type is currently strongly neglected in biofabrication. Engineering methods are improving rapidly and more emphasis needs to be put on the biology of microvasculature when designing 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.http://www.frontiersin.org/Journal/10.3389/fbioe.2014.00052/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Louise Kenny and colleagues conduct a population-based cohort study in Denmark to assess the likelihood of stillbirth, miscarriage, and ectopic pregnancy following cesarean section compared to women who gave birth by vaginal delivery. There is an error in


Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy however the gene is not frequently mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic.Cells were engineered to overexpress an enzymatically active variant of GLS1 that does not bind the previously described GLS1 inhibitor, BPTES ethyl sulfide), to validate the on-target effects of the drug and utility as a specific tool compound. A panel of NSCLC cells were screened with BPTES and the effects on proliferation were correlated with transcriptional/genetic markers.Low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, marked NSCLC cells that are particularly sensitive to inhibition of GLS1. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquired sensitivity to the GLS inhibitor. Metabolic studies suggested that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition.These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways to target in this aggressive cancer type.


The exploration and identification of new anti-cancer therapeutic indications for an approved drug already in use, or for a drug that was shelved for non-safety reasons in early clinical development, represents an attractive alternative to the costly, time-consuming and inefficient process of developing novel anticancer agents. Drug repositioning or repurposing can bypass the discovery and early clinical phases of cancer drug development, potentially reducing the hundreds of millions of dollars and more than a decade typically required to see a novel agent through to regulatory approval. Incidental clinical observations, case–control analysis of large populations and modern high-throughput drug screening techniques have been employed in identifying agents with hitherto undiscovered or ignored biological activity that could underpin effective anticancer activity.To date, the most significant example of drug repositioning in oncology has seen thalidomide and its analog lenalidomide approved for the treatment of multiple myeloma. NotoriouSarcomas are rare tumors of mesenchymal origin, encompassing over 70 histological subtypes of distinct clinical behavior and underlying biology. Metastatic disease is common and associated with poor prognosis. Diverse molecular pathology and, in many cases, lack of effective systemic therapies indicate a compelling case for drug repurposing studies in sarcoma. The characterization of recurrent mutations of cKIT in gastrointestinal stromal tumors (GIST) and TSC1/2 in malignant peripheral epithelioid cell tumors (PEComa) formed the basis for the respective repurposing of imatinib and sirolimus. Imatinib, a small molecule inhibitor originally developed to target BCR-ABL in chronic myeloid leukemia but whose kinase inhibition extends to cKIT, has dramatically improved survival outcomes in both early-stage and advanced GIST, whilst uEBioMedicine, they now describe their retrospective evaluation of a combination of propranolol and vinblastine-based chemotherapy in a small series of patients with metastatic angiosarcoma.(in vitro and in silico drug screening that exploits a burgeoning understanding of sarcoma molecular pathology can be employed to hopefully identify drugs that are candidates for repositioning studies.Propranolol has been successfully repurposed for the treatment of infantile hemangioma, based on a number of initial clinical observations and a subsequent randomized trial. Furthermosarcoma. These daThe study raises a number of relevant questions regarding research and drug development in rare cancers. Obtaining access to drugs such as propranolol for a small number of patients is relatively easy, but performing a randomized trial in a very rare and heterogeneous entity such as angiosarcoma is a considerable financial and logistical challenge. Large international multicenter studies are required to achieve sufficient recruitment, a considerable undertaking that pharmaceutical companies are unlikely to consider due to the lack of return from generically available products. There is, however, a strong track record of investigator-initiated trials within the international sarcoma community. The funding of such trials by funding agencies or charities may be limited, as such projects may be deemed less exciting or innovative compared with other studies evaluating a novel agent. In addition, drug repositioning is not risk free, and involves the use of a drug in a new patient population with potentially differing pathophysiological characteristics and also new drug formulations and dosing schedules.In conclusion, the study by Pasquier and colleagues is of interest and should be explored further in a prospective trial. Drug repositioning could potentially be of benefit in other sarcoma subtypes particularly those with currently no effective systemic therapy. Such an approach clearly requires collaboration between basic, translational and clinical researchers.The authors declared no conflicts of interest.


Innovative research on the interactions between biomechanical load and cardiovascular (CV) morphogenesis by multiple investigators over the past 3 decades, including the application of bioengineering approaches, has shown that the embryonic heart adapts both structure and function in order to maintain cardiac output to the rapidly growing embryo. Acute adaptive hemodynamic mechanisms in the embryo include the redistribution of blood flow within the heart, dynamic adjustments in heart rate and developed pressure, and beat to beat variations in blood flow and vascular resistance. These biomechanically relevant events occur coincident with adaptive changes in gene expression and trigger adaptive mechanisms that include alterations in myocardial cell growth and death, regional and global changes in myocardial architecture, and alterations in central vascular morphogenesis and remodeling. These adaptive mechanisms allow the embryo to survive these biomechanical stresses  and to compensate for developmental errors (genetic). Recent work from numerous laboratories shows that a subset of these adaptive mechanisms is present in every developing multicellular organism with a “heart” equivalent structure. This chapter will provide the reader with an overview of some of the approaches used to quantify embryonic CV functional maturation and performance, provide several illustrations of experimental interventions that explore the role of biomechanics in the regulation of CV morphogenesis including the role of computational modeling, and identify several critical areas for future investigation as available experimental models and methods expand. The heart and vasculature is the first organ system to form and the only one which is required to function successfully throughout embryonic and fetal life for survival  due to pulsatile blood flow. Over a century ago, based on observations of chick embryos, Thoma proposed that these forces influence vascular morphology Thoma, . Since tThe relationship between CV development, morphogenesis, and biomechanical events is an area of active investigation  live imaging, enhanced molecular techniques, and novel experimental interventions enable complementary, quantitative studies to link phenotypic growth, morphogenesis, and remodeling events with hemodynamic forces and fundamental biologic mechanisms. This review presents an overview of approaches to study hemodynamics and CV morphogenesis in the embryo and how they have contributed to our current understanding of developmental CV biomechanics. The development of computational models to investigate higher-order flow characteristics and mechanical loading is also addressed. Finally, the outlook for future research is discussed.in situ hybridization to create 3D reconstructions of the embryonic chick heart with segmented tissue structures  offers the greatest theoretical resolution and has been widely used to obtain 3D geometries of fixed tissues through contrast agents or polymeric casting of vascular structures  has emerged as a preferred method for live imaging of the chick embryo  has been used to measure blood flow velocities in chick and zebrafish embryos. μPIV uses cross-correlation between two consecutive images to calculate local displacements. In chick embryos, erythrocytes or injected fluorescent microspheres have been used as the seeding particles  multiplied by blood viscosity. For straight vessels, it is simple to compute the shear rate. However, for curved vessels, the vector normal to the wall must be obtained. μPIV methods performed in 2D can compute the normal vector from the microscopy image , continuum solid mechanics, and lumped parameter models. Experimental data is required to train and test the results of computational models and is often needed for initial inputs. Insights gained from modeling can, in turn, inspire new experiments. Thus, experiments and models complement each other and are both needed to study embryonic CV development.CFD is widely used to analyze hemodynamics at a higher level than Doppler velocimetry or μPIV can provide. Doppler techniques are not suited for complex flow fields and μPIV requires vessels that are accessible and not obscured. CFD models overcome these limitations by