Hemoptysis is a common complication in all kinds of surgery. However, it is rarely critical because it resolves with or without intervention.Here the authors present what is believed to be an unprecedented report of a case involving a fatal idiopathic bronchial hemorrhage complication during cardiac surgery. Eighty-five-year-old female with severe aorticvalve stenosis had elective aortic valve replacement. Subsequently, she developed diffuse bilateral severe idiopathic bronchial hemorrhage which required maximum intervention such as external bronchial ligation, V-A ECMO, coil embolization of bronchial artery and internal airway blockage by spigot.Airway bleeding is not a rare complication in cardiac surgery, but this case should increase awareness of this potentially life threatening perioperative complication.The online version of this article (doi:10.1186/s13019-016-0477-0) contains supplementary material, which is available to authorized users. Airway bleeding during or after heart surgery is common, but is rarely reported because it is generally minor and resolves spontaneously in most cases. Here the authors report an unusual case of exsanguinating diffuse airway bleeding during cardiac surgery. The complication required aggressive intervention measures such as extra-corporeal membrane oxygenation (ECMO), bronchial ligation, bronchial artery coil embolization and internal airway blockage using an intrabronchial spigot.The case involved an 85-year-old female with congestive heart failure caused by severe aortic valve stenosis. Aortic valve replacement with a bioprosthetic valve was conducted electively. After the ascending aorta was unclamped, anesthetist noticed blood coming up from the endotracheal tube. Intraoperative flexible bronchoscopy confirmed massive hemoptysis coming up from all peripheral bronchi and mainly from the bilateral inferior lobes. The left chest was inspected through additional anterolateral thoracotomy connected perpendicularly to median sternotomy wound at 4th intercostal space. And the left hilum was exposed and lower bronchus was ligated externally to reduce the amount of intrabronchial bleeding. Repeated bronchoscopy showed persisting large-volume airway bleeding  have beThere is no doubt that systemic heparinization in cardiac surgery triggered the diffuse bronchial hemorrhage. Complete heparin reversal and expeditious weaning from cardiopulmonary bypass is reported to be the best and the only way to manage major hemoptysis –5. HowevUnderlying pulmonary disease such as bronchiectasia or tuberculosis can also cause perioperative hemoptysis. Pulmonary capillaritis such as Wegener’s granulomatosis or Goodpasture syndrome are also known to be related to pulmonary hemorrhage . AlthougIn summary, this was a rare case of diffuse exsanguinating hemoptysis during cardiac surgery. Even after all the aggressive intervention and intensive care administered, the patient’s respiratory status failed to show improvement in terms of lung compliance and oxygenation. Practicing cardiothoracic surgeons should note this unusual but catastrophic perioperative complication.All authors have approved the manuscript. Patient’s family has agreed with submission of the case report. Upon acceptance, authors will transfer copyright to the Publisher. All authors have no ethical problem or conflicts of interest to declare.


This survey sought to evaluate differences in the understanding and management of shunt-dependent hydrocephalus among the senior North American Pediatric Neurosurgery membership.Surveys were sent to all active American Society of Pediatric Neurosurgeons (ASPN) members from September to November 2014. A total of 204 surveys were sent from which 130 responses were recorded, representing 64% of active ASPN membership. Respondents were asked 13 multiple choice and free response questions focusing on four problems encountered in shunted hydrocephalus management: Shunt malfunction, cerebrospinal fluid (CSF) overdrainage, chronic headaches and slit ventricle syndrome (SVS). Qualtrics® online survey software was used to distribute and collect response data.ASPN surgeons prefer three varieties of shunt valves: 41% differential pressure, 29% differential with anti-siphon device (ASD), and 27% programmable. Respondents agree shunt obstruction occurs most often at the ventricular catheter due to either in-growth of the choroid plexus (67%), CSF debris (18%), ventricular collapse (8%), or other reasons (9%). Underlying causes of obstruction were attributed to small ventricular size, catheter position, choroid plexus migration, build-up of cellular debris, inflammatory processes, or CSF overdrainage. The majority of respondents (>85%) consider chronic overdrainage a rare complication. These cases are most often managed with ASDs or programmable valves. Chronic headaches are most often attributed to medical reasons  and managed with patient reassurance. The most popular treatments for SVS include shunt revision (88%), cranial expansion (57%) and placement of an ASD (53%). SVS etiology was most often linked to early onset of shunting, chronic overdrainage and/or loss of brain compliance.This survey shows discrepancies in shunt-dependent hydrocephalus understanding and management style among a representative group of experienced North American pediatric neurosurgeons. In particular, there are differing opinions regarding the primary cause of ventricular shunt obstructions and the origins of SVS. However, there appears to be general consensus in approach and management of overdrainage and chronic headaches. These results provide impetus for better studies evaluating the pathophysiology and prevention of shunt obstruction and SVS.


Isolated coarctation of aorta is usually diagnosed and treated surgically in most of patients during childhood, but optimal management strategies for adult patients with coarctation of aorta associated with stenosed bicuspid aortic valve are controversial.We aim to treat such rare patients successfully by less invasive two staged hybrid management.We report a case of 18 years old female with history of dyspnoea on exertion since 4 years, with complaints of headache and bilateral lower limb claudication, clinical findings revealed no characteristic clinical feature of Turner syndrome, on examination femoral pulses were delayed and diminished bilaterally, laboratory examination were unremarkable with negative karyotyping for Turner syndrome, chest x-ray showed enlarged cardiac silhouette with rib notching, echocardiography revealed severely stenosed bicuspid aortic valve with left ventricular hypertrophy, descending aortogram showed post ductal coarctation and collaterals, pressure analysis revealed gradient of 70 mmHg across coarctation. She underwent successful hybrid approach with percutaneous catheter balloon coarctoplasty (PCBC) with stenting in the first stage, followed by surgery for aortic valve replacement in the second stage.Post operatively she was asymptomatic and had uneventful recovery after each stage of management, post-operative echocardiography findings were within acceptable limits, and at 6 months follow up the mean echocardiographic aortic arch gradient was within acceptable limits.Two staged hybrid approach of (PCBC) with stenting for coarctation of aorta and aortic valve replacement for coexistent stenosed bicuspid aortic valve is a safe, less invasive and can be effective alternative to complete surgical approach, if done meticulously.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Giant cell reparative granuloma (GCRG) is a rare, benign intra osseous lytic lesion occurring especially in gnathis bone but also seen in feet and hands. It has similar clinical and radiological presentations than giant cell tumor, chondroblastoma, aneurysmal bone cyst, and hyperparathyroidism brown tumors but with specific histological findings We report a case of a GCRG of hallux phalanx in 18 years old patient appearing many years after enchondroma curettage and grafting. Radiographs showed a multiloculated osteolytic lesions involving whole phalanx with cortical thinning and without fluid-fluid levels in CT view. Expected to be an enchondroma recurrence, second biopsy confirmed diagnosis of GCRG with specific histological findings. Although if aetiopathogeny remains unknown, GCRG is reported to be a local non neoplasic reaction to an intraosseous hemorrhage. Our exceptional case claims that this tumor can appear in reaction to cellular disturbance primary or secondary. Giant cell reparative granuloma (GCRG) is a rare, benign intraosseous lytic lesion occurring especially in gnathic bones and also in hands and feet phalanx –3. It isA 13- year-old boy presented in emergency room with left hallux painful evolving for several months without any underlying trauma. The physical examination noted a localized swallowing of the dorsum of the right hallux base with no inflammatory local signs and a pain sensitivity induced by examination. Mobility of the adjacent joint was normal and no biological abnormalities were found. Radiographic examination showed a well-defined lytic lesion of the hallux proximal phalanx. Neither cortex erosion nor soft tissues abnormalities were noticed . An initInitially described by Jaffe  in 1953,This case suggests a potential connection between GCRG and enchondroma and hypothesis that GCRG could appear in reaction of cellular disturbance primary or secondary to enchondroma surgical treatment.


Praelongorthezia praelonga (Hemiptera: Ortheziidae), an important pest of citrus orchards. The data presented in this article are related to the article entitled “Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes” C. nymphaeae, emerges through the thin cuticular intersegmental regions of the citrus orthezia scale body revealing orange salmon-pigmented conidiophores bearing conidial masses, as well as producing rhizoid-like hyphae that extend over the citrus leaf. By contrast, nymphs or adult females of this scale insect infected with Lecanicillium longisporum exhibit profuse outgrowth of bright white-pigmented conidiophores with clusters of conidia emerging from the insect intersegmental membranes, and mycosed cadavers are commonly observed attached to the leaf surface by hyphal extensions. These morphological differences are important features to discriminate these fungal entomopathogens in citrus orthezia scales.We describe symptoms of mycosis induced by two native fungal entomopathogens of the citrus orthezia scale,  Specifications Table•C. nymphaeae and L. longisporum.Data show symptoms of two important fungal diseases in the citrus orthezia scale caused by •Simple and practical recognition of these entomopathogenic fungi can be achieved on the basis of their conspicuous morphological characters, including conidiophores and conidia, from mycosed scale insects.•The importance of using morphological features based on symptoms of fungal diseases can serve as a guideline to facilitate recognition and quantification of these pathogens in citrus orthezia scale populations.1C. nymphaeae and L. longisporum  of infected citrus orthezia scales with two native fungal pathogens, namely gisporum . Images 2L. longisporum or C. nymphaeae from citrus groves located in a commercial farm  were taken to the laboratory to record the morphological features of mycosis in individuals of the citrus orthezia scale. Images of mycosed insects were acquired by using a stereo-microscope at 10–30× magnification, while images of conidiospores were taken by phase-contrast and scanning electron microscopy. These morphological characters related to signs of fungal diseases were used as a guideline for identification and quantification of these fungal entomopathogens in this insect population during field surveys Field collected insects infected with


