Drosophila melanogaster and its associated powerful genetic tools have become a very attractive model system to study tumour-intrinsic and non-tumour-derived processes that mediate tumour development in vivo. In this review, we will summarize recent work on Drosophila as a model system to study cancer biology. We will focus on the interactions between tumours and their microenvironment, including extrinsic mechanisms affecting tumour growth and how tumours impact systemic host physiology.The study of cancer has represented a central focus in medical research for over a century. The great complexity and constant evolution of the pathology require the use of multiple research model systems and interdisciplinary approaches. This is necessary in order to achieve a comprehensive understanding into the mechanisms driving disease initiation and progression, to aid the development of appropriate therapies. In recent decades, the fruit fly Despite being the most studied human disease, cancer remains a leading cause of mortality worldwide. Nearly 1 in 6 deaths in 2015 was attributable to cancer, according to the World Health Organization, with an increase of 70% of new cases projected within the next two decades [ in vivo studies of non-tumour autonomous processes difficult to accomplish in conventional whole animal model systems.The organismal implications and ultimate outcome of tumour burden in patients are undoubtedly determined by a combination of tumour-intrinsic mechanisms and interactions between tumours and proximal, as well as distal tissues –4. WhileDrosophila melanogaster remains the most powerful genetic model in research. During the last decades, the development of various tumour models, including leukaemia, neuroblastoma, glioblastoma, colorectal, and ovarian cancer, has made the fruit fly an attractive in vivo model system to decipher tumour intrinsic  and extrinsic  molecular mechanisms mediating tumour growth and metastasis [ scribble-group of tumour suppressors genes , which encode key components of the basolateral polarity complex [ Ras or Src, drives tumour cell proliferation, spreading to distant tissues [ scrib-group genes as tumour suppressors in Drosophila, research has provided growing evidence that these models are directly relevant to human conditions. Indeed, scrib and dlg proteins are known targets of several oncogenic viruses, such as Human Papillomavirus, the main agent of cervical cancers. These viruses induce the degradation of the polarity complex proteins, comprising a key part of the process of malignant transformation in these conditions [ scrib has also been shown to work as a tumour suppressor in human breast, liver, skin, and lung cancers [ Drosophila homolog, scrib also cooperates with the Ras oncogene to promote tumour cell invasion [tastasis , 10. Suc complex . These m tissues , 13. Durnditions , 14, 15. cancers –19. The  cancers  and hepa cancers  and is a cancers . Moreoveinvasion , 23. Drosophila that have shed light into how extrinsic signals influence tumours, as well as mechanisms that mediate the systemic impact of tumours in the host. We focus on new findings highlighting the influence of immunity and metabolism in cancer progression and cancer-related disorders.Here, we discuss recent discoveries in Drosophila has allowed the discovery of molecular mechanisms mediating both pro- and antitumoural immunity. In contrast to mammals, which possess both innate and adaptive immunity, Drosophila only relies on innate immunity to fight against pathogens and tumours. Additionally, while mammals have numerous types of white blood cells, the cellular arm of Drosophila innate immunity includes only three main cells types—plasmatocytes, lamellocytes, and crystal cells—commonly called haemocytes. Only plasmatocytes have been currently reported to be associated with tumours [ Drosophila and mammals [Work in mammals has highlighted the immune system as a key component of the tumour microenvironment (TME), which plays a critical role in defining tumour outcome. While early studies on cancer patients support anticancer activity of the immune system , recent  tumours ; however mammals –29.α) is a major proinflammatory cytokine produced within the TME, which was originally characterised for its ability to induce tumour death [α's discovery led to great expectations for its use as a therapeutic target for cancer. However, further experiments have revealed a dual role for TNF-α as both an anti- and protumour factor [α's antagonistic actions were poorly understood. However, recent research in Drosophila has highlighted some key molecular aspects underlying this dual action of the cytokine. Drosophila possesses a single TNF-α homolog called Eiger (Egr) [ Drosophila tumour models through the use of mutants of the scrib-group of tumour suppressors genes. Egr expression is induced in tumours and tumour-associated immune cells [α, which is detected in tumour cells as well as macrophages and T lymphocytes [Tumour Necrosis Factor alpha (TNF-ur death . Consistr factor . The moler (Egr) , 33, whoer (Egr) . The impne cells , 36, mucphocytes . Given tphocytes , 37. scrib, lgl, or dlg mutant cells generated in imaginal discs, delaminate, and are mostly removed from the epithelia through cell competition [ Egr specifically within haemocytes, highlighting a conserved non-tumour autonomous anticancer function of Egr in Drosophila [α receptor Grindelwald (Grnd) in scrib mutant cells suppressed their removal from the epithelia [α is conserved in mammals, as TNF-α induces cell death through TNFR1 and subsequent JNK signalling activation [ Drosophila, including immune response proteins, may uncover new mechanisms involved in the elimination of cancer cells from a healthy tissue [ scrib-group genes, where neoplastic tumours develop from the whole imaginal disc, also show dependency from haemocyte-derived Egr to trigger JNK activation and tumour cell death [α-dependent inflammation in Drosophila models of cancer.Experimental evidence showed that immune cell-derived Egr has antitumoural activity. Patches ofpetition , 37–40. osophila , 36 . Ras is a conserved proto-oncogene mutated in many cancer types, with a 16% overall incidence rate in all analysed human tumours [ Drosophila, clones of cells mutated for scrib-complex proteins and overexpressing Rasv12 fail to be eliminated by surrounding epithelial cells. Instead, they form neoplastic tumours that can invade distant tissues [ scrib mutant clones, cooperation with Rasv12 in these clones diverts the function of JNK pathway activation toward tumour cell proliferation and invasion [ scrib, Rasv12 mutant cells to grow and invade. Strikingly, transplantation of Egr-wild-type immune cells could rescue the progression of scrib, Rasv12 tumours, as well as JNK activation, providing the final demonstration that Drosophila TNF shares protumour effects with its mammalian counterpart [ scrib, Rasv12 tumours and by data showing that Grnd knockdown in those tumours also disrupts their growth and invasive properties [scrib, Rasv12 tumours [v12-driven tumour progression requires the activation of Caspases, which function as tumour promoters. This mechanism is suggested to be one of the key mediators of the switch of Egr from an antitumour to a protumour cytokine by Ras. The protumoural function of TNF-α produced by immune cells is highly reminiscent to the one described in mammalian systems. In a mouse model of skin carcinogenesis where loss of TNF-α suppresses tumour formation [α competent mice is sufficient to restore tumour formation. However, this effect appears to be indirectly mediated through TNF-α-dependent regulation of T-cell number [α-dependent antitumoural responses in flies and humans comes from work on Kras-dependent intrahepatic cholangiocarcinoma. In this context, TNF-α produced by Kuppfer cells (liver-specific myeloid cells) drives preneoplastic lesions through JNK signalling pathway activation [In contrast to the described antitumoural functions, tumours . In Dros Ras Rasv. Ras is  tissues . While Jinvasion , 49. In nterpart  , a Syntaxin involved in the fusion of endocytic vesicles to the early endosome, is dependent on Grnd but escapes the need for Egr [ avl tumours produced high levels of Wingless (Wg) protein, which is a known target of JNK pathway activation and a key driver of compensatory proliferation, which is linked to cancer progression [ Drosophila showed that Wg dependent tumours proliferate independently of the TME and TNF-α/Grnd [v12 and hyperactivation of the nonreceptor tyrosine kinase Src, which also feature Wg overexpression [α. The expression of growth factors and activation of downstream signalling pathways in epithelial tissues in general and in Drosophila imaginal discs in particular are usually restricted to certain tissue locations [ Drosophila identified the presence of “tumour hot-spots.” Tumour hot-spots are defined as locations within tissues where neoplastic mutations are more likely to result in successful tumoural growths capable of invading normal tissues and it is a process involving differential activation of JAK/STAT signalling [α in this context.The demonstration of antagonistic actions of TNF- for Egr . Interesgression , 56. It F-α/Grnd . Similarpression , are larocations , 60. Recgnalling . It is l scrib-group mutants is the loss of epithelial cell polarity. In tumours lacking lgl, knockdown of the JNK pathway rescues loss of cell polarity [α may be a determinant in the loss of cell polarity in tissues carrying these neoplastic transformations. Indeed, Egr regulates asymmetric localisation of determinants of asymmetric division, Miranda and Prospero, in neuroblasts, supporting a role for Egr in cell polarity determination [α-dependent loss of cell polarity has been reported upon induction of chronic inflammation in the mouse intestine [α-dependent EMT increases lung cancer metastasis [α and cell polarity could also be the driving force for TNF-α's protumour effect on Rasv12 expressing cells, as Ras hyperactivation facilitates the prosurvival function of JNK signalling.A key phenotypic feature ofpolarity . Loss ofpolarity , 64. Givmination . Interesntestine . Likewistastasis . This poβ) signalling family, can promote intestinal stem cell (ISC) proliferation in response to infection [ Drosophila interleukin homologs that function as ligands of the JAK/STAT pathway [ scrib mutant larvae Upd 3 produced by the tumour induces JAK/STAT activation in the immune tissues (fat body and haemocytes), leading