As yet, no cure exists for upper-limb paralysis resulting from the damage to motor pathways after spinal cord injury or stroke. Recently, neural activity from the motor cortex of paralyzed individuals has been used to control the movements of a robot arm but restoring function to patients' actual limbs remains a considerable challenge. Previously we have shown that electrical stimulation of the cervical spinal cord in anesthetized monkeys can elicit functional upper-limb movements like reaching and grasping. Here we show that stimulation can be controlled using cortical activity in awake animals to bypass disruption of the corticospinal system, restoring their ability to perform a simple upper-limb task. Monkeys were trained to grasp and pull a spring-loaded handle. After temporary paralysis of the hand was induced by reversible inactivation of primary motor cortex using muscimol, grasp-related single-unit activity from the ventral premotor cortex was converted into stimulation patterns delivered in real-time to the cervical spinal gray matter. During periods of closed-loop stimulation, task-modulated electromyogram, movement amplitude, and task success rate were improved relative to interleaved control periods without stimulation. In some sessions, single motor unit activity from weakly active muscles was also used successfully to control stimulation. These results are the first use of a neural prosthesis to improve the hand function of primates after motor cortex disruption, and demonstrate the potential for closed-loop cortical control of spinal cord stimulation to reanimate paralyzed limbs. A long-standing ambition of neural prosthetics has been to reconnect artificially parts of the nervous system that have been disconnected as a result of injury Craggs, . One sucBrain-Machine Interface techniques developed first in monkeys  using muscimol. Spike activity recorded from ventral premotor cortex (PMv) or residual electromyogram (EMG) activity was used to control stimulation of the spinal cord.Experiments were approved by the local ethics committee at Newcastle University and appropriate UK Home Office licenses in accordance with the Animals (Scientific Procedures) Act 1986.(Macaca mulatta) were trained to perform a self-paced reach-and-grasp task . The wrist extensor ECR was chosen as a control to monitor paralysis and stimulation effects. In addition, a recording chamber was fixed over the right arcuate sulcus for intracortical microstimulation (ICMS) mapping. The hand region of PMv was identified from low threshold (<50 μA) responses in hand muscles to ICMS trains . In a second surgery, a custom-made moveable microwire array  patch electrodes were implanted over hand and forearm muscles and tunneled subcutaneously to a connector on the head  responses in hand muscles. Muscimol  was injected into hand area of M1 Figure  under keAt the beginning of each experimental session, we assessed the stimulation thresholds of the intraspinal electrodes. We used trains of 3 biphasic pulses  and increased the current up to 100 μA until we either observed movements of arm or hand muscles or stimulus-evoked potentials in any of the EMG recordings in at least 50 percent of the cases. For subsequent closed-loop stimulation, we generally chose an electrode whose stimulation caused wrist or finger flexion movements. In monkey R, where multiple electrodes were available simultaneously, the electrode evoking strongest movements at low amplitudes (~50 μA) was chosen.We recorded action potentials of premotor neurons . The neuron whose firing correlated best with onset of movement in the previous, non-stimulation session was chosen to drive ISMS. If that neuron was not available, the animal was observed attempting the behavioral task and a task-related neuron was chosen based on its evident activity pattern, i.e., consistent modulation of firing frequency during several consecutive trials. Discriminated spike events were transmitted in real time to a computer that estimated the instantaneous firing rate Figure . The firIn some experimental sessions, residual motor unit potentials were recorded from muscles mostly paralyzed due to the muscimol injection. In these cases, the action potential of a single motor unit could be discriminated using the same template matching technique as employed for cortical neurons.Efficacy of muscimol paralysis was assessed first by visual inspection and testing the monkeys' ability to grasp a piece of fruit. We then also compared the average EMG responses during grasp movements with and without paralysis.n = 10,000; α = 0.05).We measured maximum lever position during attempted trials to compare task performance between stimulation and control epochs. Since the task was self-paced, attempted trials were aligned by thresholding the firing rate of the neuron controlling stimulation. Trial epochs were 4 s long (1 s pre- and 3 s post-threshold crossing). The maximum lever position for each such epoch was determined, and the difference of mean maximum lever displacements between stimulation conditions was tested for significance using a two-tailed randomization test  on the shuffled trial assignments, using the difference of trial rates as the test statistic.As well as lever displacement , we also measured the rate at which animals performed successful trials . We divided the number of successful trials completed during stimulation and control periods by the total time of each period. In order to determine the significance of differences in trial rates between stimulation and control periods, we created surrogate shuffled data by randomly allocating each trial into stimulation or control periods. To determine the length of an individual trial, we chose the time point half-way between two consecutive trial completion events as the trial boundary. We then performed a permutation test  trials