Cancers depend on one or more ubiquitously activated oncogenes to survive and maintain their tumorigenic phenotype. The MYC oncogene is critically important in a variety of hematological malignancies, including Burkitt's lymphoma, acute lymphoblastic leukemia, and multiple myeloma. Cancers' reliance on few oncogenes, aptly termed oncogene addiction, presents an exploitable vulnerability. Indeed, targeted therapies inhibiting key oncogenes in a multitude of cancer types are routinely used in clinic . HoweverExperimental model systems of MYC oncogene inactivation in various cancers have revealed molecular mechanisms of oncogene addiction, escape from oncogene dependence resulting in tumor recurrence . InactivRecently, we gained insight into the mechanism of tumor recurrence. We found that when either of two tumor suppressor genes, ARF or p53, is lost or mutated, tumor recurrence rapidly always occurs . We prevWe found that ARF loss prevented MYC inactivation from inducing cellular senescence through a p53 independent mechanism . Our obsThe mechanism through which ARF regulates innate immunity is not clear. Analysis of gene expression revealed a signature of genes involved in innate immunity . ARF, itEmerging immunotherapies have intensified interest in the therapeutic role of the innate and adaptive immune system. Biologics targeted at tumor cells for enabling antibody-dependent cell-mediated cytoxicity (ADCC) rely on innate immune system for elimination of cancer cells . Since ACells undergoing senescence secrete factors that bolster the senescent phenotype in both autocrine and paracrine fashion by engaging innate immune system . ARF losOur research has uncovered ARF as a common regulator of senescence and tumor clearance that appears to require the innate immune system. However, there are several questions awaiting answers. First, we have identified innate immune-related gene expression changes due to ARF loss; yet direct causality of these changes in macrophage infiltration into regressing tumors has not been established. Second, the mechanism by which cellular senescence is dependent upon macrophage recruitment is unclear. Third, whether ARF loss in tumors affects other immune infiltrates, such as NK cells, monocytic and granulocytic myeloid cells, or even adaptive immune cells, needs to be investigated. Fourth whether ARF's effect on innate immune-related genes and recruitment of macrophages extends to other types of cancers remains to be determined. Finally, it is of paramount interest whether ARF status may influence efficacy of immunotherapies.


Cancer imaging is frequently at the cutting edge of new imaging techniques which are often rapidly incorporated into routine use.Skeletal metastatic disease is a frequent complication of neoplastic conditions, and results in specific challenges to the general radiologist and specialist oncological radiologist alike.Non-neoplastic conditions and normal variants may simulate skeletal metastases, and the radiologist must recognise such cases and avoid over-investigation and unnecessary treatment.This lecture will briefly review standard imaging techniques and demonstrate normal appearances, normal variants and non-neoplastic lesions that mimic primary and secondary skeletal malignancy, and will then review a spectrum of malignancy-associated bone lesions with the use of standard and more specialised imaging techniques, including PET MRI, PET CT and diffusion weighted imaging,.Expected post treatment imaging findings, and treatment-associated complications will also be discussed.


To investigate through Structural Equation Modelling (SEM), the relationship between parenting styles, teasing about body weight/shape or eating, internalization of the thin ideal and body dissatisfaction amongst eating disorders (EDs) and controls and to see whether the risk factor model differed across various European countries and ED subtypes.The total sample comprised 1373 participants(ED patients=618).The Cross-Cultural Questionnaire (CCQ) was used to assess the above-mentioned risk factors.SEM analyses showed that the best fitting model was one allowing risk paths to vary across countries . In all countries teasing about weight/shape or eating was associated with body dissatisfaction.There was a strong significant path from body dissatisfaction to ED.Teasing about weight/shape or eating also directly predicted EDs .There was however a weak effect of parenting on both teasing about weight/shape or eating and EDs directly.Risk models slightly varied across ED diagnoses.Our hypothesised model was partially confirmed; in particular the central role of teasing on EDs both directly and mediated by internalization of the thin ideal and body dissatisfaction was shown across five European countries.Conversely, the effect of parenting varied by country and therefore might have cross-cultural effects.Prevention & Public Health stream of the 2014 ANZAED Conference.This abstract was presented in the


A hallmark feature of the neural system controlling breathing is its ability to exhibit plasticity. Less appreciated is the ability to exhibit metaplasticity, a change in the capacity to express plasticity . Recent advances in our understanding of cellular mechanisms giving rise to respiratory motor plasticity lay the groundwork for (ongoing) investigations of metaplasticity. This detailed understanding of respiratory metaplasticity will be essential as we harness metaplasticity to restore breathing capacity in clinical disorders that compromise breathing, such as cervical spinal injury, motor neuron disease and other neuromuscular diseases. In this brief review, we discuss key examples of metaplasticity in respiratory motor control, and our current understanding of mechanisms giving rise to spinal plasticity and metaplasticity in phrenic motor output; particularly after pre-conditioning with intermittent hypoxia. Progress in this area has led to the realization that similar mechanisms are operative in other spinal motor networks, including those governing limb movement. Further, these mechanisms can be harnessed to restore respiratory and non-respiratory motor function after spinal injury. As with most neural systems, a hallmark of the neural system controlling breathing is its ability to express plasticity; defined as a persistent (>60 min) change in neural network function after an experience/stimulus has ended  after the stimulus is removed. Neuromodulators often work through metabotropic G protein coupled receptors, which alter cell excitability through covalent modifications of membrane channels. Modulation does confer system flexibility and can initiate cellular mechanisms resulting in plasticity  is augmented by concurrent application of another (substance P).Plasticity (long-term) is a persistent (>60 min) change in function that outlasts the initiating stimulus , a long-lasting increase in phrenic motor output observed following acute intermittent hypoxia  but otherwise the same AIH protocol  in humans (Lee et al., per se (Ling et al., Enhanced pLTF could occur and/or be modified at any site in the neural network transmitting chemosensory activity to PMN; including chemoreceptors themselves, their afferent terminations in the nucleus of the solitary tract, neurons of the ventral respiratory column, raphe neurons, spinal phrenic interneurons and/or PMN Figure . There iAlthough relatively little is known concerning sites and signaling mechanisms of CIH metaplasticity, CIH-enhanced moderate AIH-induced pLTF is still serotonin-dependent (Ling et al., Because CIH elicits respiratory metaplasticity, we initially had contemplated CIH as a treatment option to restore respiratory control in clinical disorders that impair breathing capacity; such as cervical spinal injury (Fuller et al., Less intensive rAIH preconditioning protocols retain the ability to elicit plasticity and metaplasticity without the known pathogenic effects of CIH (Lovett-Barr et al., AIH-induced pLTF is modified by a number of other pre-treatments that enhance and diminish its expression (Mitchell et al., Of these, the best characterized is enhanced pLTF following CDR. Twenty-eight days following bilateral CDR, moderate AIH induced pLTF is doubled, with an equivalent increase in serotonin terminal density in the immediate vicinity of identified PMN (Kinkead et al., One week post-CDR, ventral spinal BDNF and neurotrophin-3 concentrations are increased (Johnson et al., Although considerable effort has been devoted to investigations of detailed cellular mechanisms giving rise to AIH-induced pLTF (Devinney et al., With our significant progress in understanding cellular mechanisms of AIH-induced pLTF, it is an advantageous model to study metaplasticity in respiratory motor control. Such studies are warranted from a basic science perspective, but also because rAIH is rapidly moving towards clinical application as a treatment for respiratory insufficiency in disorders that compromise breathing capacity (Lovett-Barr 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.