to a positive feedback loop that increases Upd 3 levels in haemocytes, which is required for JAK/STAT-induced proliferation of haemocyte and subsequent tumour suppression [ scrib/Rasv12 tumour, where it cooperates with JNK to promote growth and metastasis [ Drosophila blood cell neoplasia [The discovery of other immune-derived cytokines may have implications on their role in cancer progression through the TME. Haemocyte-derived Dpp, the fly homolog of Bone Morphogenetic Protein 2/4 (BMP2/4), a member of the Transforming Growth Factor beta , and JAK/STAT signalling [ Spaetzle (spz) in haemocytes or by removing Egr from tumours, suggesting that Egr produced by the tumour promotes Spz production by haemocytes, which in turn activates the Toll pathway in the fat body [α-dependent activation of systemic Toll signalling is an important component of a nonautonomous tumour suppressor program  [ Drosophila models. These findings demonstrate the drastic effect that the perturbation of host metabolism by extrinsic factors can have on tumour growth, how tumours exert systemic effects on distant tissues, and how the genetic properties of the tumour itself are critical in mediating this crosstalk.Obesity and type 2 diabetes are common comorbidities in modern society and are characterised by systemic insulin resistance and hyperglycaemia. These conditions are associated with an increased risk of developing cancer and are a risk factor for cancer mortality –94. Insuondition . In thisondition . Tumoursondition  that actondition . Under nt (slif) . Interest (slif) . Interest (slif) , which it (slif) –101. Thi Drosophila models and those found in vertebrates. Preexisting obesity and diabetes promoted tumour growth in a rat cancer model [ Drosophila cancer models involving diet and obesity are therefore particularly relevant to the human condition, as the protumour effects demonstrated in the contexts of these studies appear to be conserved in higher organisms, and the mediating factors are environmental influences that are very common in developed societies. The studies discussed here highlight new aspects of tumour physiology, suggesting that tumours are direct competitors to host tissues for nutrients and are frequently able to outcompete them for access to metabolic resources through various means  that drive the onset of microenvironmental autophagy are not yet fully defined; however, ROS signalling is an excellent candidate for further investigation , a secreted insulin-signalling antagonist that functions by direct binding to Dilp2 [ ImpL2 in the tumour was sufficient to reduce the systemic insulin resistant phenotype and thus partially rescue the wasting phenotypes observed in peripheral tissues, without impacting the growth of the tumour [ Drosophila cancer models in the field of tumour-microenvironment interactions. Research into cachexia is an emergent field, and the identification of a tumour-derived factor that mediates a systemic effect on host tissue metabolism is an important example of the ability of Drosophila models to recapitulate and dissect complex phenotypes. Interestingly, autophagy is one of the main mechanisms of tissue degradation during cancer cachexia [ Drosophila models discussed here represent a good opportunity to answer some of these important open questions.Two independent models of cancer cachexia have shown the utility ofresearch , 120. Boto Dilp2 . These sto Dilp2 –126 (Fige tumour , 120. Thcachexia , 128. Thcachexia . Togethecachexia , 130. Th Drosophila is a relevant model for studying cancer and its interactions with the TME, with many parallels to orthologous vertebrate conditions. Research utilising Drosophila as a model system has shown that immune and metabolic processes induced in a nonautonomous manner by the presence of the tumour are sufficient to feed back to the tumour and alter its characteristics. This can be shown well in the studies of microenvironmental autophagy, which is induced in the TME by the tumour, and serves to support tumour growth and metastasis [The studies discussed here demonstrate thattastasis , 104, antastasis , 46 (Fig Drosophila. Work in human cell culture and mouse models has demonstrated that tumours can alter host immunity via directly influencing immune cell metabolism. Lactic acid secreted by the tumour into the TME changes macrophages metabolism, polarising them towards a tumour-promoting state [Given the effects observed in response to the tumour there are likely to be interactions between the immune system and metabolism in this context. Indeed, nutrient restriction in larvae inhibits TOR signalling in the fat body, leading to increased levels of circulating Egr. Egr binds to insulin-producing cells in the brain and suppresses the production and secretion of progrowth Dilp2 and Dilp5 . As prevng state , 133. Thng state . Anotherng state , 87. Drosophila cancer models represent an excellent basis for the study of the roles these factors may play, both individually and combined together, and how nonautonomous signalling inputs might influence both tumour and host tissue responses. There are likely to be inevitable questions about the ability of simple Drosophila tumour models with one or two genetic drivers to fully recapitulate the complexity of tumour burden in higher animals, including human patients. However, the simplicity of these models is likely to prove advantageous when attempting to dissect the contributing roles of the multiple interacting factors that comprise tumour-TME interactions. There is also interesting work on the generation of Drosophila “avatars,” fly lines that can generate close homologs of tumours from specific patients, including the numerous genetic aberrations that drive a particular type of tumour in humans [ Drosophila cancer models, in order to investigate whether these discoveries still hold in a more complex tumour setting, including tumour-TME responses.However, there is a lack of comprehensive understanding as to how different factors such as diet and metabolism, immune responses, and tumours interact and cooperate or synergise when presented together. This is often the case in the human condition, for example, in the case of a cancer patient with diabetes.n humans . Such av Drosophila models represent an excellent platform for the continued investigation of these complex interactions, thanks to the multiple advantages of the model system. Low genetic redundancy, powerful genetic tools, and the possibility of tightly controlling not only the genetics of the tumour but also various aspects of the tumour micro- and macroenvironment render Drosophila a strong paradigm for further work into these complex interactions that impact human health and disease.The mechanisms mediating the crosstalk between tumours and local and distal tissues are still being uncovered. Improving the understanding of the signalling pathways that may link together the complex interactions between host metabolism, immunity, and tumour growth is an essential aspect towards the unravelling of such crosstalk.


The higher prevalence of multiple sclerosis (MS) in females, along with the modulation of disease activity observed during pregnancy and the post-partum period, has suggested a hormonal influence in MS. Even if prolactin (PRL) does not belong to the sex hormones family, its crucial role in female reproduction and lactation has prompted great efforts to understand if PRL could represent a gender factor in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), the animal model for this disease. Extensive literature has documented a remarkable immune-stimulating potential for this hormone, indicating PRL as a disease-promoting factor in MS and EAE. However, recent work has pointed out that PRL is endowed with important neuroprotective and remyelinating properties and has encouraged a reinterpretation of the involvement of this hormone in MS. In this review we summarize both the protective functions that PRL exerts in central nervous system tissue as well as the inflammatory activity of this hormone in the context of autoimmune responses against myelin. Last, we draw future lines of research that might help to better clarify the impact of PRL on MS pathology. Multiple sclerosis (MS) is a chronic, inflammatory disorder of the central nervous system (CNS) that affects more than 2.5 million people worldwide, representing the most common cause of neurologic disability in the white young adult population . NeuroloDepending on geographical areas, MS has a sex ratio  of 2:1 to 3:1 . Women aPRL gene is controlled by the pituitary and extrapituitary  promoters in humans [Prolactin is a 23 kDa polypeptide hormone mainly secreted by the lactotrophic cells of the anterior pituitary gland, even though other cell types can also release this hormone, including immune cells, adipocytes, mammary and epithelial cells . Transcrn humans . The PRLn humans ,17. PRLRn humans . Broad en humans  and therIn this review, we attempt to provide an integrated overview of PRL involvement in MS and EAE, summarizing both the protective functions that PRL exerts on CNS tissue as well as the inflammatory potential of this hormone in the context of autoimmune responses against myelin. Last, we trace future lines of research aimed at further understanding the involvement of PRL in the pathogenesis of MS and EAE.In recent years a significant amount of work has documented numerous actions mediated by PRL in the CNS. Circulating PRL can cross the blood-brain barrier; however, its transport into the brain does not depend on PRLR but is mediated by a still-unidentified transport molecule .Prlr+/− mice (Prlr−/− mice are sterile), a 50% reduction of PRLR is sufficient to induce a significant reduction of proliferating neural progenitors in the SVZ and a consequent reduction of olfactory bulb interneurons. Interestingly, when adult neurospheres are cultured in vitro in the presence of PRL, the number of differentiated neurons is doubled [Several studies in the last years have demonstrated that PRL can positively affect neurogenesis in several physiological and pathological conditions. PRL is important for pregnancy-stimulated neurogenesis of the female adult brain, a process that probably supports maternal adaptation to offspring . PRLR ha doubled . PRL was doubled . Another doubled . Moreove doubled . The imp doubled . Again,  doubled .The actions of PRL in the CNS are not solely restricted to adult neurogenesis. PRL has been indicated as a promising therapeutic agent