per muscimol session and 112 (31) trials per training session. Monkey R performed 122 (18) trials per muscimol session and 260 (20) trials per training session during the same period.After muscimol injections and while the monkeys recovered from sedation, we assessed the monkey's ability to perform reach and grasp movements by presenting pieces of food. In monkey B, hand and forearm muscles were reliably paralyzed by muscimol injections, leading to severely disabled grasps. In monkey R, despite using more muscimol, the behavioral effects were less reliable and paralysis was often strongest on intrinsic hand muscles. In both monkeys, the effects persisted throughout the duration of the stimulation session up to several hours; stimulation sessions usually lasted 1–2 h.To produce controlled and repeatable motor deficits in macaque monkeys without requiring permanent injury, we inactivated the hand representation of M1 with focal injections of the GABA-agonist muscimol . Trial-averaged EMG plots revealed that stimulation restored task-modulated activation profiles in muscles paralyzed by muscimol injection , and was associated with a significant increase in trial completion rate during stimulation epochs . For comparison, the average success rates during training sessions without muscimol injection were 3.8 trials/min (monkey B) and 5.8 trials/min (monkey R).In individual sessions with both animals we were able to demonstrate improvements in performance, measured both in terms of movement amplitude and rate of successful trials. In the best session with monkey R Figure , lever dp = 0.0001, permutation test) and trial rates were higher .An increasing proportion of spinal cord injuries are motor incomplete DeVivo,  meaning p = 0.2, permutation test as before). Across 27 sessions, Monkey R performed 4671 trials in 1039.6 min with stimulation and 823 trials in 249.8 min without stimulation. This corresponded to trial completion rates of 4.5 vs. 3.3 trials per minute respectively, demonstrating a significant (p < 0.01) performance improvement due to stimulation across the combined sessions. That we were unable to demonstrate significance with the pooled analysis in monkey B may reflect our use in this animal of acutely positioned spinal electrodes which were less effective at producing robust, stable stimulation effects throughout every session. Nevertheless, the fact that we can demonstrate a significant benefit of spinal stimulation within individual sessions (e.g., Figures The behavioral effects of muscimol injections were variable across sessions, ranging from complete paralysis of the hand to relatively minor deficits. In addition, the stimulation effects in monkey B were quite variable due to the use of acutely inserted electrodes. As a result we did not expect to obtain significant performance improvements in every session. Nevertheless, we performed a combined analysis of all sessions in which closed-loop spinal stimulation was attempted, by calculating the overall rate at which successful trials were completed when stimulation was on compared with during catch trials with no stimulation. Combining over all six recording sessions, monkey B performed a total of 476 trials in 297.3 min with stimulation, and 93 trials in 49.6 min without stimulation. This corresponds to an average rate of trial completion of 1.6 trials per minute with stimulation, and 1.9 trials per minute without stimulation. This difference that was not significant (Stability of stimulation responses is a key requirement for a successful spinal implant. In monkey R we tested stimulation thresholds of all 17 electrodes on the FMA over a period from 3 to 112 days after implantation Figure . ShortlyGrouping the electrodes by their length shows that the shorter, more lateral electrodes showed lower thresholds on average, but a larger increase in threshold over time, whereas average stimulation thresholds of longer electrodes decreased over time.In monkey B we were able to follow the activity of one task-related PMv neuron over multiple sessions Figure . During These results are to our knowledge the first demonstration of successful control of a neural prosthesis in primates after disruption of primary motor cortex. Although premotor areas such as PMv have direct projections to the spinal cord (Dum and Strick, It should be noted that the reversible inactivation of M1 used in this study does not model the long-term effects of cortical or spinal cord injury, including plastic changes leading to reorganization of cortical function, up-regulation of spinal reflexes or spasticity. The success of BMI demonstrations in human patients (Hochberg et al., Closed-loop functional electrical stimulation of muscles has previously been shown to improve performance on wrist (Moritz et al., Robust responses to stimulation were observed in one animal over a 112-day period, but stimulation thresholds, particularly those of the shorter electrodes in our array, did increase slowly over the course of the experiment. This may be explained by scar tissue forming between the array and the surface of the spinal cord gradually pushing the electrodes upwards. Since spinal cord injured patients can live for many decades, the stability of electrode implants remains a major challenge for clinical applications of intraspinal microstimulation. It is possible that epidural stimulation of either the dorsal or ventral spinal cord may provide a less invasive and more stable approach for reanimating the paralyzed limb (Sharpe and Jackson, Although we only implemented closed-loop stimulation during experimental sessions, emerging evidence suggests long-term, continuous use of neural prostheses may have further applications in neurorehabilitation by driving activity-dependent plastic changes (Jackson and Zimmermann, 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.


Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome.Zac1/Plagl1 locus is abrogated in oocytes, resulting in failure of DNA methylation establishment at all CpGs of this locus. ChIP analysis in oocytes reveals H3K4me2 enrichment at the Zac1 imprinted control region when transcription is ablated, establishing a connection between transcription and chromatin remodeling at CpG islands by histone demethylases.We perform deep RNA-Seq and de novo transcriptome assembly at different stages of mouse oogenesis. This reveals thousands of novel non-annotated genes, as well as alternative promoters, for approximately 10 % of reference genes expressed in oocytes. In addition, a large fraction of novel promoters coincide with MaLR and ERVK transposable elements. Integration with our transcriptome assembly reveals that transcription correlates accurately with DNA methylation and accounts for approximately 85–90 % of the methylome. We generate a mouse model in which transcription across the By precisely defining the mouse oocyte transcriptome, this work not only highlights transcription as a cornerstone of DNA methylation establishment in female germ cells, but also provides an important resource for developmental biology research.The online version of this article (doi:10.1186/s13059-015-0769-z) contains supplementary material, which is available to authorized users. The DNA methylome is particularly dynamic during germ cell specification and gametogenesis, relating to the epigenetic reprogramming required to ensure the renewal of totipotency at each generation , 2. DNA trans-acting factors, and cross-talk between histone modifications and DNA methyltransferases (DNMTs) .


Ephrins and Eph receptors enable contact-mediated interactions between cells at every stage of nervous system development. In spite of their broad binding affinities, Eph proteins facilitate specificity in neuronal migration and axon targeting. This review focuses on recent studies that demonstrate how these proteins interact with each other, and with other signaling pathways, to guide specificity in a diverse set of developmental processes. The complexity of nervous system function reflects a vast underlying diversity of neuronal cells and their integration in precise circuitry. During development, newly born neurons migrate to their final destination, become the right type of cell, and form precise connections with their synaptic partners. Given the relatively small number of genes in our genome, how is this complexity generated? A major contributor to the formation of neural circuitry is the Eph family of proteins, which comprises Eph receptors and their ephrin binding partners. These membrane-associated proteins participate throughout neuronal development, during which they display promiscuous binding properties yet specify uniquely targeted events from proliferation to synaptogenesis.In this large family of signaling molecules, such event specificity does not generally arise from binding selectivity between Eph receptors and ephrins. Instead, multiple modes of Eph-ephrin signaling provide combinatorial codes that differentiate between groups of cells and coordinate multiple aspects of neural development, including cell migration and axon targeting.Recent studies have demonstrated that differential responses depending on ephrin expression levels and expression of ephrin-associated molecules allow cells born in one place at the same time to adopt distinct migratory routes. In addition, studies have shown how multiple signaling pathways enable distinct axons that grow through a common pathway to terminate in distinct locations.Specification of neural circuits arises from multiple interacting signaling molecules. The large family of Eph proteins uses both redundancy in expression and multiple modes of signaling to direct this specificity, causing neurons to migrate to their final locations and make synaptic connections there that are appropriate for their function. How can a large family of molecules with broad binding capabilities and redundant expression confer this precision? Here, we discuss recent work focused on factors that determine specificity during cell migration, axon guidance, and midline specializations.2. Some interesting new perspectives that take into account the unique features of Eph-ephrin signaling have emerged.Eph receptors and ephrins display broad spatial and temporal expression throughout nervous system development. During early development, they contribute to neurogenesis  bind to all ephrin-A molecules (ephrin-A1-6), and the EphB receptors (1–6) bind to all ephrin-Bs (1–3); some variability has been reported in the binding affinities of individual pairs within a class4. There is some crosstalk between classes