providing 3D data. Model geometries are often obtained from imaging techniques described in Section Introduction. Pressure and velocity measurements are used to set realistic boundary conditions. Depending on the model objectives, blood flow can be at he mean steady-state or pulsatile. CFD models of flow through the AV canal of chick embryos at different stages of development demonstrated that WSS increases monotonically and is higher on the left side of the heart Table . A CFD mComputational models that recapitulate or predict embryonic CV growth are emerging and have been developed with some success. A common approach is to use a stress-based growth law that assumes a hypothetical homeostatic stress state. A mechanistic growth field is generated in order to reach this equilibrium state. This approach was used to develop a model of growth and remodeling of the heart from its first beat to maturity , endothelin-1 (ET-1) and endothelial nitric oxide synthase (NOS-3) within the embryonic heart 1 h after right lateral VVL , is a rare but serious congenital heart defect, marked by an underdeveloped and nonfunctioning left ventricle and hypoplastic ascending and transverse aorta in association with stenosis or atresia of the mitral and/or aortic valves, and intra-uterine compensatory enlargement of right sided cardiac structures  is a method for increasing peripheral vascular resistance in chick embryos. VAL performed at HH21 resulted in reduced stroke volume and cardiac output, increased total vascular resistance, larger first harmonic and characteristic impedances, decreased arterial compliance, and transient changes in pulse pressure, hydraulic power, and oscillatory power, which all normalized by HH27  and gata2 (72% less blood cells) , which lacks a heartbeat, and cardiofunk (cfk−/−), which exhibits cardiac dilation offer novel methods to investigate embryonic development under low- to no-flow conditions  reduces ventricular contractility in the immature myocardium  HH29 chick embryos .Defining the role of biomechanics in CV development creates opportunities for new treatment strategies for CHD. The current understanding of flow and growth relationships has been translated as a rationale for cardiac intervention in the mid-gestation human fetus and intervention in humans has shown that restoration toward normal biomechanical loading partially restores the growth and remodeling of left heart structures , though recovery of fetal myocardial growth after fetal intervention remains suboptimal (Pekkan et al., 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.


Drugs that are inexpensive to manufacture and simple to administer greatly expand the potential to help tens of millions of people who need treatment for chronic hepatitis B virus (HBV) infection. Key program implementation challenges include identifying who would benefit from antiviral medication and ensuring long-term and consistent treatment to people who feel well. The best opportunities are where health systems are advanced enough to effectively address these challenges and in settings where HIV service platforms can be leveraged. Research, innovation, and collaboration are critical to implement services most efficiently and to realize economies of scale to drive down costs of health care services, drugs, and diagnostics. Drugs that are inexpensive to manufacture and simple to administer greatly expand the potential to help tens of millions of people who need treatment for chronic hepatitis B virus (HBV) infection. Key program implementation challenges include identifying who would benefit from antiviral medication and ensuring long-term and consistent treatment to people who feel well. The best opportunities are where health systems are advanced enough to effectively address these challenges and in settings where HIV service platforms can be leveraged. Research, innovation, and collaboration are critical to implement services most efficiently and to realize economies of scale to drive down costs of health care services, drugs, and diagnostics. Viral hepatitis, principally due to chronic hepatitis B virus (HBV) and chronic hepatitis C virus (HCV), claimed 1.4 million lives worldwide in 2013,The toll of viral hepatitis is now greater than that of mortality from HIV.Although HBV vaccination rates for the childhood routine hepatitis B vaccine series were 82% globally in 2014,Although chronic hepatitis B (CHB) infection is usually not curable, thankfully certain antiviral drugs are highly effective at suppressing viral replication and preventing the progress of liver damage without the development of resistance. A momentum is now building to expand access to these medications, in keeping with a new commitment within the Sustainable Development Goals to “combat hepatitis.”WHO-issued targets aim to reach 80% of people with chronic hepatitis B in need of treatment by 2030.Considering that CHB treatment is lifelong in many cases, it might at first glance seem unaffordable to seek to expand treatment access to so many. But here is the convenient truth: CHB medications are simple to administer and potentially very inexpensive.Unlike treatment for HIV or tuberculosis, in the substantial majority of CHB cases a single antiviral agent with a high barrier to resistance can effectively suppress HBV. WHO recommendations provide 2 options for preferred first-line agents, tenofovir disoproxil fumarate (TDF) or entecavir (ETV),In most cases of chronic hepatitis B infection, a single antiviral agent with a high barrier to resistance can effectively suppress the virus.TDF is already a backbone of HIV treatment at the same dosage approved for HBV treatment (300 mg/day) and had a ceiling price in 2015 of US$48 per patient year as a generic for HIVWhile it is potentially very inexpensive to manufacture generic ETV, current prices of the drug are unfortunately very high, with a lowest global price of US$427 for a generic version not approved by a stringent regulatory authority such as the U.S. Food and Drug Administration, and US$6,127 for a generic version sold in the United States.before they develop cirrhosis,WHO’s guidance appropriately recommends that treatment be prioritized first for patients who have cirrhosis. To prevent HBV-associated liver cancer and cirrhosis, however, it will be crucial to treat people Despite the low costs and clear benefits of antivirals, drugs will not be a magic bullet in and of themselves. To begin with, the proportion of people who have been tested for HBV is very low in most countries; WHO estimates that globally less than 5% of people living with chronic HBV and/or HCV are aware of their status.The proportion of people tested for HBV is very low in most countries, but rapid point-of-care tests could help.Beyond testing, other substantial implementation challenges include linkage to longitudinal care services and differentiating between patients qualifying for treatment now versus those who only need regular monitoring. In addition to providing drugs for long-term daily intake, programs will also need to convince people who feel healthy to adhere to long-term medication. While this is a challenge common to many chronic diseases, it is very salient for HBV, since only a minority of people living with chronic infection will die from its consequences, which often occur several decades after diagnosis. Engaging and retaining men into long-term treatment is also critical for CHB treatment programs, particularly because men have a more than threefold higher risk for HBV-associated liver cancer than women .Scaling up any new service entails significant organizational effort and cost. In light of these various hurdles, the best opportunities to expand CHB treatment rapidly may occur where health systems are relatively advanced, for example, in middle-income countries in Asia that have a high HBV burden. However, significant programmatic synergies with HIV platforms also exist, which could help enable service delivery even in low-income countries with less robust health systems, including many in sub-Saharan Africa that have extensive HIV treatment programs. Supply chain, laboratory testing, and longitudinal care systems for HIV treatment have great commonality with HBV treatment. Even for more complex elements of HBV care such as viral load testing, promising opportunities exist to leverage existing HIV infrastructure, as easy-to-transport dried-blood spots that are used for HIV DNA-PCR testing can also be used to quantify HBV DNASignificant programmatic synergies with HIV platforms exist, which could help enable hepatitis B treatment programs in countries with less advanced health systems.in vivo animal studies TDF produces about 50% of the levels of the active metabolites in liver cells compared with TAF on a per mg basis.Most countries with high burdens of HBV have growing economies that can mobilize their own domestic resources to support the increased costs of expanding CHB treatment, which ultimately may save costs by reducing long-term medical expenses for liver cancer and cirrhosis. But to enable optimal access to generic ETV and the variety of HBV-related laboratory tests that will be needed, countries will benefit from actively working together to achieve greater economies of scale, using approaches such as coordinated ordering and prequalification of products to address regulatory bottlenecks. New technologies will also be important to drive down costs and improve outcomes. One such example is tenofovir alafenamide (TAF), a second-generation prodrug of tenofovir recently reported to be non-inferior to TDF in phase III trials of HBV treatment.To enable optimal access to low-cost HBV drugs and laboratory tests, countries will benefit from working together to achieve economies of scale.