for spinal muscular atrophy (SMA), a neurodegenerative disorder characterized by the loss of motoneurons and progressive muscular atrophy . SystemiPrlr+/− mice. Moreover, PRL administration supports myelin repair in virgin females receiving lysolecithin lesions [Oligodendrocytes are also positively affected by PRL stimulation. Gregg and coworkers have shown that murine pregnancy promotes the increase of oligodendrocyte precursor cells (OPCs) and myelinated axons in the female adult brain . Pregnan lesions . Given iPrl transcript or protein have been detected in purified CD4+ T cell cultures of mouse origin [Prl expression in rodents, while an additional “extra-pituitary” promoter has been described in humans [PRL transcription should be taken into account when translating concepts from experimental models to humans, as a more complex regulation of PRL production might occur in humans. Regulatory T cells (Treg) represent a T cell subset with immune-suppressive functions and their expansion is severely impaired in MS patients [PRLR expression and PRL secretion following stimulation [Along with homeostatic and regenerative actions in the CNS, PRL is also endowed with immune-stimulating functions, even though most evidence relies on in vitro experiments. Several immune cells involved in the autoimmune attack against the CNS occurring in MS and EAE can be stimulated by PRL. Macrophages exposed to PRL secrete higher amounts of reactive oxygen species, and inflammatory cytokines -γ and IL-12) and chemokines -1α and monocytes chemoattractant protein (MCP)-1) ,31,32. Pe origin . In thisn humans . This dipatients . Interespatients . The actpatients . In diffpatients ,43,44,45mulation .Several groups have investigated a possible correlation between PRL levels in body fluids and MS. A recent study has reported that female MS patients have significantly higher PRL levels in serum and cerebrospinal fluid if compared to healthy donors, male MS subjects or patients with clinically-isolated syndrome . HoweverPrlr−/− and Prl−/− mice, elicited by immunization with MOG peptide 35–55 (MOG35–55). We reported that mice lacking PRLR or PRL develop chronic EAE with a modestly but significantly delayed onset and a disease severity comparable to control mice [Prlr−/− and Prl−/− mice is associated with a mild delay in the development of Th1 and Th17 autoimmune responses against myelin in draining lymph nodes [Attempts to lessen MS activity by administration of BCR were based on results in EAE, a widely used animal model of Th1/17-mediated autoimmune demyelination of the CNS. Active EAE is mostly induced in susceptible strains of rats or mice, by immunization with immunodominant epitopes of myelin antigens supplemented with adjuvants . EAE genrol mice . The retph nodes . This woph nodes . In thisph nodes . Howeverph nodes .Data discussed in this review suggest that PRL might play dual and opposing functions in both MS and EAE. Its effects are likely to be the result of a fine balance between protective actions on CNS tissue and stimulation of the immune system. On the one hand, PRL might provide regenerative signals for neurons, oligodendrocytes and adult neural stem/progenitor cells, and thus support CNS repair; on the other hand, PRL stimulation of peripheral immune cells, in particular T and B lymphocytes, might sustain autoimmune responses and negatively impact on disease pathology .35–55-induced EAE in non-pregnant mice is not substantially influenced by the absence of PRL or its receptor [The development of MOGreceptor . Given treceptor  and givereceptor .PRL transcript, which was first identified in human lymphocytes and endometrial tissue [PRL gene and all its regulatory elements, the human, but not mouse, PRL transcript was detected in lymphoid organs, such as the spleen and thymus [Callithrix jacchus) [Prl gene was replaced with human PRL [From a therapeutic perspective, the dual nature of this hormone suggests great caution when trying to manipulate PRL levels in MS. It is also possible to speculate that PRL depletion or administration might be beneficial, depending on the MS form and/or stage. Also, no indication on breastfeeding can be drawn by data produced so far in experimental models. In this regard, the interpretation of results obtained in mouse models in the context of human immune responses and autoimmunity is complicated by the relatively recent discovery in humans of a cleaved 16 kDa isoform of PRL. This PRL isoform has different properties from the full 23 kDa PRL, exerting anti-angiogenic functions and mediating cardiomyopathy in the post-partum period . It mustl tissue ,65. The d thymus . In ordejacchus) , or in auman PRL .In conclusion, further studies are still necessary to better clarify how PRL influences autoimmune responses of the CNS and whether PRL might represent a target of therapy in these disorders.


Litoria verreauxii alpina) throughout a longitudinal exposure experiment involving both infected and negative control individuals. We found that chytridiomycosis dramatically altered the organism-wide metabolism of clinically diseased frogs. Chytridiomycosis caused catastrophic failure of normal homeostatic mechanisms (interruption of biosynthetic and degradation metabolic pathways), and pronounced dysregulation of cellular energy metabolism. Key intermediates of the tricarboxylic acid cycle were markedly depleted, including in particular α-ketoglutarate and glutamate that together constitute a key nutrient pathway for immune processes. This study was the first to apply a non-targeted metabolomics approach to a fungal wildlife disease and specifically to dissect the host-pathogen interface of Bd-infected frogs. The patterns of metabolite accumulation we have identified reveal whole-body metabolic dysfunction induced by a fungal skin infection, and these findings have broad relevance for other fungal diseases.Chytridiomycosis is among several recently emerged fungal diseases of wildlife that have caused decline or extinction of naïve populations. Despite recent advances in understanding pathogenesis, host response to infection remains poorly understood. Here we modelled a total of 162 metabolites across skin and liver tissues of 61 frogs from four populations (three long-exposed and one naïve to the fungus) of the Australian alpine tree frog ( Knowledge is needed urgently to develop novel approaches for control4. While targeted metabolite studies have documented host changes associated with white nose syndrome and chytridiomycosis6, there have been no broad scale metabolomics studies investigating these fungal diseases excepting those aimed at identifying anti-microbial peptides or probiotics7. Chytridiomycosis is an often fatal fungal skin disease of amphibians, affecting approximately 42% of species examined8 and causing declines and extinctions around the world9. Fungal sporangia invade the superficial epidermal cells leading to disrupted skin functions involving ion transport10. While the proximate cause of mortality due to the pathogen, Batrachochytrium dendrobatidis (hereafter Bd) has been linked with alteration of electrolyte balance (low plasma levels of sodium and potassium) and subsequent cardiac arrest10, the accompanying host metabolic phenotype has not been well characterized. Several gene expression studies have identified pathways associated with end-stage physiology. These include increased expression of genes associated with cellular stress and disruption of skin homeostasis, detoxification, proteases, antimicrobial peptides, as well as decreased expression of cytochrome p450 genes, blood coagulation genes and immune genes 18. An improved understanding of the molecular basis underlying the pathophysiology of chytridiomycosis will be fundamental to improving management techniques and hence population outcomes3. In addition, comparing the metabolome of populations with differing long-term exposure histories and susceptibility to Bd may elucidate immune mechanisms associated with more resistant or tolerant phenotypes.Recently emerged fungal skin diseases of wildlife include chytridiomycosis in amphibians, white nose syndrome in bats, snake fungal disease and mucormycosis in platypus20), as well as exogenous influences such as occurring through exposure events. Mass spectrometry (MS) and chemometrics are the cornerstone of modern metabolomics research, capable of discriminating thousands of metabolic features to characterize a metabolic phenotype, or ‘phenome’21. Non-targeted metabolomics analyses can identify patterns of metabolite flux to elucidate biochemical perturbations induced by infection and disease21. Such insights have led to the development of numerous assays for screening biomarkers of disease, particularly in humans24. Metabolomics approaches have also been widely used in toxicological and pharmacological studies26.Non-targeted metabolomics (otherwise known as metabonomics or metabolic phenotyping when undertaken to investigate and compare disease states) seeks to measure the small molecule  component of a biological system. It investigates the precursors, intermediates and products of biochemical pathways  associated with chytridiomycosis throughout pathogenesis. Moreover, no study has yet examined differences in metabolic phenotype among different populations of a single frog species with varying exposure histories and clinically demonstrated differential susceptibility to disease.To date, numerous studies have used mass spectrometry to characterize the interaction between Bd and the amphibian host including investigations of intra and extracellular fungal metabolites, amphibian urinary metabolic markers of stress, epidermally secreted antimicrobial peptides, and the analysis of antifungal metabolites produced by symbiotic skin bacteriaIn this study we used a non-targeted metabolomics approach to examine the longitudinal metabolic phenotype of chytridiomycosis infection post exposure, as well as to compare the metabolome of different populations of the same species with differing long-term Bd-exposure histories and survival responses. We analyzed skin tissues at the site of infection, as well as liver tissues from exposed and unexposed control frogs at multiple time points throughout disease progression .Litoria verreauxii alpina) were collected by D. Hunter in accordance with Scientific License number: S12848. Animals were captively-raised by experienced animal handlers under quarantine conditions following Taronga Conservation Society Animal Ethics