in that EphB2 can bind to ephrin-A5 and EphA4 can bind to ephrin-B26.The Eph receptors are the largest known class of receptor tyrosine kinase. Together with the ephrins, they are divided into the A and B classes on the basis of sequence homology and binding affinities9. The communication between receptors and membrane ligands operates in both directions upon the binding of an Eph receptor in one cell with an ephrin on another cell. Forward signaling refers to signal transduction in a cell expressing the Eph receptor, which upon ligand binding initiates tyrosine phosphorylation. In reverse signaling, signal transduction events are initiated in a cell expressing an ephrin upon its binding with an Eph receptor. Both ephrin-A and ephrin-B proteins can mediate reverse signaling12.Unlike most ligands for receptor tyrosine kinases, the ephrins are associated with cell membranes. Ephrin-B proteins contain a transmembrane domain, and ephrin-A proteins are associated with cell membranes through a glycosylphosphatidylinositol linkage. A major consequence of this membrane association is that Eph-ephrin signaling mediates short-range cell-cell communication, although in some cases soluble forms of ephrin molecules can provide longer-range cuesIn cell migration and axon guidance, movement ultimately relies on the integration of signals that influence the cytoskeleton. Eph-ephrin signaling influences the functions of Rho GTPase proteins, which in turn regulate the actin cytoskeleton  cells. This transient population of cells plays a critical role in cortical layering through its release of reelin, which is essential for the characteristic “inside-out” formation of layers. Although apical progenitors require ephrin-B1-mediated adhesion, a recent study usedin vivo time-lapse imaging together with modeling of cell movement to show that tangential dispersion of CR cells relies on contact-mediated repulsion29. Pharmacological and genetic disruption of Eph signaling showed that both EphA and EphB signaling contribute critically to this repulsion29. Interestingly, the function of reelin in establishing the cortical layers depends extensively on ephrin-B-EphB signaling. Reelin enhances clustering of ephrin-Bs and EphBs, in addition to binding to its receptors. Mutant mice lacking ephrin-B-EphB signaling display severe migration phenotypes with inverted lamination, similar to those seen inreeler mice, and activation of ephrin-B-EphB signaling can rescue migration phenotypes inreeler mice31.The diverse and coordinated functions of Eph-ephrin signaling in cell migration are demonstrated by recent studies of cerebral cortex formation. Early in neural tube development, adhesion of neural progenitors to the apical surface is associated with symmetric cell division. A study of null mutations in ephrin-B1 found that these mice exhibited abnormal neuroepithelia and exencephaly32. The tangential dispersion of a subpopulation of these developing cortical neurons promotes crosstalk between clonal columnar units and is significantly reduced in mutant mice lacking ephrin-A signaling33.Another influence of Eph proteins occurs during radial migration. During cortical development, electrical synapses form preferentially between radial glial cells and their sister neurons, forming networks of lineage-related cells in radial columns. When inside-out radial migration is genetically disrupted, the preferential coupling between sister cells is lost34. MGE interneurons also express ephrin-As, and reverse signaling induced in ephrin-As by EphA4 enhances motility in these migrating MGE interneurons35.In addition to regulating radial migration and cell dispersion, Eph-ephrin signaling is critical for the tangential migration of interneurons. Excitatory cortical neurons are born at the ventricular zone and migrate radially. In contrast, inhibitory interneurons are generated in the basal telencephalon and migrate extensively along a tangential route. Cortical interneurons born in the medial ganglionic eminence (MGE) migrate along a deep route, whereas preoptic area (POA)-derived interneurons migrate along a superficial route. These routes exhibit complementary expression of EphA4 and ephrin-B3, respectively. A study using organotypic cultures and stripe assays together with gene knockdown and pharmacological approaches showed that forward signaling through EphA4 and reverse signaling through ephrin-B3 induce repulsion of MGE and POA interneurons in the inappropriate routes36. What accounts for these divergent effects? In cortical interneurons, EphB1-ephrin-B3 reverse signaling leads to enhanced phosphorylation of Src tyrosine kinase and of focal adhesion kinase (FAK), leading to enhanced repulsion. For striatal neurons, this same signaling leads to