Unapposed hemichannels (HCs) formed by hexamers of gap junction proteins are now known to be involved in various cellular processes under both physiological and pathological conditions. On the other hand, less is known regarding how differences in the molecular composition of HCs impact electrical synaptic transmission between neurons when they form intercellular heterotypic gap junctions (GJs). Here we review data indicating that molecular differences between apposed HCs at electrical synapses are generally associated with rectification of electrical transmission. Furthermore, this association has been observed at both innexin and connexin (Cx) based electrical synapses. We discuss the possible molecular mechanisms underlying electrical rectification, as well as the potential contribution of intracellular soluble factors to this phenomenon. We conclude that asymmetries in molecular composition and sensitivity to cellular factors of each contributing hemichannel can profoundly influence the transmission of electrical signals, endowing electrical synapses with more complex functional properties. Channels formed by connexin (Cx) or pannexin proteins  were shown to impact cellular properties and underlie various pathological processes by serving as conduits for ions and various autocrine and paracrine signaling molecules , and indirectly proportional to the area of the membrane (size and geometry) of the coupled neurons , which is directly proportional to the membrane resistance  and the antennal lobe. One forms a rather rigid reflex network, the other is a heavily modulated sensory information processor dealing with odors. In addition, we discuss the contribution of heterotypic GJs to memory formation in the fly brain and to C. elegans nervous system signaling.It was actually proposed that the formation of homomeric, homotypic channels is generally rare among fly Inxs  and passover characterized the involved gene  gene gives rise to five different splice variants, resulting in three confirmed protein isoforms: ShakB, ShakB, and ShakB, which has 16 more amino acids on the amino-terminus  variant is not  was successfully rescued by ectopic expression of ShakB in adult flies. Since homomeric ShakB HCs fail to form functional GJs , which in turn relay the olfactory information into higher brain regions Wilson, . The gloDrosophila calyx is of olfactory nature, coming from the antennal lobe. Certain Kenyon cell subdivisions exhibit preferential roles in memory acquisition and in memory retrieval. The MB is innervated by two large amacrine cells per hemisphere; the GABAergic anterior paired lateral cell (APL), which innervates all MB regions . The MB is regarded as the homologous structure of the vertebrate pallium in the brain of protostomes  isoform and UNC-9 was shown to be rectifying  in the nematode nervous system and may be a conserved functional unit for coincidence detection  between Club endings and the Mauthner cell revealed a four-fold difference between the antidromic (from the postsynaptic Mauthner cell to the presynaptic Club ending) and orthodromic (from the Club ending to the Mauthner cell) directions  rectification with characteristics of a p-n junction in semiconductors. At that time GJ channels had not yet been discovered and thus the properties of the rectifier and the electrostatic effect were assigned to the “synaptic membrane”. Nonetheless, the novel hypothesis of p-n junctions in biological membranes was examined  in some electrical synapses.The original mechanism proposed for rectification of electrical transmission was represented as a simple analogy to an electric rectifier or diode . The instantaneous Ij-Vj relationship is mostly determined by the electrical properties of the unitary conductance of the fully open state (γo) of GJ channels, which can rectify by allowing larger Ijs in one direction than in the other. The unitary conductance of the residual state  may also rectify , or simply by differences in intrinsic sensitivity to Vj , a bigger fraction of Vj drops across the aHC with higher resistance (Cx45), thus enhancing its sensitivity to Vj compared to the aHC with smaller resistance (Cx43). Therefore, differential drop of Vj in aHCs of heterotypic GJs may also result in asymmetric gating , and asymmetric gating arises from differences in intrinsic Vj-sensitivity of aHCs rather than opposite gating polarities or differences in γo,H.Analysis of heterotypic Cx32/Cx26 GJs at the single channel level revealed that γp Figure  can makep Figure . The mecIj-Vj relationship contained a voltage-dependent kinetic component with a time constant in the order of milliseconds, which was attributed to a rapid gating mechanism present in one of the aHCs. The authors proposed that this gate was set to a low open probability at resting conditions and that changing the polarity of Vj would rapidly open the gates. This “millisecond timescale” gating mechanism has not been reported in any other Cx- or Inx-based rectifying electrical synapse; hence it is unclear whether rapid gating mechanisms may contribute to the observed electrical rectification in other invertebrate and vertebrate electrical synapses.In addition to the p-n junction hypothesis for electrical rectification, a unique voltage-dependent gating mechanism was proposed after a detailed characterization of the rectifying crayfish giant motor synapse using high-quality voltage clamp at low temperatures  to exhibit significant electrical rectification or asymmetric gating, respectively. As an analogy to p-n junctions in semiconductors and silica nanochannels . Only action potentials that lower this resting Vj would produce a “forward bias” effect in the junction to allow the spread of electrotonic potentials.Gap junctions can occur in homocellular or heterocellular junctions; that is, coupled cells can be from the same or different cell types and perform similar or different functions, respectively. One remarkable similarity among electrical synapses showing steep rectification is that they occur mostly in heterocellular junctions and very often there is a difference in the resting potential of coupled neurons that give rise to a relatively constant Vj, GJ channels and HCs are sensitive to phosphorylation, lipophilic molecules and other chemical factors   and the thalamic reticular nucleus (TRN) as well as heterologous expression systems transfected with Cx36 are bi-directionally modulated by changes in intracellular concentration of free Mg2+-dependent instantaneous gj-Vj relationship, in which instantaneous gj increases over Vj under high [Mg2+]i, or remain constant over Vj under low [Mg2+]i. Interestingly, an intercellular gradient of Mg2+  produces electrical rectification  exhibited differential sensitivity to changes in [MgC. elegans  with asymmetric transjunctional current flow Figure . ElectriDespite the presence of molecularly distinct pre- and postsynaptic sites, chemical synapses are considered indivisible functional units at which both sites are required to generate synaptic function. Electrical synapses can be viewed in a similar way. The fact that the docking of two HC is required does not necessarily imply that their molecular composition and that of the hemiplaques are the same. Hemiplaques should be different, suggesting that electrical synapses in analogy to chemical synapses can have distinct pre- and postsynaptic sites, endowing electrical synapses with more complex functional properties. While we emphasize in this article asymmetries in the composition of aHCs by GJ-forming proteins, asymmetries might also include the presence of associated scaffolding and regulatory proteins. Finally, an interesting scenario would be if asymmetries could be dynamically created by post-translational modifications of Cxs in only one of the aHCs (asymmetric phosphorylation), or by differences in the intracellular concentration of soluble factors that affect channels properties as a result of metabolic changes in one of the coupled cells, providing electrical synapses with plastic rectifying properties.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.