In plant leaves, resource use follows a trade-off between rapid resource capture and conservative storage. This “worldwide leaf economics spectrum” consists of a suite of intercorrelated leaf traits, among which leaf mass per area, LMA, is one of the most fundamental as it indicates the cost of leaf construction and light-interception borne by plants. We conducted a broad-scale analysis of the evolutionary history of LMA across a large dataset of 5401 vascular plant species. The phylogenetic signal in LMA displayed low but significant conservatism, that is, leaf economics tended to be more similar among close relatives than expected by chance alone. Models of trait evolution indicated that LMA evolved under weak stabilizing selection. Moreover, results suggest that different optimal phenotypes evolved among large clades within which extremes tended to be selected against. Conservatism in LMA was strongly related to growth form, as were selection intensity and phenotypic evolutionary rates: woody plants showed higher conservatism in relation to stronger stabilizing selection and lower evolutionary rates compared to herbaceous taxa. The evolutionary history of LMA thus paints different evolutionary trajectories of vascular plant species across clades, revealing the coordination of leaf trait evolution with growth forms in response to varying selection regimes. Although leaf morphology has evolved manifold variations across vascular plant species, there is strong evidence of a universal spectrum constraining leaf functioning from rapid resource capture to efficient resource use  shaping trait evolution within lineages  from 180 published and unpublished studies and electronic databases . Second, node ages were estimated using the bladj module of the Phylocom software  + σ2dt, where the parameter α controls the rate of adaptive evolution to the optimum  to test for the existence of phylogenetic structure in the trait data. The PI model ignores phylogenetic relatedness across species as if they were placed at equal distance on a star phylogeny. It serves as a null hypothesis which supposes the absence of phylogenetic covariance in the trait distribution.λ , while a value of λ = 1 indicates that the phylogenetic pattern conforms to Brownian motion on the given phylogeny  to −1, indicating strong negative association. It has an expected value of n is the number of tips, under the null hypothesis of no correlation between trait values and phylogenetic distance. We also calculated I within different classes of divergence time  which were chosen to ensure a sufficient number of observations within each class. This was used to build a resulting phylogenetic correlogram, which represents how trait similarity among species varies with the time since their divergence. In theory, this temporal pattern allows one to discriminate the BM from the OU models : Pagel's λ Pagel , BlomberDW, which measures the degree of divergence (or trait radiation) between the child clades of each node. An ancestral node was considered as diversifying when the corresponding DW value was higher than expected at random, and as conservative when DW was lower than expected at random. Significant divergence widths were detected by testing the statistic against the null hypothesis PI using permutations (n = 105) and correcting for multiple comparisons , indicate past diversifying events (trait radiation) followed by subsequent conservatism within daughter clades. On the other hand, significantly conservative nodes correspond to divergences of low amplitude between daughter clades thus displaying similar trait values and phylogenetic conservatism thereafter.We analyzed the phylogenetic signal at clade level using the analysis of traits procedure . This analysis can detect functionally diversifying and conservative ancestral splits in trait values across daughter clades  to 1479.1 g.m−2 in Hakea leucoptera (Proteaceae). LMA ranges largely overlapped across the major clades in Tracheophyta, but on average, Lycopodiophyta and Monilophyta (ferns) exhibited the lowest LMA, while Gymnospermae showed the highest LMA compared to the other major clades . Of these 62 nodes, 12 corresponded to diversifying nodes with trait radiation , was investigated across a large set of vascular plant species. Using comparative phylogenetic methods and a global trait database of over 5000 species, we detected that leaf strategies were overall weakly, albeit significantly, conserved during the diversification of vascular plants. This low conservatism appeared to be associated with multiple regimes of stabilizing selection, that is differing adaptive optima across the major clades. Importantly, we highlight the strong interaction between the evolution of growth forms and of leaf strategies, with woody species displaying slower leaf diversification and higher conservatism in LMA than herbaceous plant species.Spermatophytae) on the other, and between Angiospermae and Gymnospermae (Fig. Angiospermae clade, diversity in LMA reflects the wide adaptive radiation of flowering plants into a range of ecological strategies, involving diverse growth forms and ecological niches both within and across habitats (Ricklefs and Renner, Angiospermae. In fossil floras, basal plant clades display a higher diversity of growth forms than is currently observed among extant species: treelike plants existed within Lycopodiophyta, as well as ruderal herbaceous taxa within gymnosperms (Rothwell et al. Leaf mass per area appeared significantly but weakly conserved. This low phylogenetic signal captures the fact that distantly related species may occupy similar positions along the leaf economics spectrum. The large variability found within clades here evidenced a pattern of functional convergence across clades over a broad phylogenetic scale. However, significant levels of trait conservatism imply that, to a certain extent, closely related species do tend to invest dry matter similarly within leaf tissues and hence occupy nearby positions along the leaf economics spectrum. In particular, evolutionary splits identified as conservative nodes were mostly found at the level of genera. As might be expected, the degree of similarity in resource-use strategies varied with the length of the period of common evolution along lineages (Hansen and Martins, mae Fig. . Within in natura set constraints on plant strategies that may directly or indirectly affect achievable LMA values. A recent study of the evolution of the leaf economics spectrum highlighted the nature of evolutionary forces on LMA (Donovan et al. Models of continuous trait evolution supported the hypothesis that stabilizing selection shaped the phylogenetic pattern in LMA, against the hypothesis of evolution by drift alone. Given the breadth of sampling in our dataset, over a large taxonomic scale and a range of ecological situations, the stabilizing selection we detected likely reflects selection against extreme phenotypes that infringe structural and physiological limitations to LMA. The phylogenetic patterns evidenced here showed simultaneously conservatism, mostly within genera, and convergence across large clades. Congruence between radiation and restrictive environmental conditions, such as within the Proteales, also suggests strong filtering effects that may lead to conservatism at larger phylogenetic scales and functional distinctiveness (Cornwell et al. Recent analyses of molecular sequences have shown that woody species have evolved more slowly than herbaceous species (Smith and Donoghue, The observed patterns in the evolution of LMA raise the issue of selective pressures resulting in different selection regimes at large evolutionary scales. Fossil evidence suggests that large spatiotemporal scale changes in climate and disturbance regime were partially responsible for the diversification of vascular plants Stebbins  and thisBy exploring the evolutionary history of LMA we have revealed different evolutionary trajectories involving coordination and trade-offs between leaf traits and plant traits related to growth form. As a consequence, different optimal phenotypes of resource-use strategies have evolved in vascular plants setting fundamental limits on the structure and physiology of organisms and therefore on ecological processes within communities. Continuing advances in genomics (Leebens-Mack et al.


The intestinal epithelia rely on resident stem cells to undergo homeostatic growth as well as tissue repair upon injury. In response to insults elicited by pathological bacterial infection or tissue damaging chemicals such as dextran sodium sulfate (DSS), intestinal stem cells (ISCs) speed up their proliferation and differentiation to effectively replenish lost cells. Despite numerous studies, the regulatory mechanisms underlying the control of stem cell activity during homeostasis and regeneration still remain poorly understood.Drosophila adult midgut provides a powerful system to address these problems because sophisticated genetic tools are available to allow the manipulation of gene function in any cell types within the intestinal epithelia as well as in surrounding tissues. Our recent study, which was published in The Journal of Cell Biology, established a critical role of Hh signaling in the regulation of regenerative stem cell proliferation in Drosophila adult midguts [The Hedgehog (Hh) pathway governs animal development in species ranging from fruit fly to human. In additional to their role as morphogen to control cell differentiation and pattern formation in embryonic development, the secreted Hh glycoproteins also function as mitogen to regulate cell proliferation in adult tissue homeostasis and tumorigenesis . Hh sign midguts .Drosophila midguts constantly turn over and are replaced by new cells derived from ISCs localized at the base of the epithelia. ISCs divide to produce themselves and enteroblasts (EBs) that subsequently differentiate into more specialized cell types. Previous studies revealed that ISC proliferation was elevated upon intestinal injury caused by DSS feeding [Similar to mammalian intestines,  feeding . Here we feeding .In searching for the effectors that mediate the Hh function in midguts, we found that Hh signaling in EBs upregulated several cytokines and mitogens, most notably, the JAK-STAT pathway ligand Upd2. Indeed, inaction of Upd2 in EBs blocked ISC proliferation induced by either DSS feeding or ectopic Hh signaling. Taken together, our study suggests that DSS stimulates Hh signaling in EBs where it increases the production of Upd2, which in turn activates the JAK-STAT pathway in stem cells to fuel their proliferation. Interestingly, a recent study revealed a similar Hh-JAK-STAT signaling axis that drives tumorigenesis in mouse skin , suggestupd2 or indirectly through an intermediate pathway. In this regard, it is interesting to note that the Hippo (Hpo) pathway is also involved in DSS-stimulated ISC proliferation [Drosophila midguts and perhaps also in other settings.How Hh production is upregulated upon injury remains poorly understood but our study revealed that DSS increased Hh expression in part through the JNK pathway although the precise mechanism remains to be further explored. Another unresolved issue is how Hh signaling regulates the production of Upd2. Hh could directly regulate the transcription of feration , raising


Journal of Experimental Botany (pages 2573–2586), Rebetzke et al. take the debate forward, showing that wheat awns are associated with specific grain traits in different environments.Awns, which are derived from floral structures in grasses, are known to be critically important for photosynthesis and transpiration. However, arguments about the costs and benefits of their growth and development for crop grain yield are still ongoing. In this issue of Triticum dicoccoides (Körn. ex Asch. & Graebner) Schweinf.], the awns flex as humidity levels change, which can help bury the seeds (T. durum and T. aestivum) have shorter or non-existent awns to facilitate grain harvesting, handling and storage . Therefore, raising grain yield is still the main goal for wheat breeding, particularly as recent decades have seen a reduced rate of increase in wheat yields globally. Increasing assimilate partitioning into wheat’s grain-bearing structures, the spikes, is an important strategy for improving yield. The introduction of dwarfing Reduced height (Rht) genes greatly enhanced the partitioning of assimilates towards spikes . HoweverAwns can be considered an alternative target for the improvement of wheat grain yield through their known functions, including photosynthesis, carbohydrate storage and increased water-use efficiency  four spring wheat populations with a range of diverse genetic backgrounds were developed containing multiple Near-Isogenic Line (NIL) pairs with variation for the presence and absence of full awns; (2) four separate sets of experiments representing a total of 25 environments  were conducted depending on the germplasm and environments sampled; and (3) for comparisons a number of important agronomic traits were measured in multiple years and environments while some traits were related to spike morphology. Separate glasshouse studies were also undertaken.Manipulating source–sink relations is an important strategy for wheat breeding. Spike morphology is a crucial phenotype for displaying the distribution of assimilates. Spike morphology-related traits  demonstrate great variation under variable growth conditions. Therefore, environmentally induced plasticity of spike morphology-related traits can in principle show the redistribution of assimilates.The influence of wheat awns on grain yield can be summarized as a balance between the costs of awn setting and the benefits of awn functions. As hypothesized by Flowering time is commonly considered to be an important determinant for grain yield.