Committee guidelines (4c/01/10). Experimental protocols involving animals were similarly carried out in accordance with the approved guidelines and protocols under permits issued by James Cook University (A1408) and Taronga Conservation Society (4c/01/10) Animal Ethics Committees. Animals were individually examined at least once daily for clinical signs of chytridiomycosis infection , and humanely euthanized if clinical signs were detected 34.Wild alpine tree frog eggs . In brief, 61 chytridiomycosis-naïve adult alpine tree frogs were captively-raised from wild-caught eggs collected from four geographically distinct populations in Kosciuszko National Park, New South Wales, Australia. Batrachochytrium dendrobatidis had emerged at three of these sites  over two decades earlier than the time of egg collection (as determined via long-term monitoring33). The remaining site (hereafter Grey Mare) was known to be Bd-naïve at the time of egg collection. Forty-six of these frogs were individually exposed to 750,000 zoospores of Bd using the AbercrombieNP-L.booroolongensis-09-LB-P7 isolate in a topical bath of dilute salts solution (DSS)33, while the remaining 15 frogs were sham-exposed with only DSS (unexposed negative control group). Seventeen animals  at each of three subclinical sampling sessions  were skin-swabbed to quantify Bd infection intensity, euthanized and then had skin and liver tissues sampled. The remaining 10 animals were swabbed, euthanized and sampled at the onset of clinical signs  derivatised in preparation for gas chromatography mass spectrometry (GC-MS) as previously described33. Five µL of hexane containing a series of straight-chain alkanes was added to samples for the calculation of retention indices (RIs). The GC-MS  analysis of the metabolite derivatives was described by Grogan et al.33, with data acquired and analysed using GCMSsolution 2.61  and AnalyzerPro 2.7.0.0 . A preliminary scan acquisition (m/z 50–650) was followed by a simultaneous full scan and selective ion monitoring (SIM) mode of acquisition. Gas Chromatography Mass Spectrometry analyses of skin samples were not obtained for seven frogs  due to a precipitate that formed in the derivatised samples (we could not identify the cause for the precipitate). All analytes were relatively quantified using a characteristic quantifier ion selected for each analyte, and metabolite identities assigned by comparison to authentic metabolite standards or by reference to an external mass spectral library  or Wiley Registry™). Metabolites without a match were labelled with the prefix ‘unknown’, followed by the observed retention time (RT) and calculated RI. Quality control and technical validation procedures are described in detail by Grogan et al.33.Details of the metabolomics preparative methods are provided by Grogan 35, subsequently log transformed36 (log10(x + 1)) and pareto-scaled . Statistical analyses were performed with SPSS , R (Bell Laboratories), and Metaboanalyst 3.638. For ease of interpretation, data from sham-exposed negative control group frogs  were designated Group 0, regardless of whether they were sampled at 4, 8 or 14 days post-exposure. Exposed/infected frogs sampled at various times post-exposure were designated Groups 1, 2, 3 and 4, corresponding to being sampled at 4, 8, 14 and 28–30 days post exposure, respectively. The numbers of samples included in each group for each analysis have been described below, and varied depending on original sample size and taking into account any missing values.Measured analytes of non-biological origin were determined by comparison to no-tissue control preparations, which were prepared identically to the sample extracts, but without skin or liver tissue. Any non-biological analytes were subsequently removed from the data matrices. Peak areas were normalized to the summed total ion intensity39, and Significance Analysis of Microarrays (SAM40). In all cases, an alpha error level of 0.05 corrected for multiple testing via Bonferroni method was considered acceptable . Multivariate analyses included unsupervised Principal Components Analysis (PCA) and supervised Partial Least Squares Discriminant Analysis (PLS-DA) providing Variable Importance in Projection scores (VIP) with model quality assessed by 10-fold cross-validation based on Q2 and goodness of fit evaluated with permutation testing using 2,000 iterations. We also used the Omics Dashboard41 to explore patterns of metabolite accumulation and depletion across metabolic pathways and cellular systems in response to chytridiomycosis. Population comparisons excluded data on the two frogs from Ogilvies due to small sample size, however these were included in the sample period based analysis. Metabolites found most influential within the data modelling were searched for in intracellular and extracellular metabolite analyses of Bd sporangia and culture medium (data not shown), to determine if of frog or fungal origin.Traditional univariate statistical analyses included Analysis of Variance with post-hoc Tukey’s Honestly Significant Difference (HSD) tests, a pattern matching correlation analysis with Pearson r correlation43.All 61 experimental animals survived the duration of the experiment until they were euthanized for sampling. As expected, the 10 frogs sampled between 28 and 30 days post-exposure were demonstrating clinical signs of chytridiomycosis  at the time of euthanasia. Demographic data on the sampled frogs, together with infection intensities as assessed via qPCR can be found in Table et al.33). While we refer to acid analytes by their library standard name in the results, in the discussion we instead refer to the equivalent anion salt with the suffix ‘-ate’ , as this is the biologically relevant form.Overall, 23,868 MS features were resolved by GC-MS across both liver and skin tissues with a total of 2,177 zero values and no missing values. The MS features were deconvoluted into peaks representing individual metabolites and the relative quantitation of 162 metabolites was obtained, with 72 of these metabolites identified or putatively identified. Complete metabolite accumulation data have been archived at the MetaboLights data repository and Dryad Data Repository . It was also clear from this analysis that the metabolite profiles of skin tissue samples clustered separately from liver samples, as expected with differential underlying tissue physiology. Skin and liver samples from the frog designated Lva259 from the Grey Mare population were consistently found to be marked outliers in the PCA results  as differing significantly in relative concentration between sampling periods . Tukey’s HSD post-hoc tests identified the 10 most significantly differing (FDR < 0.05) metabolites between sampling groups to include α-ketoglutaric acid, serotonin, 5-hydroxyindole-3-acetic acid (5HIAA), isoleucine, glutamic acid, ornithine, tartaric acid, urea, threonine, and serine  included ornithine, urea, threonine, serine, adenine, putrescine and lysine . The top 10 identified and most significantly differing metabolites via Tukey’s HSD post-hoc tests included isoleucine, glutamic acid, pyroglutamic acid, leucine, putrescine, fructose-6-phosphate, citric acid, fumaric acid, glucose-6-phosphate and γ-aminobutyric acid  demonstrated marked separation between clinically diseased frog tissues (Group 4) and control and subclinically infected tissues (Groups 0–3), an effect most pronounced within the liver tissue samples Fig. . The top41) of skin and liver tissue metabolite accumulation data grouped by sampling period revealed substantial alterations in cellular system profiles in clinically diseased frogs. Most notably, numerous metabolites associated with biosynthetic, degradation and energy pathways were depleted in moribund frogs (Group 4) compared with control and subclinically infected frogs . Those metabolites identified included pantothenic acid, myo-inositol, leucine, isoleucine and creatine .One-way ANOVA of ine Fig. . The t-tine Fig. . SAM resine Fig. . There wpopulation clusters was apparent with supervised PLS-DA for both skin and liver tissue samples. Clustering was more pronounced with the skin samples, where samples from Grey Mare (Bd-naïve population) projected between clusters of samples from the two long Bd-exposed populations (Kiandra and Eucumbene)  Fig. . Top ran10. Clinical chytridiomycosis dramatically interrupted tissue homeostasis; affecting numerous biosynthetic and degradation pathways, and causing pronounced dysregulation of cellular energy metabolism in both the skin and liver tissues. We identified several key metabolites that may provide targets for research and intervention, and may help explain differences in susceptibility. The 20 key differentially abundant metabolites identified included α-ketoglutarate (2-oxoglutarate), isoleucine, pantothenate, serotonin, glutamate, myo-inositol, putrescine, 5HIAA, adenine, galactose, leucine, pyroglutamate, creatine, threonine, citrate, aspartate, tartrate, ribose, guanine, and adenosine  or effects on plasma electrolyte balance that had previously been describedL. v. alpina metabolome of clinically diseased frogs was distinctly divergent from both unexposed control and subclinically Bd-infected frog groups, likely associated with end-stage pathophysiological changes occurring in the tissues shortly prior to disease-induced mortality10. The most important effect was the organism-wide disruption of cellular energy pathways in moribund frogs, particularly the tricarboxylic acid  cycle, glycolysis, and anaerobic fermentation pathways  from the oxidation of acetyl-CoA44 which is primarily derived from dietary sources  in the healthy fed state. Here we found that several key intermediates of the TCA cycle and their anaplerotic precursors were significantly depleted in skin and liver tissues from frogs of all populations showing clinical signs of chytridiomycosis (sampled at 28–30 days post-exposure), relative to negative control and subclinically infected frogs. These metabolites included α-ketoglutarate, glutamate, citrate, fumarate and aspartate  is one of the first clinical signs of chytridiomycosis47. Together with infection-associated energy losses48, anorexia could lead to energy dysregulation in diseased frogs, and may contribute to the observed lethargy of the moribund state.Within liver samples of clinically diseased frogs we also identified significant reduction in the relative concentration of three glucose precursors ogs Fig. . As the 49 for gluconeogenesis