Different compounds have been studied for the treatment and/or symptoms amelioration of Familial Amyloidotic Polyneuropathy (FAP). We previously showed that Tauroursodeoxycholic acid (TUDCA), a biliary acid with anti-oxidant properties known to reduce non-fibrillar TTR aggregates is also capable of diminishing ER stress by its action on Bip in young transgenic Val30Met mice. This compound has already proved to be neuroprotective in several studies using genetic animal models of Huntington's and Parkinson's disease although the mechanisms underlying its neuroprotection action are still unknown.Other cellular mechanisms are being explored for possible actions of TUDCA, with special highlight to autophagy, a cellular mechanism that involves the delivery of large protein aggregates, defective organelles and other cellular debris to lysosomes for degradation that has lately been linked to several degenerative diseases where it appears to be impaired.The objective of our work is to investigate whether improvements previously observed in TUDCA-treated Val30Met mice involves autophagy.For this study, nine months old mice bearing the human TTR Val30Met mutation, in a TTR null background-hTTR Met30 were used. Animals were treated with TUDCA (500 mg/kg/day) in the drinking water, for a 3 month period after which the animals were sacrificed and organs from gastrointestinal tract were collected. An age-matched control group of animals was maintained in the same conditions, drinking regular tap water.Immunohistochemistry analyses were performed in order to evaluate the expression levels of an autophagic marker, p62, a protein naturally degraded in the final steps of the autophagic flux and that typically accumulates when autophagy is impaired.Our preliminary results point to a significant reduction in p62 accumulation in colon samples of TUDCA treated mice.TUDCA is a promising compound that has already been proved to significantly reduce TTR toxic aggregates in vivo which points to its capability of modulating TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis. Our results indicate that this modulation involves the autophagic machinery where it seems to enhance/restablish the autophagic flux that in turn may be directly involved in the pathological cascade in FAP, thus TUDCA contributes to the clearance of Val30Met TTR aggregates.


Mecopoda elongata, where some males persistently signal as followers although this reduces their mating success.Insect sounds dominate the acoustic environment in many natural habitats such as rainforests or meadows on a warm summer day. Among acoustic insects, usually males are the calling sex; they generate signals that transmit information about the species-identity, sex, location, or even sender quality to conspecific receivers. Males of some insect species generate signals at distinct time intervals, and other males adjust their own rhythm relative to that of their conspecific neighbors, which leads to fascinating acoustic group displays. Although signal timing in a chorus can have important consequences for the calling energetics, reproductive success and predation risk of individuals, still little is known about the selective forces that favor the evolution of insect choruses. Here, we review recent advances in our understanding of the neuronal network responsible for acoustic pattern generation of a signaler, and pattern recognition in receivers. We also describe different proximate mechanisms that facilitate the synchronous generation of signals in a chorus and provide examples of suggested hypotheses to explain the evolution of chorus synchrony in insects. Some hypotheses are related to sexual selection and inter-male cooperation or competition, whereas others refer to the selection pressure exerted by natural predators. In this article, we summarize the results of studies that address chorus synchrony in the tropical katydid  Grasshoppers, crickets, and katydids usually produce sound by stridulation, that is using a striated file-like body structure and associated structures that vibrate when they are rubbed across a sclerotized plectrum (peg). While crickets and katydids rub their forewings against each other, grasshoppers move their hind legs across a peg located at the base of their wings. The sound signals generated can be as short as 0.5 ms  or can last for many minutes or even longer . Acoustic signals can also be classified according to the responses they evoke from conspecific receivers: signals that are generated in aggressive interactions with conspecific rivals are termed aggressive songs, whereas calling songs are used to attract mates Heller, . When wisyllables  at fixed time intervals, the signal period could be a cue that indicates species identity  is puzzling. Mechanisms that would ultimately favor the evolution of chorus synchrony are thought to be diverse and may have evolved in response to selective forces either driven by other chorus members, through female choice  or natural predators . Males that advertise themselves in a chorus may gain one or more of the following mutual (group) benefits by timing signals . Instead, certain signals can more strongly stimulate the sensory system in receivers, increasing the likelihood of mating Ryan, . For exaIn male aggregations of anurans and katydids, females often select males on the basis of relative signal timing rather than other signal features   that is associated with calling energetics  leks, for which such a prerequisite does not exist .An inherent problem encountered when interpreting many group effects is the dilution of per capita mating success as compared to that of lone singing males. However, the increased amplitudes of group displays may enhance the mating probabilities of individual males if one considers the noisy background against which acoustic communication often takes place. Given these complex acoustic conditions, overlapping signals may allow individuals to increase the conspicuousness of their rhythmic signals in a group. Additionally, enhanced group signals were more attractive for females as compared to the solo song of a male , a tachinid fly that parasitizes field crickets, selectively orients toward the leading of two—otherwise identical—sound sources, while the lagging source had a minimal influence on the orientation of the fly. Therefore, the parasitoid fly homing in on M. elongata males may exhibit a similar leader preference as Mecopoda females, and these males would consequently suffer higher costs when signaling as leaders  [P21808-B09].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.


Evidence-based medicine is an important approach to avoiding care that is unlikely to benefit patients in both the treatment and the diagnostic context. The medical evidence alone may not determine the most appropriate care decision. Patient interests are best served when the advantages and risks of a diagnostic test are viewed through the lens of the patient’s values. That is, the paradigm of evidence-based medicine should be complemented by the paradigm of shared decision making.Diagnostic testing may offer physiological and psychological benefits. Clinicians should also discuss the potential harms, however, which may be physiological (e.g. radiation or scarring), psychological (e.g. anxiety), and financial (e.g. cost-sharing burdens). All three of these concerns are compounded by the risk of false positives or incidental findings that are not serious, but which require decisions about further testing or treatment.medically appropriate? Does the available evidence documenting short- and long-term risk and benefits support the test for its intended use, given the patient’s characteristics and symptoms?Is the test for this patient? Has the provider initiated a conversation about tradeoffs that helps the patient evaluate whether the balance of risks and benefits is consonant with the patient’s own values and preferences? Potential benefits and harms to consider include the physiological, the psychological, and the financial.Is the test appropriate We suggest that patient-centered decision making around diagnostic testing involves a two-step inquiry: The Excellence in Diagnostic Imaging Utilization Act of 2013 (H.R.3705) was introduced in the United States Congress in December of 2013. If passed, the Act would do three things: (1) direct professional medical societies to define standards of appropriate use for advanced diagnostic imaging technologies; (2) require practicing physicians to consult clinical decision support tools based on those standards (though not necessarily to follow the recommendations); and (3) establish data collection mechanisms on adherence to standards and usage of the decision support tools.Like therapeutic interventions, diagnostic tests are sometimes