a dephosphorylation of Src and FAK, which leads to attraction36. These divergent effects might arise from endogenously high levels of phosphorylated Src and FAK in striatal cells and/or to distinct combinations of transcription factors in these two cell populations that can influence elements of signal transduction pathways.The POA also generates striatal neurons, which are generated at a similar time and also express ephrin-B3. However, striatal neurons traverse an intermediate route and terminate in the striatum, which expresses EphB1. Reverse signaling through ephrin-B3 elicited by striatal EphB1 is repulsive for migrating cortical interneurons but is an attractive stop signal for migrating striatal cells37. These authors used chromatin immunoprecipitation and luciferase assays to show that the expression of EphB1 and EphB3 is enhanced by Nkx2-1, which is expressed in striatal but not in cortical interneurons born in the MGE.Striatal interneurons are generated in the MGE along with cortical interneurons. They are attracted to the striatum through Nrg1/ErbB4 signaling and are simultaneously repelled from the adjacent cortex through EphB forward signalingEph-ephrin signaling thus provides both repulsive and attractive cues for tangentially migrating neurons. The determination of migratory route depends on the ensemble of Eph receptors expressed along with the molecular context within cells destined for different routes.40. Since then, further evidence has shown that Eph-ephrin signaling is critical for establishing topographic projections in the auditory system42, somatosensory system43, olfactory system44, and others47. Both forward and reverse signaling play a role, as do attractive and repulsive interactions and interactions incis.The function of Eph-ephrin signaling in axon guidance was originally discovered in the context of topographic mapping. Graded expression of ephrin-As in the optic tectum and opposing gradients of EphA receptors were found to be necessary for forming the high degree of topography seen in this pathway48, laminar49, or ipsilateral versus contralateral50 axon targeting decisions.Formation of topography thus uses graded, or continuous, targeting signals. However, Eph-ephrin signaling also contributes significantly to selection of discrete, or discontinuous, synaptic targets, and these modes of targeting often occur together in a single neural pathway. Interestingly, the function of individual Eph or ephrin family members is specialized within a structure, so that family members regulating topography are distinct from those regulating modular51. This phenotype is recapitulated when the cytoplasmic domain of EphB1 is deleted, suggesting that reverse signaling is not necessary for this targeting choice. Conversely, overexpression of EphB1 in mouse embryos is sufficient to direct retinal ganglion cell axons to the ipsilateral trajectory52. Interestingly, loss or gain of EphB1 reduces or increases ipsilateral targeting, respectively, whereas changes in EphB2 and EphB3 are relatively ineffective, even though these receptors are co-expressed to varying degrees in retinal ganglion cell axons52. Analysis of the effectiveness of overexpressed chimeric receptors suggests that unique sequences in both the juxtamembrane and the extracellular domains of EphB1 work together to direct axons ipsilaterally. The selective role for EphB1 might result from differences in its ability to engage downstream signaling pathways. Additionally, crossing axons might express additional proteins that normally overcome this ipsilateral cue. In this study, the authors overexpressed the zinc finger transcription factorZic2, which is expressed in retinal ganglion cells and which activates EphB1 and regulates numerous other genes as well. They found that early exogenous expression ofZic2 was significantly more effective at inducing ipsilateral projections than EphB1, consistent with the view that a network of genes is needed to balance responses to ipsilateral versus contralateral cues. The identification of these genes and the integration of their roles in target selection, which may require a computational modeling approach, will greatly facilitate our understanding of how Eph-ephrin signaling leads to precision in axon targeting.Eph-ephrin signaling plays an important role in axon targeting at choice points, where axons select between two alternative routes. For example, null mutations in EphB1 result in reduced numbers of retinal ganglion cells that project ipsilaterally through the optic chiasm to the ipsilateral region of the lateral geniculate nucleus56. Eph family molecules play key roles in establishing the crossed projections of the central nervous system57. Repulsion from ephrins expressed at the