High-density lipoprotein (HDL) and its principal apolipoprotein A-I (ApoA-I) have now been convincingly shown to influence glucose metabolism through multiple mechanisms. The key clinically relevant observations are that both acute HDL elevation via short-term reconstituted HDL (rHDL) infusion and chronically raising HDL via a cholesteryl ester transfer protein (CETP) inhibitor reduce blood glucose in individuals with type 2 diabetes mellitus (T2DM). HDL may mediate effects on glucose metabolism through actions in multiple organs  by three distinct mechanisms: (i) Insulin secretion from pancreatic beta cells, (ii) Insulin-independent glucose uptake, (iii) Insulin sensitivity. The molecular mechanisms appear to involve both direct HDL signaling actions as well as effects secondary to lipid removal from cells. The implications of glucoregulatory mechanisms linked to HDL extend from glycemic control to potential anti-ischemic actions via increased tissue glucose uptake and utilization. Such effects not only have implications for the prevention and management of diabetes, but also for ischemic vascular diseases including angina pectoris, intermittent claudication, cerebral ischemia and even some forms of dementia. This review will discuss the growing evidence for a role of HDL in glucose metabolism and outline related potential for HDL therapies. There is accumulating experimental and clinical evidence that HDL particles can control glucose metabolism via a variety of mechanisms . At a meThe action of HDL to move glucose from the circulation and into tissues has potential clinical relevance in terms of both reducing vascular complications by removing excess glucose from the circulation in the setting of T2DM, and also in providing adequate glucose to tissues for energy production, particularly in the context of ischemia. HDL therapeutics may therefore have application not only for the prevention and management of T2DM , but alsThis review will discuss the growing evidence for a role of HDL in glucose metabolism in the context of the evolving HDL hypothesis. It is now recognized that the main clinical variable used to assess HDL, HDL cholesterol, fails to provide an accurate representation of the multiple functions of heterogeneous HDL particles, which are composed of many hundreds of lipids and multiple proteins . The rolT2DM, insulin resistance and glucose intolerance are associated with low HDL cholesterol levels . While tOn the other hand, a recent Mendelian randomization study showed that genetically reduced HDL cholesterol does not associate with increased risk of T2DM . While tHigh-density lipoprotein has been implicated in the modulation of insulin secretion in cellular and animal studies with corroborating evidence from human intervention trials. The diverse functionality of HDL to combat cellular lipid accumulation, endoplasmic reticulum (ER) stress and apoptosis are three potential mechanisms that may preserve pancreatic beta cell function.Pancreatic lipid accumulation and lipotoxicity have been well-documented to inhibit insulin production and secretion . We haveAt the signaling level the HDL transporters ATP-binding cassette, sub-family A, member 1 (ABCA1) and ATP-binding cassette, sub-family G, member 1 (ABCG1) have both been implicated in HDL-mediated effects on insulin secretion. Beta cell-specific deletion of the ABCA1  and ABCGHDL may also influence insulin secretion via mechanisms other than cholesterol depletion, including via actions on insulin transcription . At doseThe ability of HDL to inhibit ER stress-induced beta cell apoptosis could be another important mechanism by which HDL may ameliorate beta cell dysfunction . Bioacti2+ influx and activation of calcium/calmodulin activated protein kinase kinase (CaMKK)  . Subsequ (CaMKK)  and faciin vivo effects of HDL on glucose disposal in the absence of a change in circulating insulin. Such effects may be mistakenly attributed to increased insulin sensitivity.Beyond glucose uptake, HDL has been shown to activate Akt in ApoA-I transgenic mice and directly enhance both glycolysis and glucose oxidation in mouse C2C12 muscle cells . These iTissue lipid deposition and chronic inflammation associated with obesity, metabolic syndrome and T2DM establishes a functional deficit in skeletal muscle and the heart, impairing how these metabolically active tissues respond to insulin. The insulin resistance associated with lipid accumulation and a pro-inflammatory milieu promotes lipolysis, increasing circulating free fatty acids, thus perpetuating dyslipidemia and insulin resistance . It is sin vivo is more complex due to the fact that increased glucose disposal relative to insulin may not necessarily reflect increased insulin sensitivity. As discussed in the previous section, such observations may actually be explained by direct actions of HDL on glucose disposal by mechanisms which do not involve insulin. Nevertheless, there are two recent in vivo studies consistent with the cellular studies, which suggest that HDL/ApoA-I can increase peripheral insulin sensitivity. The first of these employed intraperitoneal glucose and insulin tolerance tests in high fat diet fed mice treated for 2–4 weeks with lipid-free ApoA-I (8 mg/kg body weight) and showed improved glucose and insulin tolerance compared to untreated mice on the same high fat diet  testing or hyperinsulinemic-euglycemic clamp studies to examine direct effects on insulin sensitivity. Definitive evidence for an effect of HDL on insulin sensitivity in humans is also lacking. However, HDL cholesterol elevation by CETP inhibition has been shown to improve glycemic control in the face of reduced plasma insulin in people with T2DM in the ILLUMINATE trial . As evidThe actions of HDL on glucose metabolism have potential benefit beyond vascular protection. The ability of HDL to stimulate tissue glucose uptake has implications for cellular metabolism in the brain, as well as for cell viability in other organs under ischemic/anoxic conditions where glucose becomes the predominant metabolic substrate. This latter mechanism may have particular relevance to ischemic vascular conditions, including coronary, cerebral and peripheral artery diseases.We have recently established that HDL increases insulin-independent glucose uptake in rat cardiomyocytes via an Akt signaling pathway . In thisInsulin resistance may directly contribute to the increased risk of ischemic heart disease and poorer outcomes after infarction in patients with T2DM . It is pThe emerging actions of HDL and its bioactive components on glucose metabolism in the myocardium may contribute to the demonstrated protective effects of HDL in the context of ischemia-reperfusion injury . Such acFor the same reasons that HDL-mediated effects on glucose metabolism may protect the ischemic myocardium, these actions may equally act in the brain to protect against transient ischemic attacks and stroke. In fact, the reliance of the brain on glucose for ATP production means that mechanisms controlling glucose uptake may be of even greater significance than in other organs. Even minor changes in cerebral blood flow have the potential to impact on neuronal viability via reduction in glucose supply. Indeed cerebral hypoperfusion and hypometabolism coincide in patients with dementia .The implications of reduced blood flow, and thus glucose supply to the brain, extend beyond acute stroke risk to Alzheimer’s disease, which has a significant vascular component . In thisGiven the well-established positive actions of HDL on vascular function, it is not surprising that several co-morbidities associated with increased Alzheimer’s disease risk are themselves associated with decreased HDL function . A role in vitro efficacy of ApoA-I at similar concentrations. More recently, in vitro primary cell culture experiments, suggest that plasma ApoA-I gains access to the CNS primarily by crossing the blood-CSF barrier via specific cellular mediated transport  barrier . ApoA-I ransport . The bloransport , which mThere is now convincing evidence that HDL modulates glucose metabolism in multiple tissues. These actions have deepened our understanding of the pathophysiology of a variety of disease states associated with low or dysfunctional HDL. While there are still many unanswered questions relating to the underlying mechanisms and key HDL component(s) responsible for the metabolic effects, this opens up the possibility of targeting glucose metabolism with HDL therapeutics currently in development. Future preclinical investigations and clinical trials will determine the relevance of HDL-mediated modulation of glucose metabolism to both glycemic control as well as tissue glucose supply to vital organs including the heart and the brain, especially under ischemic conditions.AS, SH, BK drafted, critically revised and approved the final version of this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. BK has received product from CSL Behring and patient plasma from Hoffman La Roche both for investigator initiated clinical trials, with no associated research funding. She has also partnered with Resverlogix to fund an investigator-initiated clinical trial.


Plasmodium falciparum-infected children with cerebral malaria (CM) die from respiratory arrest, but the underlying pathology is unclear. Here we present a model in which the ultimate cause of death from CM is severe intracranial hypertension. Dynamic imaging of mice infected with P. berghei ANKA, an accepted model for experimental CM, revealed that leukocyte adhesion impairs the venous blood flow by reducing the functional lumen of postcapillary venules (PCV). The resulting increase in intracranial pressure (ICP) exacerbates cerebral edema formation, a hallmark of both murine and pediatric CM. We propose that two entirely different pathogenetic mechanisms—cytoadherence of P. falciparum-infected erythrocytes in pediatric CM and leukocyte arrest in murine CM—result in the same pathological outcome: a severe increase in ICP leading to brainstem herniation and death from respiratory arrest. The intracranial hypertension (IH) model unifies previous hypotheses, applies to human and experimental CM alike, eliminates the need to explain any selective recognition mechanism Plasmodium might use to target multiple sensitive sites in the brain, and explains how an intravascular parasite can cause so much neuronal dysfunction.Most  P. falciparum causes injury to a sensitive location such as the brainstem, where a small lesion could have fatal consequences  infected mice, an accepted model for experimental cerebral malaria (ECM), in the context of recent advances in the understanding of the pathogenesis of human cerebral malaria (HCM). For the purpose of this Perspective, we focus on pediatric HCM, because African children with HCM tend to exhibit more BBB dysfunction, monocyte and platelet accumulation in the brain, and intracranial hypertension (IH) compared to adult HCM patients in Southeast Asia  patients die from respiratory arrest  cause death is not completely understood. Several models have been proposed over the decades to explain the pathogenesis of HCM  expressing iRBC that bind to the endothelial protein C receptor (EPCR)  ExtP. yoelii XL (PyXL)-infected mice, which develop hyperparasitemia without neurological signs , we found more platelets, CD8+ T cells, neutrophils, and macrophages accumulated in postcapillary venules (PCV) from PbA-infected mice with ECM compared to Arteriolar vasospasms during ECM  with poor outcomes in pediatric HCM patients in Kenya  Ute Frevert, the review process was handled objectively. 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.