ordered in cases where there is little likelihood of benefit to the patient, or where the expected benefits come with the risk of potential harms, 2. The medically appropriate? Does the available evidence documenting short- and long-term risk and benefits support the test for its intended use, given the patient’s characteristics and symptoms?Is the test for this patient? Has the provider initiated a conversation about tradeoffs that helps the patient evaluate whether the balance of risks and benefits is consonant with the patient’s own values and preferences?Is the test appropriate The medical evidence supporting a given clinical decision is only the first step of the clinical inquiry, however. Patient interests are best served when the advantages and risks of the test are viewed through the lens of the patient’s values. Evidence-based medicine is by no means antithetical to those goals, and is most effective when complemented by the paradigm of shared decision making. The medical community is actively working on the challenge of integrating these two decision making paradigms in order to improve the quality of care. Much ofIn recent decades, clinicians have faced growing pressure to base their treatment decisions on rigorous research rather than local custom. Proponents of evidence-based medicine see it as a way to address unexplained geographical variance in care, improve patient wellbeing, and potentially even curb rising health care costs. The ChoA much larger and more nuanced set of scenarios involves decisions about diagnostic or therapeutic options that offer potential benefits that may or may not justify associated risks. The US Preventive Services Task Force (USPSTF) issues recommendations about who should receive screening and how often for these and other tests, and they recently updated their guidelines to recommend against both PSA screening for prostate cancer and routine mammography for breast cancer in younger women. These changes prompted an outcry from some physicians and from disease-specific advocacy groups, who noted that these guidelines rely on quantitative metrics about the “average” patient without accounting for patient-specific preferences. A 45-year-old woman whose close friend recently passed away from breast cancer may feel very differently about mammography than a 85-year-old woman with several chronic conditions who would likely opt for palliative measures even if cancer were diagnosed.Diagnostic decision making should start with an assessment of the testing options that are clinically indicated for the patient’s symptoms, signs, and other characteristics. It should not end there, however. It has become a mantra that clinicians should “treat the patient, not the disease”. Diagnosis should be similarly holistic: we urge clinicians to “diagnose the patient, not the symptom”.and potential harms and what they would mean for that particular patient’s wellbeing.The shared decision making approach involves eliciting patient-centered goals and values in order to reach a treatment plan that is consistent with the patient’s own priorities and preferences. Currently, many informed consent conversations focus primarily on the expected benefits of the recommended course of action. This is particularly likely with medications or screenings that impart low risk compared to major surgical interventions. Under aInformation about patient-centered outcomes can help patients envision their quality of life following various interventions. In addition to the risk of medical complications, patients may want to know about expected levels of pain, or the potential for incapacitation that would undermine their ability to participate in work or daily activities. Clinicians should also be mindful of patients’ financial situations. Patients with no insurance or large cost-sharing burdens may benefit from considering upfront the out-of-pocket costs for different care options.Armed with this information, some patients may wish to forego a procedure with a large price tag or impact on quality of life, choosing instead to pursue a more conservative treatment or to simply live with the underlying condition, 7.These treatment-related concerns remain relevant in the diagnostic context, since testing is often a predicate to treatment decisions. Before prescribing a test, clinicians should consider talking through the possible outcomes with the patient; it may well be that the differential diagnosis is populated with conditions whose treatments are unproven, unpalatable to the patient, or identical to the recommended course of action without a definitive diagnosis (conservative treatment as part of a “wait and see” approach).Clinicians and patients should also consider together several factors specific to the diagnostic decision itself (see below list for a summary). Except in rare occasions, tests do not themselves make patients feel better. The potential benefits of testing derive from the possibility of a diagnosis being made, since an accurate assessment of patients’ complaints often facilitates effective treatment. Aside from the potential physiological benefits gained by therapeutic intervention, the patient may also experience psychological benefits: worrying about symptoms can produce stress and anxiety, which caPotential benefits of testing:○ Consider whether the patient would actually want to undergo the recommended treatment for the conditions in the differential diagnosis.Physiological: A symptomatic patient may receive a diagnosis, thereby enabling treatment to address the underlying conditionPsychological: The patient may finally get an answer about worrying symptoms, or may rule out a worrisome possibility.Physiological: Some tests impart medical risks, such as radiation exposure from imaging, or complications and scarring from biopsies or other invasive testing.Financial: The patient may have cost-sharing burdens.All three of these concerns are compounded by the risk of false positives or incidental findings that are not serious, but which require decisions about further testing or treatment.Potential harms of testing:The potential harms of testing typically receive less attention in informed consent conversations, but theyThese three distinct types of potential burdens are all magnified by the potential for false positives or incidental findings, which rePatients may be acutely sensitive to any or all of these potential harms. Imagine George, a single father with low income who presents with persistent sciatica. His doctor suggests lumbar MRI to check for disc protrusion. However, George faces a hefty copay, and worries about missing time from work during his imaging appointment. Even with a confirmed diagnosis of disc protrusion, George would likely opt for conservative treatment with exercises and anti-inflammatory medications over discectomy or an epidural steroid injection. These preferences reflect his limited insurance coverage, his desire to remain fit for work and parenting, and a general aversion to invasive interventions. If George’s clinician helps him fully understand the costs and benefits, he may choose to forego the MRI and simply begin conservative treatment.We note that some health care facilities assist in arranging social services to address many of these types of patient concerns. Providers who practice shared decision making can proactively identify such needs by inquiring upfront about patient preferences. This approach is preferable to discovering addressable problems only when a patient resists the clinician’s recommendation or simply fails to complete prescribed testing and treatments.Shared decision making has received growing attention in recent years, particularly in the emotionally fraught area of cancer screening. Even there, however, patients often report that they did not discuss the trade-offs of the full range of risks and benefits. We shouThe Excellence in Diagnostic Imaging Utilization Act relies on the tenets of evidence-based medicine to address the stubborn problem of medically inappropriate diagnostic testing decisions. Shared decision making is much harder to mandate, however, since it does not depend on externally verifiable metrics like matching a diagnosis or biomarker to a recommended treatment. Clinical decision tools can include prompts for conversations about patient-centered outcomes and financial burden, but it is ultimately up to individual providers to elicit each patient’s values and preferences to help guide decisions.in general for a patient with this set of characteristics and symptoms. Clinicians should then consider which test or other course of action is appropriate for this particular patient in light of her personal goals and preferences. Indeed, the process of deciding on a diagnostic test requires the same consideration and patience necessary for determining a medical diagnosis itself.To ensure appropriate diagnostic care decisions, clinicians should begin by asking what tests are appropriate