or through toxic effects. Isoleucine was the most significantly elevated metabolite in the liver samples of clinically diseased frogs, but it was also significantly elevated in skin samples. Leucine was significantly elevated in the liver of clinically diseased frogs , and these metabolic disorders have been linked with neurotoxicity and ketoacidosis among other signs51.The accumulation of essential branched-chain amino acids (BCAAs) such as isoleucine and leucine in the moribund state is likely a by-product of tissue protein breakdownogs Fig. . Unlike 41 indicated that other amino acids were predominantly depleted in the moribund state and that their biosynthetic and degradation pathways were affected, particularly in skin tissues. In most cases this was linked with the depletion of α-ketoglutarate and glutamate. Additionally, the function of numerous other biosynthetic and degradation pathways was found to be compromised in clinically diseased animals, suggesting generalized failure of cellular homeostatic mechanisms and secondary functions. The most significantly affected pathways included nucleotide, carbohydrate, secondary metabolism, cofactor, and hormone biosynthesis and degradation as well as metabolic regulatory pathways . Although studies have not demonstrated effects of chytridiomycosis on blood acid-base balance57, the evidence for reduced function of acid resistance pathways may be due to over utilization of buffering mechanisms due to excess BCAAs. Our results may indicate that systemic metabolic disruption due to infection with Bd may contribute to disease signs.In contrast to elevated BCAAs, pathway analysisays Fig. . Metabol58. As such, increased concentrations of putrescine may be an indicator of catabolism and were likely responsible for the putrid smell of infected individuals . Although putrescine could be associated with post-mortem autolysis during tissue collection, this cause is unlikely as putrescine was significantly positively correlated with sampling group . Putrescine is also active in immune processes and basic homeostatic mechanisms60. Interestingly, ornithine, and the polyamines putrescine and agmatine, are potent inducers of a trichothecine mycotoxin of Fusarium graminearum, whereby mycotoxin production is linked to the natural accumulation of these metabolites within the host61. These metabolites may thus also be tightly coupled to clinical signs of disease in moribund animals.As an example of toxin accumulation, putrescine was found to be significantly elevated in the skin and liver of clinically diseased frogs in comparison with control and subclinically infected frogs Fig. . Putresc66. Depletion of these metabolites may be both causal and a consequence of multiple pathophysiological features of clinical chytridiomycosis in the amphibian host, although it is as yet unknown whether depletion due to utilization by Bd may also be involved.Common to several of the pathway disruptions we have discussed thus far is the severe depletion of both α-ketoglutarate and the amino acid glutamate. These metabolites were significantly depleted in both skin and liver tissues of clinically diseased frogs, with α-ketoglutarate identified as the metabolite with the most significantly decreased concentration in the skin samples, and glutamate the second most significantly reduced in the liver samples. Enzymatically linked pyroglutamate was also significantly depleted in both skin and liver tissues Fig. . Alpha-k67. Although we did not examine brain tissues, if glutamate depletion is systemic this could contribute to the loss of alertness and righting reflex observed in clinically diseased animals68. Alpha-ketoglutarate and glutamate are also precursors for GABA which was similarly found depleted in both skin and liver tissues  when compared with frogs from the other populations. In this study we found that pantothenate, myo-inositol, isoleucine, leucine, and creatine demonstrated highly significantly different concentrations in skin samples between populations  is an essential water-soluble vitamin for the synthesis of coenzyme-A (CoA) which is principally involved in energy (tricarboxylic acid cycle) and fatty acid metabolism. This metabolite is also essential for the growth of pathogenic organisms, including fungi83, and has been previously measured in developing frogs84. Analogues of pantothenate have even shown antimicrobial capabilities, as they prevent the uptake of the naturally occurring endogenous and essential metabolites86. Whilst there is no determined time series association, different amounts within the skin among frog populations may imply a selective pressure towards this difference. It is unclear, however, how differences in pantothenic acid relate to the clinical evidence of survival between the populations, since in the clutches we compared, frogs from Eucumbene were more susceptible to infection than those from Kiandra.Pantothenate demonstrated the most significant difference  in relative concentration between source populations, with skin samples from Eucumbene frogs having higher concentrations than samples from both Grey Mare and Kiandra. Pantothenate  were also identified in the L. v. alpina skin.Interestingly, the metabolite composition of the skin (site of Bd infection) and liver tissues differed substantially, consistent with the underlying differential physiology and biochemistry of these tissues, regardless of health or disease status  or gastrointestinal tissues. Metabolites of non-host origin could not all be unambiguously distinguished from the endogenous host metabolites with the methods utilized here yet this distinction in future pathogenesis studies may be worthwhile . Here, we sought to clarify the origin by comparing the metabolite profiles of the host to that of Bd in culture (data not shown) for an exclusionary approach to dissecting the origin of metabolites as frog or fungal, although the expression of the fungal metabolome will likely differ between an 91. The inclusion of fluxomics data would also likely provide further detail of metabolite origin. Further work on assessing the importance of the specific metabolites  could include examination of the tissue metabolome of other amphibian species. Further studies could also include targeted functional validation studies to characterize metabolite role by blocking production within the host, inhibiting activity within skin explants ex vivo, or testing the effect on Bd growth in vitro92.Depending on the regulatory mechanisms, and proximate metabolite source or relationship to gene and protein expression, a systems biology approach may greatly assist in understanding these complex host-pathogen relationships. Future approaches could include (1) comparing the metabolomes of infected frogs, with those that have been treated with an antibiotic to reduce or eliminate bacterial load, (2) biolabeling , or alternatively (3) mass spectrometry imaging to examine the source of metabolites. The concentration of metabolites in tissues is also reliant on their flux; the rate of production (upstream processes), but also their rate of modification, anabolism or catabolism to other metabolites, or removal from the systemL. v. alpina skin and liver tissues. Metabolites relating to differences in the population of origin, which may be associated with variation in phenotypic resistance or tolerance between populations were also uncovered. Most notably, the differences in metabolite expression that we have identified may be characteristic of processes involved in chytridiomycosis across host species, and may have broad relevance to understanding the pathogenesis of fungal skin diseases and future control of devastating fungal pathogens such as Bd.To our knowledge, this study was the first to apply a non-targeted metabolomics approach to a fungal wildlife disease and specifically to dissect the host-pathogen interface through analysis of host tissues. We demonstrated that the metabolome of clinically diseased frogs diverged distinctly from that of subclinically Bd-infected frogs and unexposed control groups. We found that chytridiomycosis dramatically affected organism-wide homeostatic mechanisms, including interruption of biosynthetic and degradation pathways as well as causing dysregulation of cellular energy metabolism. Depletion of α-ketoglutarate and glutamate appeared central to these changes. This was also uniquely the first metabolomics investigation of the alpine tree frog, and as such we have revealed numerous interesting observations on the composition of the Supplementary Information


Lack of uniform diagnostic criteria often poses a challenge in the diagnosis and management of tubercular uveitis. The index case describes an unusual presentation of tubercular panuveitis initially misdiagnosed as sympathetic ophthalmia, where the appropriate diagnosis was made using various imaging and laboratory investigations.A 52-year-old Indian woman underwent multimodal imaging, extensive clinical and laboratory work-up, and analysis of microbiological and histopathological specimens. At presentation, her best-corrected visual acuity (BCVA) was 20/30 in OD and no perception of light in OS. Ocular examination revealed multiple grayish-yellow choroiditis lesions resembling Dalen-Fuch’s nodules, vitritis, and disc edema. Diagnosis of sympathetic ophthalmia was made and patient treated with intravenous and oral corticosteroids and immunosuppressive therapy. After an initial favorable response, the lesions progressively increased with worsening of vitritis. Due to worsening of chorioretinal lesions which were atypical for sympathetic ophthalmia, further investigations were performed that revealed positive tuberculin skin test and contrast-enhanced computerized tomography chest showed calcified mediastinal lymph nodes. Enucleation of OS confirmed acid-fast bacilli on Ziehl-Neelsen staining, tubercular granulomas on histopathology, and positive polymerase chain reaction. Anti-tubercular therapy and oral steroids were started with good healing response.Tubercular uveitis may have protean clinical manifestations. Thorough clinical evaluation and molecular/histopathological evaluation helps in establishing the diagnosis and the institution of appropriate therapy. Mycobacterium tuberculosis organisms on smear microscopy or culture from ocular samples difficult. Nucleic acid amplification techniques such as multi-targeted polymerase chain reaction (PCR) have greatly enhanced the sensitivity and specificity of