Foot ulceration is one of the most significant complications of diabetes, and will affect 15-20% of people with diabetes at some point in their lives. Such ulcers frequently become infected with very serious sequelae which can often lead to amputation making diabetes is the most common cause of lower extremity amputations. Infections cause increased morbidity  which means that they represent significant clinical events, requiring immediate attention in relation to local and systemic complications thus requiring well-coordinated management. Unfortunately diabetic foot infections (DFI) frequently fail to display overt signs and symptoms of infection including purulence, erythema, pain, tenderness, warmth and induration. This makes it difficult for clinicians to detect infection, and to make timely interventions to limit the highly undesirable consequences of DFIs. Alternative means of rapidly diagnosing infection are urgently required.To determine if the presence/absence of microorganisms, and ultimately the presence of infection, are affected by diabetic foot ulcer (DFU) wound fluid pH.DFUs of patients (n=55) were assessed in terms of presence/absence of clinical signs of infection as part of their routine clinical appointment at a High-Risk Foot Clinic. Wound fluid samples were also collected from the DFUs by filter paper absorption and/or pipette aspiration. The pH of samples was determined using a micro-electrode pH meter. Bacteria in the wound fluid were recovered by 24hr incubation in Tryptone Soya Broth, and plating on selective agars which included; MacConkey Agar , Pseudomonas Agar Base (Pseudomonas spp). Chromocult Agar , Baird Parker Agar (Staphylococcus spp) and Columbia Blood Agar (Streptococcus spp). Organisms identified as Staphylococcus Aureus cultured on Muller Hinton Agar and and MRSA present detected using Oxacillin and Cefoxitin antibiotic disks.Sample pH values ranged from 6.2 to 8.5. Recovered bacteria included Pseudomonas, Enterobacter, Staphylococcus and Streptococcus spp. Correlations were observed between DFU fluid pH values, the presence/absence of these species and the presence/absence of clinical signs of infection. This presentation will discuss the potential clinical implications of these findings.pH conditions within DFUs influence bacterial presence/absence in these wounds. pH conditions also influence the presence/absence of clinical signs of infection. Timely monitoring of DFU fluid pH could enhance clinicians’ abilities to rapidly detect and more effectively manage DFU infections. An improved understanding of the interactions between DFU pH and bacterial metabolism may identify ways to limit the duration and wider impact of DFU infections.Governance compliance statement - Ethical approval and research governance has been granted for the clinical study to take place and all research governance procedures are being adhered to during the completion of the study.


Cerebral collateral circulation is a subsidiary vascular network, which is dynamically recruited after arterial occlusion, and represents a powerful determinant of ischemic stroke outcome. Although several methods may be used for assessing cerebral collaterals in the acute phase of ischemic stroke in humans and rodents, they are generally underutilized. Experimental stroke models may play a unique role in understanding the adaptive response of cerebral collaterals during ischemia and their potential for therapeutic modulation. The systematic assessment of collateral perfusion in experimental stroke models may be used as a “stratification factor” in multiple regression analysis of neuroprotection studies, in order to control the within-group variability. Exploring the modulatory mechanisms of cerebral collaterals in stroke models may promote the translational development of therapeutic strategies for increasing collateral flow and directly compare them in term of efficacy, safety and feasibility. Collateral therapeutics may have a role in the hyperacute  phase of ischemic stroke, prior to recanalization therapies. Cerebral collateral circulation is a subsidiary vascular network which is dynamically recruited after arterial occlusion and may provide residual blood flow to ischemic areas. Cerebral collateral flow during acute ischemic stroke is highly variable among different individuals and is emerging as a strong prognostic factor either in unselected stroke patients and in patients treated with intravenous rtPA or endovascular recanalization therapy . ExperimHere, we review the current methods for assessing cerebral collaterals during acute ischemic stroke and the most promising collateral therapeutic strategies, focusing on experimental stroke models.Many similarities, with some notable differences, exist between humans and rodents in term of cerebral collateral circulation. The circle of Willis includes the anterior communicating artery in humans, while this vessel is totally absent in rodents, whose proximal segments of anterior cerebral arteries (ACA) converge to form one single median artery called Azigos ACA. In case of occlusion of cervical arteries, the circle of Willis represents a compensatory system to rapidly redistributing blood flow in both species. In rodents, the pterygopalatine artery originates from the proximal internal carotid artery (ICA) and provide extracranial collateral connections between external carotid artery and ICA via many arterial branches to facial, orbital and meningeal districts. In both humans and rodents, each cerebral artery provides blood flow to its vascular territory ramifying along the cortical surface to form a pial arteriolar network, creating anastomotic connections among different vascular territories, known as leptomeningeal anastomoses (LMAs). LMAs are mostly developed between cortical branches of middle cerebral artery (MCA) and ACA or posterior cerebral artery. In case of proximal occlusion of a cerebral artery, dynamic blood flow diversion through these anastomoses may provide residual (retrograde) blood flow to the cortical surface of the occluded artery territory, distally from the occlusion.The anatomy of cerebral collaterals in acute stroke patients can be assessed using conventional digital subtraction angiography (DSA), CT angiography (CTA) or MR angiography (MRA), while their functional performance can be studied through tissue perfusion evaluation, via CT and MR perfusion techniques (PCT and PWI). At present, there is no agreement in clinical practice on which imaging should be performed, when after stroke and which patients would benefit most from cerebral collateral imaging. DSA is the gold standard for evaluating the recruitment of cerebral collaterals, but it is invasive and usually reserved for patients selected for endovascular procedures. CTA is able to provide direct visualization of collateral flow after arterial occlusion Fig.  3]. How. How3]. In experimental stroke models, both the site and the duration of arterial occlusion are controlled. Continuous or repeated assessment of cerebral collateral flow could be performed, including pre-stroke assessment. For these reasons, preclinical research could play a crucial role for a deeper understanding of collateral response during cerebral ischemia and promote the translational development of collateral-based therapies. However, both cerebrovascular differences between different species and strains and inter-individual variability need to be meticulously considered to achieve effective results in this field .Although some techniques used in stroke patients, such as DSA or MRI, could be used in stroke models for assessing cerebral collateral flow –14, signLaser speckle contrast imaging (LSCI)  providesIn contrast to LSCI, two photon laser scanning microscopy (TPLSM) is an optical technique providing quantitative measure of blood flow velocity and direction in single vessels, with depth resolution up to 1 mm. Single arterioles, venules and capillaries of both surface and subsurface vasculature are resolved after intravenous injection of dextran conjugated with a fluorescent dye. A cranial window is required and scanning procedure is time-consuming. Collateral response after occlusion of both pial and penetrating arterioles in rats  were stu3 volume. Real-time relative cortical CBF values are obtained, while absolute CBF quantification cannot be achieved [Laser-Doppler flowmetry (LDF) is a well-established technique for tissue perfusion monitoring and is recommended to confirm successful occlusion and exclude subarachnoid hemorrhage in experimental ischemic stroke . Opticalachieved . Our groachieved . A custoThe use of any of these methods (or a combination of them) to monitor arterial occlusion and collateral perfusion cannot be over emphasized to improve accuracy of pre-clinical stroke research. Advantages and drawbacks, in terms of temporal and spatial resolution, invasiveness and affordability of each technique are shown in Table Despite over 1000 putative neuroprotective agents obtained promising results in experimental stroke models , no succA well-recognized limitation of preclinical stroke models is outcome variability , particuThe variability of cerebral hemodynamics during ischemia has been largely neglected in preclinical research, as well as the influence of drugs on CBF . MonitorAnimal stratification by collateral flow during MCAO represents a promising tool to adjust for outcome variability in experimental stroke studies. Using cerebral collateral flow during MCAO as a covariate in multiple regression analysis may represent a simple method to stratify animals in term of pre-treatment perfusion deficit, reducing the within group variability and improving efficacy analysis in preclinical neuroprotection studies. Further studies are needed to determine the more suitable method, timing and statistical tool for collateral flow assessment in pre-clinical neuroprotection trials.Intravenous thrombolysis with rtPA (Alteplase) within 4.5 h from symptom onset (for any vessel occlusion) and endovascular thrombectomy within 6 h from symptom onset (for large vessel occlusion) are currently the best therapeutic options for acute ischemic stroke , 39. UnfIncreasing systemic blood pressure represents a first strategy. Phenylephrine, a selective α1-adrenergic receptor agonist, causes systemic vasoconstriction with very limited effects on cerebral vessels. A 30 % augmentation of blood pressure obtained through phenylephrine infusion after distal MCAO induction in mice enhanced cortical CBF both in core and penumbra . In smalIncreasing intravascular volume may represents a second strategy. Cerebral blood volume augmentation by plasma expansion and haemodiluition could improve cerebral perfusion in experimental stroke models . HoweverInduction of selective cerebral vasodilation is a third strategy. Nitric oxide (NO) is a strong endogenous vasodilator with therapeutic potential for ischemic stroke . NO inha2 levels, causing pial arteriolar vasodilation and increased cortical perfusion in piglets [Selective cerebral arteriolar vasodilation could be obtained using acetazolamide, which inhibits carbonic anhydrase and consequently augments CO piglets . In clin piglets . Quite s piglets , was perCerebral flow diversion is a fourth strategy. Gravitational influences of head positioning after acute vascular occlusion may affect pressure gradients in cerebral circulation, which enhancement may promote leptomeningeal recruitment. Augmentation of cerebral perfusion and increased MCA blood flow velocity has been reported in stroke patients after flat head positioning , 61 and A limited number of clinical and preclinical stroke studies focused on cerebral collateral circulation. Generally, neuroprotective effects are being sought, whereas the contribution of collateral blood flow is rarely considered or just inferred. Preclinical stroke research has the potential to directly study the adaptive capacity and modulatory mechanisms of cerebral collateral flow during focal cerebral ischemia, using different methods and in different experimental conditions. These preclinical efforts are likely to be worthwhile and may produce useful translational concepts and direct comparisons of the different strategies to enhance cerebral collateral flow, including some therapeutic approaches which did not prove successful in past clinical trials conducted in the pre-thrombolysis era.