South Africa is committed to eliminating malaria with a goal of zero localtransmission by 2018. Malaria elimination strategies may be unsuccessful if theyfocus only on vector biology, and ignore the mobility patterns of humans,particularly where the majority of infections are imported. In the first study inMpumalanga Province in South Africa designed for this purpose, a metapopulation modelis developed to assess the impact of their proposed elimination-focused policyinterventions. A stochastic, non-linear, ordinary-differential equation model isfitted to malaria data from Mpumalanga and neighbouring Maputo Province inMozambique. Further scaling-up of vector control is predicted to lead to a minimalreduction in local infections, while mass drug administration and focal screening andtreatment at the Mpumalanga-Maputo border are predicted to have only a short-livedimpact. Source reduction in Maputo Province is predicted to generate large reductionsin local infections through stemming imported infections. The mathematical modelpredicts malaria elimination to be possible only when imported infections are treatedbefore entry or eliminated at the source suggesting that a regionally focusedstrategy appears needed, for achieving malaria elimination in Mpumalanga and SouthAfrica. Mathematical modelling is an integral tool aiding our understanding of the dynamics ofinfectious diseases .MathemaMalaria prevalence and control in Mpumalanga has been documented extensively –13. The Maputo Province, which shares the eastern border of Mpumalanga, has also experienced asharp decline in malaria cases since 2002 but still has substantially higher malariaincidence. The LSDI malaria control programme was a regional collaboration between SouthAfrica, Swaziland and Mozambique that aimed to decrease malaria in the areas surroundingthe Lubombo Mountains .InterveMetapopulation modelling is one method to describe movement between geographical areaswith several applications in malaria and other infectious diseases. The metapopulationconcept has been used to examine the spread of chloroquine resistance , model mEthical approval for use of the data was obtained from the Mpumalanga Department ofHealth and the University of Cape Town Human Research Ethics Committee.β, the number of infectious bites, multiplied by theproportion of infectious humans, delayed by four time steps. This delay takesaccounts for life cycle of the mosquito from the first human bite to the second humanbite.Metapopulation models divide a population into a number of discrete patches under theassumption that the sub-populations are well-mixed/homogenous. This structure allowsfor the modelling of transmission within and between different populations. The areaunder consideration is divided into six patches: five patches for the fivemunicipalities in Ehlanzeni District (Thaba Chewu (TC), Mbombela (MB), Umjindi (UJ),Nkomazi (NK) and Bushbuckridge (BB)) and one patch for Maputo Province. Each patch isfurther divided into three sub-patches representing (1) the local population of thepatch currently in the patch, (2) the local population of the patch having returnedfrom travel to a foreign place and (3) the population from the foreign place currently in the patch . In eachHuman movements are incorporated in two ways. Local movements are allowed between thefive Mpumalanga patches .Foreign travel is allowed between the Maputo patch and any of the five Mpumalangapatches  as illustrated in et al.(2013) describes in detail the characteristic triple peaked pattern in the Mpumalangacase data with peaks occurring during October, December/January and April/May whileMaputo Province exhibited the December and April peaks only. Seasonal forcing,described as a function of rainfall, geography and source of infection, thatdetermines the behaviour of transmission for the six patches is derived from the datausing Seasonal decomposition of Time series by LOESS (STL) methods for extractingtime series components [The metapopulation model is fitted to weekly case-notification data of treated casesfrom Mpumalanga and Maputo from 2002 to 2008, and then cross-validated against thedata from 2009 to 2012. In Southern Africa, most malaria transmission occurs duringthe summer rainfall season between October and May. Silal mponents .ACT druλ as the number oftreated cases per week. Several parameters are estimated through the data-fittingprocess using the particle swarm optimisation routine, a population-based globalsearch algorithm [The model is run deterministically from 1990 to reach a steady state before beingfitted to data from 2002. The model-predicted weekly treated malaria cases are fittedto the data from 2002 to 2008 using the maximum likelihood approach by assuming anunderlying Poisson distribution with rate lgorithm , 30. Thelgorithm . The parlgorithm , 33.The interventions to be tested include: scaling-up of vector control from currentlevels, mass drug administration in three local patches, a focal screen and treatcampaign at the Mpumalanga-Maputo border, foreign source reduction through vectorcontrol and MDA in Maputo Province only and relaxing vector control during the FSATcampaign. The scale up of vector control is assumed to occur uniformly within eachpatch (if vector control is being performed in the patch). MDA is modelled at acoverage below 100%. Being a metapopulation model, individuals are not tracked,therefore all members of a metapopulation are equally likely to receive MDA, thoughno member can receive it twice in the same round. With FSAT, all border-crossers areequally likely to receive FSAT, and they are tracked as a group until such time asany future infection will be a locally sourced one. Source reduction in MaputoProvince is conducted at the metapopulation level only. Thus it is assumed that thevector control and MDA are performed uniformly throughout the province. A fulldescription of the interventions modelled can be found in etal. (2008) conducted household surveys in Mozambique and South Africabetween 2001 and 2002 to evaluate malaria-related treatment-seeking behaviour andfound that 100% of respondents in Mpumalanga and 99% of respondents in Mozambiquewith recent malaria sought treatment. Hlongwana et al. (2011)conducted a study on malaria-related knowledge and practices in BushbuckridgeMunicipality in 2008 and reported that 99% of respondents would seek malariatreatment . Different rates are fitted for theforeign treatment proportion and foreign movement rate before and after April 2005when the South African and Mozambican governments waived short-stay visa requirementswhich subsequently led to increased movement between the two countries [The proportion of infections treated varies widely across Africa with some estimatesas low at 10% and others as high as 90% . In Soutountries .t as a random realisation of a Poisson process with rateλ, the deterministic flow value at that time, and bysimulating the parameter values from their 95% confidence intervals. Eight randomseeds were selected per parameter set and 150 parameter sets were simulated. Theparameters were simulated from uniform distributions with ranges in parentheses inInterventions are tested on a stochastic version of the fitted model; the sameintervention is applied to multiple model runs such that its impact on localinfections can be described with a mean effect and a 95% confidence interval.Stochastic uncertainty and parameter sensitivity has been accounted for as follows.The model is run stochastically by treating each flow between compartments at eachtime point βi, the number oflocal human contacts with infectious mosquitos. Scaling up vector control inNkomazi, Bushbuckridge and Mbombela municipalities i.e. decreasingβi by a further 10%, ispredicted to result in a decrease in local infections inall municipalities , nine months from September to May (green), all year round (blue) andall year round at 100% coverage (purple) compared to no FSAT (black). The impacton local infections is predicted to be substantial regardless of the duration ofFSAT. At 70% coverage, FSAT is predicted to be most effective when performedthroughout the year (blue) as even a few imported cases over the non-malariaseason can maintain local transmission. Even when performed throughout the year at70% coverage, FSAT is insufficient to eliminate local infection. Elimination ispredicted if FSAT is performed continuously throughout the year at 100% coverage.This is of course unrealistic but serves to illustrate the prediction that ifimported infections are treated before entry intoMpumalanga , elimination of localmalaria becomes a possibility. Like MDA, FSAT has a