detection from ocular tissues [Intraocular tuberculosis (IOTB) is a form of extrapulmonary TB and has been reported to be the most common etiology of infectious uveitis in TB-endemic regions –3. It ca tissues –6.Accurate diagnosis of IOTB often needs a multimodal approach with a combination of a comprehensive clinical evaluation and targeted laboratory investigations. The index study describes an unusual case where the diagnosis of IOTB was confirmed with the help of microbiological and histopathological evaluation of the enucleated specimen from the fellow non-seeing eye.A 52-year-old woman of Indian origin presented in November 2015 with complaints of decreased vision in right eye (OD) for the past 1 month. She had a history of trauma to the left eye (OS) with a stone in 2013. At the time of presentation, the best-corrected visual acuity (BCVA) was 20/20 in OD and no perception of light in OS. On ocular examination, anterior segment examination of OD revealed 1+ cells and 1+ flare, and granulomatous keratic precipitates (KPs) in the anterior chamber. Posterior segment examination showed presence of round-to-oval outer retinal/choroidal grayish-yellow lesions in all the quadrants with presence of peripapillary fluid Fig. , d showeHowever, on her follow-up visit at 3 months, the fundus lesions had increased in number and size with recurrence of vitritis Fig. . Her BCVLaboratory tests revealed a positive tuberculin skin test (18 × 18 mm) and QuantiFERON TB Gold® test. Contrast-enhanced computerized tomography of the chest revealed multiple calcified lesions in both the upper lobes of the lung with small mediastinal lymph nodes . Whole body positron emission tomography (PET) scan revealed fluorodeoxyglucose (FDG) avid subcentimetric mediastinal lymph nodes and FDG avid patchy consolidatory changes in both lung upper lobes likely to be post-granulomatous changes. An FDG avid lesion was present in the left orbit.In order to rule out the presence of lymphoma and to determine if the etiology of the fundus lesions was related to TB, the patient was advised enucleation of the non-seeing eye (OS). The patient agreed for the procedure and enucleation of OS was performed. Histopathological examination revealed presence of multiple choroidal granulomas with caseous necrosis Fig. , b. TB PThe index case presented as a diagnostic challenge since granulomatous panuveitis in a monocular patient with a history of trauma in the fellow eye was highly suggestive of sympathetic ophthamia. Our patient demonstrated interval improvement in vitritis and optic disc edema, and the choroidal lesions seemed to heal during the initial follow-up after initiation of intravenous methylprednisolone, oral corticosteroids, and azathioprine Fig. a. HoweveAn important learning point in our case was the detection of choroidal granulomas on EDI-OCT and identification of FAF lesions Figs.  and 3. WThus, the diagnosis of IOTB is often challenging due to its protean clinical manifestations. However, once the diagnosis is confirmed, treatment with ATT and corticosteroids may result in resolution of the choroiditis lesions with progressive hypo-autofluorescence on FAF imaging. The limitations of our case were the non-availability of indocyanine green angiography to detect the choroidal granulomas. However, choroidal granulomas can be well demonstrated on EDI-OCT. , 13 Our The index case report demonstrates challenges encountered in diagnosing and managing IOTB. Our patient presented with granulomatous panuveitis that was initially misdiagnosed as sympathetic ophthalmia. Techniques such as histopathology, multimodal imaging including FAF and EDI-OCT, as well as PCR analysis greatly aid in arriving at the accurate diagnosis in such challenging cases.


Plant associated microbes rely on secreted virulence factors (effectors) to modulate host immunity and ensure progressive infection. Amongst the secreted protein repertoires defined and studied in pathogens to date, the CRNs (for CRinkling and Necrosis) have emerged as one of only a few highly conserved protein families, spread across several kingdoms. CRN proteins were first identified in plant pathogenic oomycetes where they were found to be modular factors that are secreted and translocated inside host cells by means of a conserved N-terminal domain. Subsequent localization and functional studies have led to the view that CRN C-termini execute their presumed effector function in the host nucleus, targeting processes required for immunity. These findings have led to great interest in this large protein family and driven the identification of additional CRN-like proteins in other organisms. The identification of CRN proteins and subsequent functional studies have markedly increased the number of candidate CRN protein sequences, expanded the range of phenotypes tentatively associated with function and revealed some of their molecular functions toward virulence. The increased number of characterized CRNs also has presented a set of challenges that may impede significant progress in the future. Here, we summarize our current understanding of the CRNs and re-assess some basic assumptions regarding this protein family. We will discuss the latest findings on CRN biology and highlight exciting new hypotheses that have emanated from the field. Finally, we will discuss new approaches to study CRN functions that would lead to a better understanding of CRN effector biology as well as the processes that lead to host susceptibility and immunity. Phytophthora genus rank amongst the most devastating pathogens, collectively affecting virtually every dicotyledonous crop plant  that suppress immunity and trigger susceptibility in a process dubbed Effector-Triggered Susceptibility (ETS) . AcquisiMany cellular processes contribute to host immune signaling or PTI . Given tPhytophthora genus, two predominant classes of cytoplasmic effectors have been identified and studied, namely the RXLR and CRN effector protein families. Both protein classes feature modular architectures, featuring motifs or domains required for delivery situated at the N-terminus (the RXLR motif for RXLR effectors and the LXLFLAK motif for CRN proteins), followed by C-terminal domains that carry effector functions  factors in the presence of specific (intracellular) receptor-like resistance (R-) genes have prompted and driven the discovery of a plethora of effector targets, virulence functions and molecular strategies within this family  phenotype when systemically expressed in plant tissue  sequencing approach and found to have a predicted signal peptide. Subsequent application of a high-throughput functional expression assay in planta led to the identification of proteins that induce cell death upon expression in plants, two of which (CRN1 and CRN2), were found to be related on the sequence level    were found to be dispensable for cell death induction and CRN effectors thus seemingly acted inside plant cells, it was suggested that CRN N-termini specify the secretion and translocation of effector domains into the host  . Aiming sequence . This obn family . From thgure 1A) . In linethe host .P. infestans mycelia revealed that 98% of all annotated CRNs are expressed and 66% of those were amongst the top 10% when assessed for array signal intensities. These results were similar to those found for RXLR effectors, where 66% of the genes were expressed and 4% were in the top 10%  transcriptional regulation, translational control and post-translational modifications form important means by which level of functional effector proteins could be controlled. A quantitative phospho-proteomics study in P. infestans revealed that CRN proteins are phosphorylated across distinct life cycle stages . Whilst computational and deletion analyses pointed at a role for CRN C-terminal domains inside the host cell and implicated N-termini in delivery, more concrete evidence emerged from functional studies in nfestans  and sugga system . The abia system . ImportaFigure 1B) (in vivo.A recent study shed a different light onto the supposed requirement of LXLFLAK motifs in CRN translocation . Genome gure 1B) . With a in planta  respectively  accumulation. PsCRN63 was shown to increase H2O2 accumulation while PsCRN115 was shown to suppress this process. It was also suggested that PsCRN63 recruits plant catalases into the host nucleus leading to catalase destabilization while PsCRN115 inhibits these events  induced cell death when over-expressed in N. benthamiana leaves. The remaining 9 were able to suppress cell death caused by PsojNIP; 8 by PsCRN63; 5 by Avr3a + R3a; and 3 by Avh241  motif that inhibits the expression of Heat Shock protein (HSP) genes in A. thaliana, N. benthamiana and soybean. This is achieved by the binding of PsCRN108 to conserved promotor regions of HSP genes named heat shock elements (HSEs). HSEs are bound by heat shock transcription factors (Hsf’s) leading to tight regulation of HSP expression. PsCRN108 was shown to be able to inhibit the binding of the Hsf AtHsfA1a which induces HSP gene expression in response to stress (Aphanomyces euteiches (AeCRN13) and from the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13) also showed that both these effectors directly interact with DNA. These two cell death inducing effectors contain an HNH-like endonuclease motif that triggers plant DNA damage response (DDR). Mutation of key residues in the AeCRN13 HNH-like endonuclease motif abolished AeCRN13 capacity to interact with DNA, to induce DDR and to increase the susceptibility of Nicotiana Benthamiana to P. capsici. Thus the function of the HNH-like endonuclease motif on inducing DDR has been connected to AeCRN13 virulence function  . From this, the authors suggest that the LXLFLAK motif is important for translocation as they are important for Ubl domain structure. This Ubl N-terminal domain is significantly related to those found in fungal signaling proteins, namely SSK1/Mcs4. SSK1 orthologs play important roles in stress responses in various true fungi, and in some cases, are known to do so in a phosphorylation-dependent manner, employing an interaction between their N-terminal domains and a MAPKKK heteromer  . In thisgure 1B)  remains eteromer . From theteromer . To whatAngomonas, shows a hydrophobic region implying possible membrane interactions and secretion. Moreover, CR proteins from diverse eudicot plants contain CR headers that contain helix-turn-helix (HTH) domains also found in the Myb transcription factor family. In Myb transcription