HEart and BRain interfaces in Acute ischemic Stroke (HEBRAS) study aims to assess whether an enhanced MRI set-up and a prolonged Holter-ECG monitoring yields a higher rate of pathologic findings as compared to diagnostic procedures recommended by guidelines .An effective diagnostic work-up in hospitalized patients with acute ischemic stroke is vital to optimize secondary stroke prevention. The Prospective observational single-center study in 475 patients with acute ischemic stroke and without known atrial fibrillation. Patients will receive routine diagnostic care in hospital as wells as brain MRI, cardiac MRI, MR angiography of the brain-supplying arteries and Holter-monitoring for up to 10 days. Study patients will be followed up for cardiovascular outcomes at 3 and 12 months after enrolment.By comparing the results of routine diagnostic care to the study-specific MRI/ECG approach, the primary outcome of HEBRAS is the proportion of stroke patients with pathologic diagnostic findings. Predefined secondary outcomes are the association of stroke localization, autonomic dysbalance and cardiac dysfunction as well as the effect of impaired heart-rate-variability on long-term clinical outcome.The investigator-initiated HEBRAS study will assess whether an enhanced MRI approach and a prolonged ECG monitoring yield a higher rate of pathological findings than current standard diagnostic care to determine stroke etiology. These findings might influence current diagnostic recommendations after acute ischemic stroke. Moreover, HEBRAS will determine the extent and clinical impact of stroke-induced cardiac damage.NCT02142413.Clinicaltrials.gov  Variations and delays in the diagnostic procedures during hospitalization after acute ischemic stroke are common –3. At thRecent studies have shown that magnetic resonance imaging (MRI) and MR angiography are able to detect cardiac , 6, aortHEart and BRain interfaces in Acute ischemic Stroke (HEBRAS) study therefore aims to assess the detection rate of pathologic findings relevant to stroke etiology as obtained by an enhanced MRI set-up  and a prolonged, non-invasive, commonly available Holter monitoring (of up to 10 days duration) in comparison to findings obtained by routine diagnostic procedures after acute stroke. In addition, the underlying pathophysiological mechanisms and the prognostic impact of stroke-induced cardiac damage are still poorly understood  or the Find-AFRANDOMISED trial [Besides feasibility and diagnostic value of enhanced MRI, HEBRAS will assess the ability of extended systematic analysis of ECG-recording to improve AF detection compared to the current clinical standard, as similarly done in currently ongoing trials such as the ED trial , as wellED trial , 29. DetED trial . A fasteAnother unique feature of the investigator-initiated HEBRAS study is the systematic assessment of stroke-induced autonomic dysfunction and cardiac damage by using a laboratory data. It has long been recognized that acute cerebrovascular events may coincide with or trigger cardiac dysfunction and elevation of cardiac biomarkers such as troponin. Several studies reported on elevation of cTn in patients with acute ischemic stroke –32. HoweIn addition, natriuretic peptides  have been shown to be independently associated with atrial fibrillation in stroke cohorts –36, but In summary, the HEBRAS study aims to clarify whether recent technical advantages can improve etiologic work up after acute ischemic stroke. This study has the potential to set essential modifications of current diagnostic standards after acute ischemic stroke, aiming at improving secondary stroke prevention. Moreover, the HEBRAS study will hopefully elucidate mechanisms of stroke-induced autonomic dysfunction and cardiac damage.


Autism spectrum disorders (ASD) are characterized by two seemingly unrelated symptom domains—deficits in social interactions and restrictive, repetitive patterns of behavioral output. Whether the diverse nature of ASD symptomatology represents distributed dysfunction of brain networks or abnormalities within specific neural circuits is unclear. Striatal dysfunction is postulated to underlie the repetitive motor behaviors seen in ASD, and neurological and brain-imaging studies have supported this assumption. However, as our appreciation of striatal function expands to include regulation of behavioral flexibility, motivational state, goal-directed learning, and attention, we consider whether alterations in striatal physiology are a central node mediating a range of autism-associated behaviors, including social and cognitive deficits that are hallmarks of the disease. This review investigates multiple genetic mouse models of ASD to explore whether abnormalities in striatal circuits constitute a common pathophysiological mechanism in the development of autism-related behaviors. Despite the heterogeneity of genetic insult investigated, numerous genetic ASD models display alterations in the structure and function of striatal circuits, as well as abnormal behaviors including repetitive grooming, stereotypic motor routines, deficits in social interaction and decision-making. Comparative analysis in rodents provides a unique opportunity to leverage growing genetic association data to reveal canonical neural circuits whose dysfunction directly contributes to discrete aspects of ASD symptomatology. The description of such circuits could provide both organizing principles for understanding the complex genetic etiology of ASD as well as novel treatment routes. Furthermore, this focus on striatal mechanisms of behavioral regulation may also prove useful for exploring the pathogenesis of other neuropsychiatric diseases, which display overlapping behavioral deficits with ASD. The earliest clinical descriptions of autism highlighted two symptom domains, focusing on social behaviors and the regulation of motor output. Kanner's seminal article “Autistic Disturbances of Affective Contact” carefully described the profound social deficits of his patients, concluding “these children have come into the world with innate inability to form the usual biologically provided affective contact with people…” Kanner, . ShortlyFrom the outset, it is worthwhile considering the utility of exploring nervous system disease pathophysiology from the vantage of neural circuit dysfunction. This approach seeks to uncover alterations in defined, reproducibly interconnected sets of neurons that are responsible for discrete behavioral phenomenon seen in neuropsychiatric diseases such as schizophrenia , which can prevent excessive computational demands on cortical structures  supporting goal-directed behavioral responding, the dorsal lateral striatum  supporting automated behaviors and the nucleus accumbens mediating motivational states and reward processing , together with whole-exome and genome sequencing, has demonstrated a substantial amount of genetic heterogeneity underlying ASD etiology, with some estimates predicting that 300–800 loci will eventually be associated with increased risk for ASD , a key repressor of translation at central synapses, has been a central model for exploring the pathogenesis of mental retardation and accompanying developmental disorders including autism  in the development of ASD-associated behaviors.Through a series of experiments, a unifying mechanism emerged in which oxytocin signaling regulated the release of serotonin in the nucleus accumbens, which subsequently modulated excitatory synaptic strength by acting at presynaptic serotonergic receptors on excitatory afferent fibers Figure . DespiteApplying a similar “circuit dissection” approach, we attempted to explore how ASD-associated mutations could promote development of the restricted and repetitive behaviors so frequently observed in ASD patients  neurons either uniquely or more highly express ASD-associated genes such that loss-of-function is more acutely sensed, or (2) neurons do not express alternative family members of ASD-associated genes that would typically allow for genetic compensation. Using a computational approach that analyzed autism genetic datasets to correlate mutations to known biological networks, it was found that autism-associated mutations are preferentially found in genes whose expression levels are enriched in both populations of striatal medium spiny neuron, as well as cortical inhibitory and projection populations, deep cerebellar nuclei and layer 6 corticothalamic neurons  examining heterozygous mutations, disease-associated point mutations or genetic modifiers, (2) exploration of environmental interactions with mutations (3) physiological analysis of behaviorally relevant circuits through selective optogenetic recruitment and (4) highly quantitative analysis to detect subtle changes in discrete components of complex behaviors. Even with these improvements, it is important to acknowledge the intra-species differences in brain complexity, genomic regulation and behavioral repertoire, which may place limits on the generalizability of rodent-based research findings  and a McCabe Fund Award (University of Pennsylvania).The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Colorectal Carcinoma (CRC) is a heterogeneous disease with different molecular characteristics associated with the sites from which, the tumours originate. Such heterogeneity is compounded by the multitude of genetic and epigenetic variations acting as passengers or drivers of the tumour. Majority of CRC develops via chromosomal instability (CIN) pathway. CIN is often exacerbated by inactivation of the Wnt signalling pathway "master regulator" APC gene, activating mutations of KRAS or BRAF oncogenes, or deletions of the 18q, and 17p chromosomal regions with deleterious effects on the tumour suppressor genes TP53 and DCC. Defective Mismatch Repair (MMR) pathway results in a subtler form of genomic instability, namely Microsatellite Instability (MSI). High levels of MSI (or MSI-H) in sporadic CRC are usually caused by hypermethylation of the MLH1 promoter. In terms of methylation, the CpG island methylator phenotype (CIMP) pathway is the second most common pathway in sporadic CRC. CIMP-positive (CIMPp) CRC tumours are usually associated with the proximal colon of older females. CIMPp CRC tumours have better prognosis if the tumours are also MSI-H. However, CIMPp CRC tumours that are Microsatellite Stable (MSS) have poor clinical outcome. To gain insight into the molecular mechanisms underpinning CRC in Saudi Arabian patients, we profiled the DNA methylation frequency of key genes  in 120 sporadic CRC cases. CRC tumours originating from the rectum, left, and right colons are represented in this cohort. Expression patterns of different proteins playing important role in CRC carcinogenesis also studied by using Immunohistochemistry (IHC) technique and their impact as CRC prognosticators was evaluated.