short-lived benefit as themodel predicts a reversion to previous levels approximately three years after theend of the intervention.The FSAT campaign is modelled at a border entry point between Mpumalanga andMaputo. Thus the proportion of the population targeted in this intervention issubstantially lower than the MDA campaign previously modelled, where threemunicipalities were targeted. Focused Screen and treat campaigns have been predicted to be effective in reducinginfections substantially if sustained for a long period. In dedicating resourcesto this intervention, it may be tempting to relax the vector control effort in anattempt to shift resources rather than procure additional resources. In 2012, 87% of all reported malaria cases in Mpumalanga were imported, and Scaling-up vector control will decrease prevalence but noteliminate malaria in the presence of imported infections.Mass interventions lead to large and immediate decreases inprevalence but will result in a rebound in prevalence three yearsafter the intervention has stopped.Smaller scale interventions such as FSAT at the border have thesame large but short-lived impact of mass interventions and aremost effective if conducted throughout the year as the presence ofeven a few imported infections, leads to onward transmission.Reducing vector control in favour of FSAT dampens the impact ofFSAT in both the short and long terms.Source reduction is likely to be effective in decreasing localprevalence, whether through better control or elimination at thesource.There is no “one size fits all” strategy to achievemalaria elimination and a tailored approach is needed to addresslinkages between populations. For example, in the case ofMpumalanga province, the high level of imported infections suggeststhat a regional approach to malaria elimination will be moresuccessful than a nationally focused one.The stochastic metapopulation transmission model developed to simulatetransmission in Ehlanzeni District, Mpumalanga, and Maputo Province,Mozambique has made predictions that lead to the following conclusions:South Africa aims to achieve malaria elimination by 2018. A malaria-elimination strategyshould aim to interrupt the transmission cycle and prevent it from being reestablished.An elimination strategy that employs a ‘more of the same’ approach maydecrease the malaria burden, but will be insufficient to eliminate it as the focus needsto shift from better overall control to the identification of residual transmission focileading to the last few infections. The interruption of the transmission cycle andprevention of its reestablishment theoretically requires three elements: (1) theelimination of the mosquito vector to prevent onward transmission, (2) inhibiting theinflow of imported infections and (3) reduction of infections at their source . The firet al. (2014)predicted that in the absence of imported infections, MDA applied continuously over sixmonths at 80% coverage would be sufficient to eliminate local malaria, but even repeatedannual rounds of MDA for seven years is insufficient to eliminate local malaria at thecurrent level of imported infections because MDA does not interrupt the inflow ofimported infections. The stochastic metapopulation model presented in this paper alsopredicts that MDA applied in the three municipalities with the highest incidence has alarge impact but this is short-lived because there is no impact on the flow of importedinfections. A Focused Screen and Treat campaign focused on treating infections at theborder control point between Maputo Province and Mpumalanga is also predicted to have alarge impact, but is not enough to eliminate local malaria on its own unless theunlikely scenario of all imported infections being screened and treated is achieved. Assoon as the FSAT campaign is stopped, infections revert to previous levels within threeyears. This suggests that screening and treating infections at the border would need tobe intense indefinitely (in the absence of new interventions) to minimise the impact ofimported infections on malaria transmission.Mass Drug Administration is an intervention that is resource intensive in terms oflabour and the cost of drugs. It is also an intervention that needs to be acted outquickly and efficiently to achieve desired target coverage rates. In a single patchdeterministic model of malaria in Mpumalanga, Silal et al. (2014) but the metapopulation model has predicted thatincreasing vector control in Nkomazi, Mbombela and Bushbuckridge municipalities onlyalso leads to a knock-on decrease in malaria cases in the other two municipalities.Another recent modelling study found that at low receptivity levels, case managementalone could not reliably prevent the reestablishment of transmission in the presence ofmedium to high importation rates [In Mpumalanga, vector control has been conducted using high-coverage IRS withdichorodiphenyltrichloroethane (DDT) and larviciding at identified breeding sites.Scaling-up vector control as an elimination intervention may include intensifying thealready extensive spraying programme, increasing the distribution of insecticide-treatedbednets and the identification and larviciding of additional breeding sites. The purposeof a scale-up in vector control would be to decrease the potential for onward localtransmission, though it is impossible to reduce this to zero. While effective if themajority of infections are locally sourced, the model predicts that increasing vectorcontrol alone will not eliminate local infection if the stream of imported infections isleft unchecked. These predictions are in line with the single patch model in Silalon rates .While scaling-up vector control alone in Mpumalanga may not be enough to interrupttransmission and prevent its reestablishment, vector control at the source of importedcases has a large effect. The model predicts that if vector control is continued atcurrent levels in Mpumalanga, but is scaled up in Maputo, the related decrease in localinfections in Mpumalanga will be substantial. This decrease results because a smallerproportion of the population that travels into Mpumalanga will be infected and hence theinfectious reservoir in Mpumalanga is reduced. These knock-on decreases in localinfections in Mpumalanga are also predicted if MDA is performed in Maputo Province,although infections revert to previous levels a few years after the end of the MDAcampaign. The model also predicts that if malaria were to be eliminated in Maputo,malaria would also be eliminated in Mpumalanga. These predictions highlight the need forand pivotal importance of cross-border/regional collaboration. The Lubombo SpatialDevelopment Initiative, a trilateral agreement between South Africa, Mozambique andSwaziland, was initiated in 1999 and successfully reduced malaria cases by 78 to 95% inthe border areas of South Africa and Swaziland within five years of the start of IRS andthen ACT deployment in Maputo Province . In Septenmasse through drug distribution orscreening in the case of FSAT.Varying levels of population immunity in Mozambique can impact results where decreasesin immunity may lead to an increase in symptomatic cases or a greater proportion ofinfections are treated in South Africa. This could result in a reduction in the numberof secondary cases if these infections are treated routinely. The impact of massinterventions is unaffected by varying population immunity in Mozambique asinterventions are deployed etal. (2014), mass interventions if administered, will most likely beperformed in smaller hotspot areas within a municipality and may be more accuratelymodelled if patches are disaggregated further or agent-based modelling is used toincorporate heterogenous behaviour among individuals. This metapopulation comprised sixpatches and while this methodology may theoretically be extended to any number ofpatches, several aspects must be taken into account. Computationally, extending themethodology to a large number of patches n with This paper presents the findings of a stochastic, metapopulation, non-linear,differential equation model of five municipalities in Mpumalanga, South Africa and inneighbouring Maputo Province, Mozambique. While the current metapopulation structureallows for more disaggregated modelling than the model presented in Silal To eliminate malaria by 2018, the government of South Africa will need to design andimplement an elimination strategy tailored for a country with a high level of importedinfections. A regionally focused strategy may stand a better chance at achievingelimination in Mpumalanga and South Africa compared to a nationally focused one in theface of frequent population movement between the pre-elimination area and neighbouringhigh transmission intensity regions . MathemaS1 Text(PDF)Click here for additional data file.