factors, these domains are implicated in DNA-binding, suggesting that these eudicot proteins might not be secreted but target intracellular invasive DNA . Although high levels of diversity are known to be present, classification led to a limited set of domain configurations that were found to be prevalent. For example, CR C-termini containing a P-loop NTPase domain, combined with a nuclease domain of the restriction endonuclease (REase) superfamily were found to account for slightly more than one-fourth of all CR C-termini. In addition, CR C-termini in which a REase superfamily domain is coupled to protein kinase domain was found to account for approximately one-sixth of the C-termini domains present in the dataset. In both cases, the toxicity function is believed to be specified by the REase domain, whilst the NTPase and Kinase domains would regulate REase activity or affinity toward nucleic acids (such as DNA) . Indeed, this model is consistent with exclusive localization of CRN proteins to the nucleus and importantly, two recent reports demonstrating binding of CRN effectors to DNA . Thus, the prediction of enzymatic domains in CR or CRN proteins represents one important mean by which new hypotheses about CRN function can be constructed and subsequently tested  biology. In contrast to CRN N-termini and consistent with previous observations, C-terminal domains are highly diverse and often resemble enzymes ( as DNA) . This vi as DNA) . Moreoves to DNA . Severals to DNA  . Furthermore, we suggest new ways of tackling these areas, by taking advantage of our knowledge on CRN domain structures, plant–pathogen interactions and effector classes that are better understood.With the increasing availability of pathogen genomes, understanding effector mode of action remains a major challenge and bottleneck. Despite recent efforts, new and more systematic ways are required to further understand CRN effector biology. Here we describe the areas where our knowledge on CRN effector biology remains poor or new opportunities have arisen for further exploration . One way of confirm this notion is to create Phytophthora strains, unable to form haustoria, by disrupting factors required for their formation, such as Haustorial Membrane Protein 1 (HMP1)  . The sucCRN N-termini and CR-header domains have been divided into a diverse set of sub families raising the possibility that not all CR or CRN N-termini facilitate translocation . To helpP. infestans, CRNs that share predicted effector (sub) domains, feature contrasting cell death inducing activities define the (cell death) activities of this protein family in more detail.With CRN effectors being highly expressed and having the ability to cause host cell death, it seems it would be necessary for CRNs to be tightly regulated during the infection process. As discussed above, post-transcriptional and post-translational control could be associated with this regulation. A better understanding on the control of CRN activity on both the transcript and protein level should allow key insights into effector as well as pathogen biology. As stated above siRNAs and PTMs, namely phosphorylation, could be responsible for CRN regulation. However, these two processes have not been connected with the control of CRN function to date. Understanding Post-translational modifications, in particular those that occur in in planta should further rationalize intense future studies on CRN effector complex function in host-microbe systems.An important factor, complicating the interpretation of cell death or virulence phenotypes is the apparent ability of CRN proteins to form homo-dimers or dimerize with other CRN effectors . While iDespite the valuable efforts aiming for the identification and characterization of CRN virulence functions, CRN effector biology remains still largely uncharacterized. However, with our available knowledge on CRN distribution and structure, and with the ever improving techniques that enable an efficient study of plant-pathogen interactions we are on the verge of truly unveiling the role of CRN effectors and their biology. Indeed, CRN effector biology is emerging as a fertile research area where new and possibly game-changing concepts in effector biology may be discovered.TA and EH wrote the paper. GT and GM contributed with comments and suggestions.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.


Psychotic experiences (PEs) are relatively common in childhood and early adolescence, being present in 17% of children aged 9 to 12 . Research suggests that young people who experience PEs are more vulnerable to psychopathology later in life, despite PEs being transient in 78.7% of cases . While childhood PEs are associated with poorer functioning , it has not yet been established whether the impact of PEs on functioning persists into later life.52 participants from a prospective cohort study (retention rate: 60.4%) of Irish young people were included on the basis that they had completed a clinical interview at all three data-collection time points . Following each interview, participants were scored on the Global Assessment of Functioning (GAF) scale, and given a Current (C-GAF) score and a Most Severe Past (MSP-GAF) score. Fixed-effects repeated-measures models were used to compare the scores of those with a history of PEs at T1 (n=18) to those without (n=34), accounting for age, gender, and childhood functioning. Secondary analyses investigated whether differences in functioning were evident in those who reported transient PEs .Overall, participants who had reported childhood PEs (T1) received significantly lower C-GAF scores  and MSP-GAF scores  than those without PEs. Simple effects analysis indicated that deficits in the PE group were evident at each time point for both C-GAF scores  and MSP-GAF scores , indicating poorer functioning from childhood, through adolescence, into early adulthood. There was no significant effect of the co-variates.When the analysis was restricted to a comparison of participants who reported PEs at T1 only (i.e. transient PEs) and those with no history of PEs, the PE group had poorer functioning scoring than their peers across the three time points .The analysis provides longitudinal evidence that the presentation of PEs is associated with persistent poor global functioning throughout adolescence and into early adulthood, even when the phenomena are transient. PEs appear to be a marker for vulnerability that extends beyond mental disorder. These results tentatively suggest a causal link between PEs and poorer functioning later in life, as the difference in functioning between the groups in early adulthood was still evident after accounting for childhood functioning. Moreover, the disparity between the groups is clinically relevant, with the PE group scoring one to two categories lower than their peers on the GAF scale even into early adulthood. Childhood PEs are an excellent prognostic marker for future functioning and providing targeted early intervention for these individuals may reduce the likelihood of developing a significant clinical disorder later in life.


Normal pregnancy corresponds to a procoagulant state. Acute myocardial infarction during pregnancy is rare, yet considering the low non-pregnant risk score of childbearing women it is still surprisingly frequent. We report a case of postpartum recurrent non-ST elevation myocardial infarction in a 40-year-old caucasian woman with essential thrombocythaemia in the presence of a positive JAK-2 mutation and an elevated anti-cardiolipin IgM antibody titer. In the majority of cases of myocardial infarction in pregnancy or in the peripartal period, atherosclerosis, a thrombus or coronary artery dissection is observed. The combination of essential thrombocythaemia and elevated anti-cardiolipin IgM antibody titer in the presence of several cardiovascular risk factors seems to be causative in our case. In conclusion, with the continuing trend of childbearing at older ages, rare or unlikely conditions leading to severe events such as myocardial infarction must be considered in pregnant women. Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder characterized by a sustained elevated platelet count with a tendency to both thrombosis and hemorrhage ,2. In ETPregnancy is an acquired risk factor for thromboembolism associated with increased coagulation and decreased fibrinolysis . HemodynPregnancy-related complications in patients with ET remains a challenge as platelet count has not been shown to represent a risk factor for pregnancy complications, nor the use of aspirin has been demonstrated to influence pregnancy outcome . We descth week  monotherapy with 50 mg metoprolol was started due to postpartal hypertension.A 40-year-old caucasian woman, gravida 3 para 2, developed postpartal arterial hypertension. Her first pregnancy, three years earlier, had been complicated by an early spontaneous abortion at gestational week 7. In the following pregnancy, one year later, while on prophylactic low-molecular weight heparin due to the previous miscarriage, a cesarean section was performed at gestational week 32 due to intrauterine fetal growth restriction (IUGR), infant birthweight 810 g, 1-, 5-, and 10-min Apgar scores of 7, 9 and 10, respectively; umbilical cord arterial blood pH: 7.00. During the present pregnancy, low-dose aspirin was given from gestational week 13 to week 37 due to IUGR in the former pregnancy. Shortly after an uneventful elective cesarean section performed in the 39The patient had the same partner since the first pregnancy and no history of spontaneous bleeding, thrombosis nor had she been diagnosed to have elevated platelet counts requiring treatment. During the present pregnancy platelet counts were initially elevated but continuously decreased from 598 G/L to 346 G/L at the time of caesarean delivery.2). No medication or illicit drugs were taken. The patient was breastfeeding.Risk factors for coronary heart or thromboembolic diseases, including smoking, hyperlipidemia, diabetes mellitus or atrial fibrillation were absent except for a positive family history of coronary artery disease and overweight . With the clinical and laboratory findings suggesting anterior wall myocardial infarction, the patient was started on aspirin, intravenous heparin and nitroglycerin, and a coronary angiography was performed -mutation, a matching bone marrow finding without evidence for iron deficiency or infection. Potential hereditary risk factors for thrombophilia , factor