Arc-indexed odor networks of awake rats. Arc-indexed networks are sparse and distributed, consistent with current views. However Arc provides representations of repeated odors. Arc-indexed repeated odor representations are quite variable. Sparse representations are assumed to be compact and reliable memory codes. Arc suggests this is not necessarily the case. The variability seen is consistent with electrophysiology in awake animals and may reflect top-down cortical modulation of context. Arc-indexing shows that distinct odors share larger than predicted neuron pools. These may be low-threshold neuronal subsets. Learning’s effect on Arc-indexed representations is to increase the stable or overlapping component of rewarded odor representations. This component can decrease for similar odors when their discrimination is rewarded. The learning effects seen are supported by electrophysiology, but mechanisms remain to be elucidated.We first review our understanding of odor representations in rodent olfactory bulb (OB) and anterior piriform cortex (APC). We then consider learning-induced representation changes. Finally we describe the perspective on network representations gained from examining  Arc-indexed odor representations. These data modify our view of odor representations and their modulation by reward. How Arc expression is recruited by odor is also considered.Here we first characterize our understanding of odor representations in the olfactory bulb (OB) and anterior piriform cortex (APC). We next review learning-related modulation of those odor representations. Finally, we discuss data using It has long been recognized that odor representations require across-fiber coding  technique to visualize Arc mRNA and characterize odor representations. Arc, as one of the immediate early genes, offers an important feature for examining representations repeatedly in the same animal  early odor preference learning in rat pup Figure ; and (2)In the rat pup model  vs. untrained rats (5%), with a reduction in the variable neuronal component. The overlap component did not show an absolute increase with training, but became a larger proportion of the representation. Thus, as in the rat pups (Shakhawat et al., Electrophysiology had not revealed learning differences in APC firing patterns for simple discriminations (Chapuis and Wilson, In the mixture discrimination, the two individual odor components were responded to as rewarded odors during behavioral probes, suggesting rats learned the components during mixture training or pattern-completed from partial cues. The degree of overlap between peppermint and vanillin in control rats was ~20% while overlap was ~45% in trained rats, consistent with the two odors becoming representationally highly similar (Shakhawat et al., In the difficult discrimination, the odor representation size was ~3% and did not change with learning (Shakhawat et al., The mechanisms for associating odor and reward, presumably resulting in the increases in stability components seen here, have been examined earlier. In the rat pup, norepinephrine paired with odor can alter behavior and AMPA receptor-mediated connectivity in OB (Cui et al., Arc imaging made possible was the finding of high variability in repeated odor representations and the finding of a high degree of overlap between representations of distinct odors. If cells encoding distinct odors were drawn at random from populations with replacement such that the same cell could participate in multiple representations, then the predicted overlap would be the representational size squared. In Arc studies, representational size varies from 1% to 10%, consistent with other estimates (e.g., Lin da et al., Arc.The two novel results that Arc itself indicates. While it is related to neuronal activation, neural firing cannot be its sole initiator. Mitral cells fire at high spontaneous levels (Rinberg et al., Arc activation. Arc recruitment is likely linked to glutamatergic excitatory synaptic driving (Cole et al., Finally we come to the issue of what Arc recruitment. Odor activation firing patterns in OB and APC are dynamic spatially and temporally (Friedrich and Laurent, Arc represent the initial pattern and its variability or the combined early and later patterns and their cumulative variability? This would be another contribution to the high variability observed.But even if that is the case, one must ask which driving patterns are encoded by Arc patterns, we would predict a larger stable component with increasing concentrations supporting more rapid discriminations.How many neurons are excited in a single synchronized odor representation? The size of odor representations appears to be normalized and varies less for concentration increases Cleland,  than oneArc-indexed cell networks portray odor representations in both OB and APC as sparse and distributed consistent with current understanding. Arc-indexed networks also reveal a considerable variability in the awake mammalian odor representation consistent with the electrophysiological evidence. This methodology further reveals a larger component of common neuronal activation for distinct odors than predicted by theory. Appetitive learning modifies odor representations to increase the proportion of stable neurons. Network representations can also decrease the proportion of stable neurons when increased behavioral discrimination is required.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.


In August 2016, more than 10,000 athletes representing over 200 countries will converge inRio de Janeiro for the 'biggest sporting event on the planet'This special issue on SPORTS in the Brazilian Journal of Physical Therapy comes at anopportune time for practitioners who work with athletes of all levels of ability andthroughout various phases of their rehabilitation recovery. With contributions by aninternational group of 'elite' practitioners in their field, readers will be sure to findtheir topic of interest in this special SPORTS issue. These topics range from expertclinical commentaries and critical reviews to presentations of original research related tocommon sports injuries.While the World Cup generates an international enthusiasm for soccer, it also informs theviewer about the injuries to soccer players. The article "SPORTS INJURIES PROFILE OF AFIRST DIVISION BRAZILIAN SOCCER TEAM: A DESCRIPTIVE COHORT STUDY" by Reis and colleaguesprovides information from a descriptive cohort study regarding injury profiles in firstdivision Brazilian soccer players, including the influence of player's age and position oninjuries. An appropriate and relevant follow up to this manuscript is presented byHespanhol Junior and colleagues: "MEASURING SPORTS INJURIES ON THE PITCH: A GUIDE TO USE INPRACTICE." This paper reviews the basic concepts of injury monitoring systems in sportsparticipation and encourages the implementation of these concepts in practice.Runners are featured in the manuscript "MALE AND FEMALE RUNNERS DEMONSTRATE DIFFERENTSAGITTAL PLANE MECHANICS AS A FUNCTION OF STATIC HAMSTRING FLEXIBILITY". In this article,Blase Williams and colleagues assess the effect of hamstring length on running mechanics inboth male and female runners and discuss the implications for injury. For athletes who havesustained a knee injury, the timing for when it is safe to return to sports can bechallenging. In the manuscript "A CONCEPTUAL FRAMEWORK FOR A SPORTS KNEE INJURY PERFORMANCEPROFILE (SKIPP) AND RETURN TO ACTIVITY CRITERIA (RTAC)," Logerstedt and colleagues discussa comprehensive system that focuses on specific indicators of rehabilitation progression,and present criteria for safe return to sports following knee injury.Patellar tendinopathy is a common problem in athletes whose sports require jumping. In thearticle "PHYSICAL THERAPISTS' ROLE IN PREVENTION AND MANAGEMENT OF PATELLAR TENDINOPATHYINJURIES IN YOUTH, COLLEGIATE, AND MIDDLE-AGED INDOOR VOLLEYBALL ATHLETES" Kulig andcolleagues discuss intervention strategies that include education, rehabilitation, trainingand return to sport that are athlete-specific. Patellar tendinopathy is not the onlycondition linked to sports involving jumping. Achilles tendinopathy is also common andhighly problematic in jumping activities. In the manuscript "CLINICAL COMMENTARY OF THEEVOLUTION OF THE TREATMENT FOR CHRONIC PAINFUL MID-PORTION ACHILLES TENDINOPATHY" Alfredsondiscusses the results of research that evolved and changed practice, including the newesttreatment for Achilles tendinopathy.Given that Golf will be returning to the 2016 Summer Games for the first time in 112 years,Evans and Tuttle's "IMPROVING PERFORMANCE IN GOLF: CURRENT RESEARCH AND IMPLICATIONS FROM ACLINICAL PERSPECTIVE" is a well-timed contribution. Using best evidence from biomechanicaland motor control research, the manuscript offers a pragmatic approach to enhancing golfperformance.Core stability is frequently a focus of an athlete's rehabilitation program, yet there islittle evidence to support the link between core stability and injury. The article bySilfies and colleagues, "CRITICAL REVIEW OF THE IMPACT OF CORE STABILITY ON UPPER EXTREMITYATHLETIC INJURY AND PERFORMANCE" provides an in-depth review of the existing scienceregarding core stability and its association between upper limb injuries and athleticperformance. The upper limb is also featured in the article by Cools and colleagues,"PREVENTION OF SHOULDER INJURIES IN OVERHEAD ATHLETES: A SCIENCE BASED APPROACH." Theauthors discuss the key risk factors that may be used to guide injury prevention and returnto sports after shoulder injury.Finally, an in-depth commentary regarding the importance of study design for injury riskprediction is presented in the manuscript by Hewett and colleagues "MULTI-CENTER TRIAL OFMOTION ANALYSIS FOR INJURY RISK PREDICTION: LESSONS LEARNED FROM PROSPECTIVE LONGITUDINALLARGE COHORT COMBINED BIOMECHANICAL -EPIDEMIOLOGICAL STUDIES." In their paper, the authorsillustrate the research process and emphasize the need for continued, collaborative work inprospective study designs.Passion and Transformation: this is the essence of theemblemParabéns Brasil! Guest Co-editor, SPORTS Special IssueBrazilian Journal of Physical TherapyDeborah A. Nawoczenski PT, PhD


The mixing problem of electroencephalography (EEG) in the presence of common source could affect directed functional connectivity measures, resulting in an incorrect directionality of information flow between two signals. Here we introduce directed Weighted Phase Lag Index (dWPLI), a signed version of Weighted Phase Lag Index (WPLI) and compare this measure to Granger Causality (GC), Symbolic Transfer Entropy (STE), Phase Slope Index (PSI), and directed Phase Lag Index (dPLI) under common source effects. Robustness of the measures was tested in both analytic and simulating ways.For the simulated time series, signals were generated from unidirectionally coupled autoregressive model and linearly mixed to achieve volume conduction and common noise effects.As expected from the analytic calculation, dPLI and dWPLI were unaffected by the volume conduction while PSI, GC and STE were largely affected. Figure . The meaFor the common noise case, only dPLI and dWPLI had their signs preserved. Figure . The meaFurthermore, the dWPLI outperformed dPLI for the common noise case  which was also predicted from analytic calculation.Present study shows the common source effects might lead to biased results with incorrect directionality of information flow. Among the five directed functional connectivity measures, dWPLI is much less affected by common source effects.