The burden of comorbidity in Autism Spectrum Disorder (ASD) is substantial. The symptoms of autism overlap with many other human conditions, reflecting common molecular pathologies suggesting that cross-disorder analysis will help prioritize autism gene candidates. Genes in the intersection between autism and related conditions may represent nonspecific indicators of dysregulation while genes unique to autism may play a more causal role. Thorough literature review allowed us to extract 125 ICD-9 codes comorbid to ASD that we mapped to 30 specific human disorders. In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders, and found 1031 genes involved in ASD, among which 262 are involved in ASD only, with the remaining 779 involved in ASD and at least one comorbid disorder. A pathway analysis revealed 13 pathways not involved in any other comorbid disorders and therefore unique to ASD, all associated with basal cellular functions. These pathways differ from the pathways associated with both ASD and its comorbid conditions, with the latter being more specific to neural function. To determine whether the sequence of these genes have been subjected to differential evolutionary constraints, we studied long term constraints by looking into Genomic Evolutionary Rate Profiling, and showed that genes involved in several comorbid disorders seem to have undergone more purifying selection than the genes involved in ASD only. This result was corroborated by a higher dN/dS ratio for genes unique to ASD as compare to those that are shared between ASD and its comorbid disorders. Short-term evolutionary constraints showed the same trend as the pN/pS ratio indicates that genes unique to ASD were under significantly less evolutionary constraint than the genes associated with all other disorders. Autism Spectrum Disorder (ASD) is a heritable developmental disorder that affects one in sixty-eight children . Its preThere has already been a massive sequencing effort which has led to the discovery of many genetic biomarkers of Autism Spectrum Disorder –6, as weOne way to perform a cross-disorder analysis of the results is through systematic screening of all genes already associated with ASD and its comorbid disorders. Identifying genes uniquely related to ASD will allow us to better analyze gene functions and identify complex disease genes by describing metabolic and regulatory pathways unique to this condition.But beyond the description of functionally distinct pathways underlying autism, can we find an evolutionary signature intrinsic to the sequences unique to ASD in comparison to those of comorbid diseases? By comparing the genes involved in ASD and its closely related disorders, we can determine whether the sequence of these characterized genes have been subjected to differential evolution constraints, either purifying or positive selection during the evolutionary timeframe for mammals or in the more recent evolution of humans. Such analysis would not only define evolutionary trends of ASD genes, but also allow us to relate this to the molecular pathology of autism.In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders using a published tool ,7,8, in We used the results of three research studies that investigate comorbid disorders that occur at a significantly higher frequency in individuals diagnosed with ASD than in an age-matched control population, as based on a population derived sample ,3,9, eleet al., [et al., [To conduct PubMed queries to retrieve disorder-related genes, we used two robust disease-gene text mining tools, Phenopedia ,4,8 and [et al., . Briefly[et al.,  correspo[et al., , as well[et al., .http://www.rstudio.org/). Clustering analysis between disorders was performed on a presence-absence matrix of all the genes involved in all the comorbid disorders by calculating distances using a binary method and generating a hierarchical clustering using Ward D2 method (library pheatmap). To consolidate the clusters, we calculated bootstrap values using 1000 iterations. We also computed p-values with multi-scale bootstrap on 1000 resamplings using the package pvclust   . We extrg/rest/) , and estWe performed a network analysis using GEPHI and the dual-circle layout using the circular layout plug-in .et al., [http://mendel.stanford.edu/SidowLab/downloads/gerp/). We used the gene file indicating each coordinate made available by NCBI to map, in R, our gene lists to the corresponding regions showing significant evolutionary constraint .For our GERP analysis, we downloaded the orthologous alignment scores used by Cooper et al.,   to synonymous (dS) nucleotide substitutions of the orthologous coding sequences (CDS) present in either the gene sets associated with ASD only or with comorbid disorders, in order to assess the evolutionary characteristics of each of these genes in the primate lineage. The primate dN/dS ratios were measured with codeml from the PAML software by using best reciprocal coding sequence alignments of 9 primates including human, chimpanzee, gorilla, orangutan, gibbon, macaque, baboon, marmoset and bushbaby. To obtain the alignments of best reciprocal CDS we used a combination of Blat  and PRANhttp://www.1000genomes.org/data). pN indicates the degree to which two sequences differ at non-synonymous sites within a population (here the African human genome from 100genomes project), i.e. the number of non-synonymous polymorphisms per gene. pS is a measure of the degree to which two homologous sequences differ with respect to silent nucleotide substitutions (substitutions that do not cause an amino-acid substitution). To calculate the pN/pS ratio, we divided the mean of all the pN values of the gene sets considered by the mean of all the pS values of the gene sets being compared , which we consolidated into 31 disorders . Using aThe identification of genes uniquely related to ASD allowed us to distinguish metabolic and regulatory pathways putatively specific to this condition. To do so, we used the 1031 symbol gene IDs retrieved from our literature search; 737 mapped to KEGG Orthologs (KO) involved in 194 KEGG pathways  Tables. Among these pathways, 113 were extracted from the 262 genes involved in ASD only, and we observed thirteen pathways not involved in any other comorbid disorders and therefore unique to ASD. While we did not detect an entire pathway associated exclusively with ASD, Mucin type O-Glycan biosynthesis shows several genes associated with ASD. Mucins are glycoproteins that are ubiquitous in the human body. They are present in mucous secretions on cell surfaces and in fluids and interact with α-Neurexins, a type of presynaptic cell surface molecule essential for neurotransmission and linked to neuro-developmental disorders such as autism and schizophrenia . SphingoInterconnected genes unique to ASD are of high interest, as any variation in their coding sequence will impact the function of pathways detected with this analysis, including several of the pathways uniquely connected to ASD. To determine whether genes unique to ASD appeared to be involved in specific pathways, we performed a networks analysis . K02649 In order to determine whether the regions involved in ASD only have been subject to purifying selection and are enriched for functional elements, we compared orthologous genomic DNA sequences by using Genomic Evolutionary Rate Profiling (GERP) . This meHuman and other primate sequences corresponding to these genes were used to calculate the dN/dS ratio of the orthologous coding sequences present in either the gene sets associated with ASD only or with comorbid disorders. Comparing the dN/dS ratio of genes involved in ASD with the ones involved in several disorders shows a significant p-value of 0.0306 ; this raWhile the dS/dN ratio reflects the evolutionary history among mammals, it does not inform us about the more recent evolution among humans. To analyze recent human-specific constraints, we used the pN/pS ratio. We compared this ratio for genes uniquely associated with autism to those genes shared between ASD and other disorders , panel AWe extended the analysis to any genes involved in comorbid disorders associated with cognitive function, i.e.: Conduct Disorder, Agoraphobia, Developmental Coordination Disorder, Intellectual Disability, Tourette Syndrome, Tic Disorder, Learning disorder, Communication Deficit, Attention Deficit Hyperactivity Disorder, Epilepsy, Depressive Disorder, Down Syndrome, Schizophrenia, Panic Disorder, Obsessive Compulsive Disorder, and Anxiety Disorder. We explored the pN/pS ratio comparing this set of cognitive disorders with the set of genes involved in ASD only, and obtained a p-value of 0.1218. The same ratio of all cognitive disorders to non-cognitive disorders  did not show any differences . We performed the same analysis for dN/dS ratio and did not find any significant differences.Our search for genes using a previously published literature mining tool  found 10Of the pathways previously linked to ASD, we identified 13 pathways uniquely involved in ASD. While the pathways related to neural functions seemed to be associated with disorders comorbid to ASD, lipid biosynthesis and regulation pathways were associated with autism only. We also observed that basal cellular functions  appeared to be more specific to ASD. The coverage of the pathway involved in ASD was less extensive than that of pathways involved in several disorders, but the network analysis reveals two KEGG Orthologs with ubiquitous functions that could constitute ASD biomarkers: K04369  and K02649 (PIK3R gene). The MAPK pathway was previously reported to be involved in ASD and other disorders; here we have shown that it evidence for its unique association to autism, in comparison to with disorders comorbid with autism.Evolutionary history of genes involved in ASD: We generated a set of genes and pathways implicated in only autism, all involved in basal cellular functions. This was in contrast to gene sets shared by ASD and comorbid disorders, which we found to be associated with neurological function. This suggested that the pathways specifically underlying autism were functionally distinct, and may have evolved (or be evolving) under different evolutionary constraints than genes shared between autism and comorbid conditions. To determine if these two gene sets showed discrepant evolutionary signatures, we looked for potential purifying or positive selection events during both mammalian evolution and more recent human history.To characterize the evolution history of the genes involved in ASD, more specifically at the levels of evolutionary constraints, we first compared evolutionary constraints within mammalian phylogenies using GERP . We alsoThe pN/pS ratio was significantly different between the gene set within the cluster containing ASD and the set outside the cluster . Taking The literature has shown that coding sequences expressed in the brain evolved at a slower rate than in the rest of the genome . This evS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.