XIII, and PAI-1) were excluded. Cytogenetics for BCR-ABL, t, were negative. IgM anticardiolipin antibodies were transiently elevated and ANA was 1:160 with negative anti-dsDNA and anti-Histone antibodies.Oral hydroxyurea was added to aspirin, but had to be discontinued due to severe alopecia. In response to alternate treatment with low dose peg-interferon α-2a, platelet count normalized. At the one-year follow-up, the patient presented with normal blood pressure and remained in remission for hematological and cardiac disease while remaining on aspirin.Pregnancy is not commonly considered a risk factor for acute myocardial infarction, however pregnancy increase the risk of acute myocardial infarction 3- to 4-fold ,15. ManyCardiac function and hormonal milieu are unfavorably altered in pregnancy whereas cardiac output is increased in the presence of elevated levels of estrogen and progesterone ,13. The Superimposed hypertension, as in the reported case may further damage blood vessels already weakened by hemodynamic stress and hormonal alterations. Overall, an increasing prevalence of cardiovascular risk factors with advanced maternal age contributes to pregnancy-associated complications ,19. MoreIn a recent retrospective review of 228 reported cases of pregnancy-related acute myocardial infarction, morphology of the coronary arteries was present in 164 cases. Atherosclerosis with or without intracoronary thrombus was found in 70 cases (43%), and definite or probable coronary thrombus without evidence of atherosclerotic disease was present in 22 (13%). Coronary artery dissection was verified in 24%, spasms and embolus in 2% and 1% respectively. Normal coronary arteries were found in 20% [Published studies on ET pregnancies report live birth rates of 50-70% and spontaneous abortion rates of 25-50% ,24. In aDespite the fact that a decrease in platelet count during pregnancies is well documented, pregnancies in ET patients frequently end in early spontaneous abortions, during the first trimester . Their oOur patient's history of previous early spontaneous abortion, IUGR and the presence of anticardiolipin antibodies suggest the possibility of an incomplete antiphospholipid antibody syndrome, which represent the most common acquired thrombophilia of pregnancy and has been associated with myocardial infarction ,29.Antiphospholipid antibody syndromes may also be associated with autoimmune diseases such as systemic lupus erythematosus, which can cause pericarditis and myocarditis . In the Our patient had experienced a severe migraine attack which has also been found to be a risk factor for myocardial infarction during pregnancy . The posDespite the fact that ET-pregnancies carried to term are rarely complicated by thrombohemorrhagic events our patient had experienced recurrent postpartal ACS in the presence of essential thrombocytosis and elevated antio-cardiolipin IgM antibodies. Since patients with ET seems to have an increased prevalence of antiphospholipid antibodies which may be associated with thrombosis  it is noThere are several recommendations  that womET: essential thrombocythaemia; AMI: acute myocardial infarction; ACS: acute coranary syndrome; LAD: left anterior descending; MTHFR: methylenetetrahydrofolate reductase; CK: creatine kinase.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SA, MGM, RL, DS, made substantial contributions to patient care and to the preparation of the manuscript. IS and VL contributed to the manuscript preparation. All authors read and approved the final manuscript.Appendix 1.Diagnostic approach of suspected acute myocardial infarction (AMI) in pregnant womenSymptoms mimicking myocardial ischemia may also be present during healthy pregnanciesST-segment depression may also be present due induction of anesthesia for cesarean section and may persist after elective cesarean sectionTroponin I levels: is the most sensitive marker for AMI. The majority of healthy pregnant women remain below the upper limit of normal Troponin I levels after delivery.Creatine kinase/CK-MB: can be significantly elevated up to 24 h after delivery in healthy pregnant womenSafe and accurate for evaluating wall-motion abnormalitiesSubmaximal protocol evaluation  with fetal monitoring for the diagnosis of myocardial ischemia or risk stratification following AMIBoth modalities may add further information with overall small or none fetal exposure to radiationPossible increased risk of coronary dissection and great risk of fetal exposure to radiationBoth the cardiologist and obstetrician should establish the treatment plan


Even though organic light-emitting device (OLED) technology has evolved to a point where it is now an important competitor to liquid crystal displays (LCDs), further scientific efforts devoted to the design, engineering and fabrication of OLEDs are required for complete commercialization of this technology. Along these lines, the present work reviews the essentials of OLED technology putting special focus on the general working principle of single and multilayer OLEDs, fluorescent and phosphorescent emitter materials as well as transfer processes in host materials doped with phosphorescent dyes. Moreover, as a prototypical example of phosphorescent emitter materials, a brief discussion of homo- and heteroleptic iridium(III) complexes is enclosed concentrating on their synthesis, photophysical properties and approaches for realizing iridium based phosphorescent polymers. Since the first observation of organic electroluminescence  in the 1960s by applying hundreds of volts to an anthracene single crystal . BoostedAlthough OLEDs already meet the requirements for some practical applications in, e.g., portable electronics like cellular phones or digital cameras  with opeet al. in 1987  immobilized on a transparent carrier material such as glass or a flexible polymer . Al. Al37·nHtris-cyclometalated iridium(III) compounds was published in the early 1980s being observed as a byproduct of the synthesis of di-μ-chlorotetrakis-2-phenyl-pyridine)diiridium(III) . Ho. Hotris-operties –63 and uoperties , therefotris-cyclometalated iridium complexes, an alternative synthesis approach is based on incorporating ancillary ligands such as acetylacetonates  complexes has received considerable attention because of a series of practical applications such as flat-panel displays. Even though numerous organoiridium compounds have been reported giving efficient electroluminescence in the red , green  and blueThe absorption and emission spectra of phosphorescent metal complexes are in general influenced by various parameters such as the valence electron configuration at the metal, the type of the electronic transitions or the correlation among lower lying electronic excited states . Similar6 and d8 transition metal complexes and play, therefore, a major role in the photophysics of iridium(III) compounds. Intraligand (IL) π–π* excited states originate from electronic transitions of the ligand. If the metal perturbation upon coordination is minimized, their spectral properties often closely resemble the free ligand states. Finally, ligand-to-metal charge transfer (LMCT) excited states are occasionally observed in complexes with metal atoms in high oxidation states or in d10 complexes [Among them, metal-centered (MC) excited states are typically present in metal complexes with partially filled d shells at the metal center. The corresponding d-d transitions are Laporte-forbidden and, consequently, exhibit very low transition probabilities. Metal-to-ligand charge-transfer (MLCT) states involve electronic transitions from metal based d orbitals to a ligand centered π* antibonding orbital. Emissive MLCT states are particularly observed in domplexes , 78, 79.All these transitions determine the photophysical properties of transition metal complexes and can be used for the interpretation of experimentally observed spectra or prediction of absorption and emission characteristics of novel compounds , 78, 79.−1 cm−1  complexes typically display absorption bands with extinction coefficients between approximately 50000 and 6000 L molvia changing the ligand structures  luminophores applying the above described tuning procedures giving materials with a broad range of excited state lifetimes  and quantum yields approaching 100% . As a coEven though many phosphorescent dyes are very promising candidates for realizing highly efficient OLEDs, for numerous phosphorescent small molecules serious problems such as the complexity of device fabrication or insufficient fine-dispersion of the dopant in the host material have been encountered. Especially phase separation is concerned as critical drawback, thus, covalent incorporation of the phosphorescent chromophores into polymers has received considerable attention.As a result, numerous concepts for the preparation of phosphorescent polymers have been proposed, among them utilization of ring-opening metathesis polymerization (ROMP), click-chemistry, the use of cross-linkable iridium complexes, grafting approaches or radical polymerization techniques –96. In aHerein the essentials of OLED technology have been reviewed putting special focus on the working principle of single and multilayer OLEDs as well as the use of fluorescent and phosphorescent emitter materials. After discussion of the advantages of phosphorescent dyes doped into suitable host materials and the corresponding energy transfer processes, the synthesis and photophysical properties of phosphorescent iridium(III) complexes have been enclosed. It has been shown that this class of materials exhibits photophysical characteristics readily tuned by changes of the cyclometalating and/or ancillary ligands, thus being ideal candidates for applications in, e.g., full color displays. Furthermore, different strategies for incorporating iridium complexes into polymers have been presented giving phosphorescent polymers with reduced phase separation in combination with facilitated device fabrication (e.g. solution processing).The development of OLEDs has undoubtedly been empowered by different applications in display technology. As a consequence, tremendous scientific efforts have been devoted to this topic concentrating on the design, engineering and fabrication of OLEDs. Nevertheless, the intrinsic limits of OLED technology have not been reached yet. Hence, further progress in device physics and materials science is necessary to provide a step forward to the complete commercialization of this highly promising technology.