Regular physical activity (PA) in the early school years is recommended by several scientific associations for primary prevention of cardiovascular disease. Long-term observational studies have shown that subjects who exercise regularly have significantly less coronary heart disease (CHD) and a reduced risk of cardiovascular disease (CVD). Exercise reduces serum triglycerides, increases serum high density lipoprotein-cholesterol, lowers the blood pressure in patients with primary hypertension . RegularRegular exercise, on the other end, is associated with potential adverse effects ; however, the absolute risk of kidney disease during exercise is low . ConsensExercise induces profound changes in the renal haemodynamics and in electrolyte and protein excretion. Proteinuria, hematuria and changes in serum electrolyte balance have been reported during intense PA; the increase in glomerular filtration may explain these transient alterations but the “nutcracker” compression on the renal vein may have a role. Haemoglobinuria and myoglobinuria may be observed under special exercise conditions .PA does not worse nor reverse kidney disease but may reduce cardiovascular risk in chronic renal disease . Childre


Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future. Regenerative medicine offers the promise of regaining organ function after acute or chronic injury. Regenerative approaches aim to promote, enhance and re-establish organ-specific repair processes to reconstitute organ structure and function after injury or in the setting of disease progression or treatment. Currently, the impact of the field of regenerative medicine in clinical practice is limited to a few specialized although highly successful practices, such as autologous bone marrow transplantation , partialRegeneration  identified essential genes, and gave insight into the physiology and pathophysiology of a variety of disease states. These mutants also frequently highlighted the genetic diversity contributing to morphologically similar disorders, potentially providing insight into the varied responses to current therapeutics or providing a platform for rational development of treatment options. For example, many diseases affecting blood formation and red blood cell physiology have been either explained by or modeled in zebrafish mutants, including the identification of previously uncharacterized genetic defects causing human disease  with disease phenotypes, including many tumor suppressors and oncogenes. The first iteration of these ‘reverse genetic’ approaches, known as TILLING  , took adic level . Furtheric level , as wellic level . These iic level . Althougex utero enables efficient introduction of foreign nucleic acids by microinjection. Long used as an effective means to ‘rescue’ mutant phenotypes to prove causation and produce transgenic fluorescent-tagged reporters of select genes of interest, microinjection has also enabled both transient and targeted gene knockdown. Although not without the caveats of potential nucleotide toxicity or off-target effects, morpholino oligonucleotide (MO) injection has been effectively used to antagonize translation in an antisense manner, thus blocking or diminishing protein production and revealing gene function. Furthermore, the procedure is titratable, providing the ability to bypass phenotypes associated with early embryonic lethality. For example, in zebrafish, just as in mice, complete loss of Wnt signaling   . APAP ovcetamol) . In thisIn addition to those discussed above, an increasing number of groups have utilized zebrafish to study regenerative repair after injury induced by a variety of methods in a broad array of organs, ranging from muscle repair after crush- or laser-induced injuries  and retiAlthough many zebrafish studies have the goal of therapeutic relevance, actual translational application of findings from zebrafish investigations is still in its infancy relative to mammalian models. That said, recent investigations stemming from chemical screening approaches focused on highly conserved aspects of regenerative biology of a select organ system, as discussed above, have directly shown the full potential of the zebrafish model for discoveries in the field of regenerative medicine. Here, we summarize the major attributes of two such studies from our own work. These investigations exploited conserved regenerative models (blood and liver) and a chemical screening approach, combined with the vast array of established tools for modulating the pathways of interest. This facilitated translational testing in mammalian systems, enabling fast translational application of the results from zebrafish to humans.S-nitrosoglutathione reductase (GSNOR), which negatively regulates protein nitrosylation, was shown to mediate the effects of NO on liver growth. To determine conservation of effect in organ regeneration, models of both physical  and chemical (acetaminophen) liver injury were used. Treatment with a novel GSNOR inhibitor (GSNORi) after liver resection in adult fish enhanced cellular proliferation and regeneration. In acetaminophen-exposed larvae and adults, GSNORi significantly prevented hepatocyte necrosis, enhanced proliferation, and improved survival alone and in combination with the current clinical therapeutic intervention, N-acetylcysteine. Significantly, the impact of GSNOR modulation is evolutionarily conserved, as GSNOR knockout mice and GSNORi-treated wild-type mice were similarly protected from acetaminophen-induced liver injury. GSNORi combines hepatoprotective and pro-proliferative properties, and represents a novel therapeutic approach for patients with toxic liver failure.As mentioned above, liver injury induced by acetaminophen exposure is the leading cause of acute liver failure. Identifying factors involved in regulation of embryonic liver growth could reveal conserved pathways with the potential to enhance liver regeneration after injury. We performed a chemical genetic screen in fluorescent reporter embryos , which rin vivo HSC reporters. In contrast, cyclooxygenase inhibition using both non-selective and specific inhibitors reduced HSC number. Adult irradiation recovery assays showed maintenance of the effect in regenerative repair, and murine embryonic stem cell differentiation studies showed conservation of function across vertebrate species. Long-term hematopoietic repopulation of the murine bone marrow following irradiation injury and limiting-dilution competitive transplantation of PGE2-exposed donor cells revealed increased in vivo regenerative potential in mammalian models. Subsequent investigations in zebrafish and mice indicated that PGE2 enhanced HSC function through cAMP-mediated enhancement of Wnt signaling, providing a useful biomarker for translational applications . In this study, both neutrophil engraftment and bone marrow chimerism will be assessed as primary clinical end points. Together, these examples from our own collaborative investigations indicate that zebrafish can be effectively used as a relevant preclinical therapeutic screening and regenerative model system that enables direct application and efficient translation to the clinical setting.Following collaborative discussions with transplant physicians and acquisition of toxicity profiles from earlier clinical endeavors, translational application of PGE2 in hematopoietic transplantation therapy clinical trials was approved by the FDA, the first such study to arise from a chemical genetic screening approach in zebrafish. In phase 1 trials primarily designed to establish safety, PGE2-treated HSC samples showed substantial changes in clinical end points, with both predominant engraftment of PGE2-treated cord blood samples and accelerated recovery of the blood counts in patients receiving the transplants compared to historical controls ; there wRegenerative medicine holds great promise for the alleviation of morbidity and mortality associated with organ failure or injury. Tissue repair and regeneration can be driven by modulation of the pathways that govern stem cell behavior and organ development. The zebrafish has traditionally been an excellent model to study early development and organogenesis, demonstrating high genetic and functional conservation with mammals. Given the inherent connection between developmental pathways and organ repair, this strength combined with a growing list of innovative regenerative models makes the zebrafish an ideal system to study regenerative processes, with the potential to translate relevant findings across species and toward clinical application. In light of the many promising projects mentioned here, developed over such a brief time frame, we anticipate an increasing number of valuable studies and novel therapeutics inspired by and discovered through zebrafish research will be developed in the field of regenerative medicine.


TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called  Personalised medicine aims to stratify patients’ cancers into new molecular subtypes who can benefit from individualised therapy –3. The tExtracting DNA and RNA from tumor cells in the context of FFPE samples is not straightforward. Most tissues containing tumor also contain a mixture of cell types such as non-neoplastic epithelial cells, mesenchymal tissue, inflammatory cells and acellular material such as mucin which has an influence on subsequent processes including nucleic acid isolation, PCR amplification and next generation sequencing . TherefoRAS, EGFR, BRAF mutational analysis, commercial tests such as Oncotype DX, Mammaprint, Prolaris and more recent clinical sequencing panels (e.g. Foundation One). Given that this role falls primarily to the pathologist, the rapid rise in the investigation of solid tumor molecular tests and their delivery in diagnostic molecular laboratories is putting a strain on pathology services. Molecular labs without in-house pathology competency, must transport slides to a qualified pathologist for review and manual annotation. The centralization of some molecular tests, despite having clear sample guidelines on their test request forms, must review the slides centrally and ensure sufficient tumor is present before macrodissection and analysis.Macrodissection is a manual process in which the region of tumor is physically scraped from the slide using a scalpel into a receptacle for subsequent nucleic acid extraction and molecular analysis. Marking the regions of viable tumor within tissue samples relies on the visual assessment of haematoxylin and eosin (H&E) tissue sections, usually by an experienced pathologist and typically performed at low power magnification. Manual macrodissection aims to enrich the proportion of neoplastic cell nucleic acid by removing non-tumor containing regions, not only increasing the likelihood of detecting a relevant mutation if it exists but also increasing the certainty that a mutant signature originates from the malignant cell isolate. Percentage tumor evaluation and macrodissection underpin most of the new and emerging molecular tests for solid tumors including et al . Us. UsTissuTissueMark image analysis method can overcome the shortfalls of visual estimation and provide a more objective measure of tumor cell sufficiency. In order to benchmark this, subjective visual estimations of tumor cell percentage accuracy were not sufficient. Instead we hand-counted a series of tissue images to get precise numbers of tumor and non-tumor cells and an absolute, gold standard measurement of percentage tumor nuclei. Similar types of manual cell counts were carried out by Smit et al  was also computed for each tile to indicate the likelihood for tumor or non-tumor class membership. This could be presented as a ROC) curve, (iii) concordance index and (iv) false discovery rate (FDR). These are fully described in It was important to compare manually drawn tumor boundaries with boundaries generated using automated image analysis. Visual assessment by an experienced pathologist allowed us to determine the quality and accuracy of automated tumor annotation as well as a visual comparison between manual and automated annotation and an evaluation on whether deviations would have impact on molecular testing. These were largely subjective evaluations. In addition, however, we devised four methods to measure boundary differences: (i) inclusion rate vs. exclusion rate, (ii) receiver operating characteristic , previously genotyped for