midline may serve to limit crossing projections spatially58 or temporally53. Eph-ephrin signaling is a significant factor in determining whether axons make ipsilateral or contralateral synaptic target selections. This role has been demonstrated in the auditory brainstem pathway from the cochlear nucleus to the medial nucleus of the trapezoid body, a strictly contralateral projection in the normal brain. Mutations that reduce reverse signaling through ephrin-B proteins59 and null mutations in ephrin-A2 or ephrin-A5 (or both) similarly reduce the specificity of this pathway, resulting in a significant ipsilateral projection50. The similarity in these phenotypes suggests crosstalk between the classes and redundancy in cues for generating the crossed projection. Unlike the optic chiasm, in which a subset of axons is selectively targeted ipsilaterally by EphB1, this auditory projection uses multiple Eph-ephrin signaling molecules to prevent the formation ofany ipsilateral projections. In this case, downstream signaling molecules might be similarly engaged by both classes of Eph proteins.Several other studies highlight the broad significance for Eph-ephrin signaling in determining whether axons cross the midlineM) motor neurons innervate ventral limb muscles, whereas the lateral LMC (LMCL) motor neurons innervate the dorsal limb muscles60. The axons of the LMC motor neurons grow out of the spinal cord together in one fascicle and make a dorsal versus ventral choice as they enter the limb. Another group of motor neurons in the medial portion of the medial motor column (MMCM) at the level of the hindlimb initially project axons together with the LMC axons but abruptly change course toward dorsal axial muscle targets. Both LMCL and MMCM axons express EphA4 and encounter ephrin-A5 along their trajectories.In ovo electroporation studies in chick embryos revealed that the two populations have opposite responses to ephrin-A5: LMCL axons avoid ephrin-A5 in the limb, whereas MMCM axons grow through ephrin-A5-positive somite regions61.Recent studies of motor neuron axon guidance have shed new light on the molecular mechanisms by which Eph-ephrin signaling coordinates distinct choices. Two groups of motor neurons of the lateral motor column (LMC) in the spinal cord innervate the limb. The medial LMC . This syndrome is characterized by abnormally large distance between the eyes, a central nasal groove, cleft palate, and skeletal/sternum abnormalities, along with other midline distortions in the body. CFNS is also associated with agenesis of the corpus callosum, a large neural tract that interconnects the cerebral hemispheres66.Eph proteins play a key role in establishing midline structures. In humans, mutations in65, which depends on ephrin-B1 reverse signaling67. Deeper examination of corpus callosum formation in several Eph family mutant mice revealed axon outgrowth defects near the midline, after axons have progressed out of cortical layers and traveled medially toward their contralateral journey. These axons coalesce and turn to project longitudinally, not medially via the commissure, similar to the human CFNS phenotype54. Axon guidance across the midline is largely dependent on ephrin-Bs and EphBs, which are expressed in growing callosal fibers. Furthermore, abnormal glial proliferation at the midline in mutant mice brains suggested that Eph family proteins regulate this population through growth suppression54. These studies suggest that agenesis of the corpus callosum in CFNS results from defects in axon guidance regulated by Eph-ephrin signaling.The mouse model for CFNS parallels many of the cranial deformities and also exhibits incomplete formation of the corpus callosumEFNB1 mutation in CFNS, investigations into the mechanisms have focused on earlier developmental stages. Indeed,in vivo manipulation of ephrin-B1 expression in the developing mouse results in perturbations of cephalic neural crest cell precursors68, which give rise to the bone and cartilage of the head. Whereas neural crest cell migration is known to be regulated by Eph proteins69, craniofacial defects in ephrin-B1 mutants largely arise from impaired cell proliferation in the developing palate68 as well as from impaired cell survival70. Craniofacial development relies on ephrin-B1 reverse signaling67. Interestingly, loss of ephrin-B1 in the developing palate leads to increased expression of EphB3, as forward signaling normally modifies EphB3 so as to promote endocytosis and degradation68. Together, these studies show that loss of ephrin-B1 affects several aspects of central and peripheral development through multiple molecular interactions.Owing to the large morphological impact of the71, characterized by dysfunction in social interactions, repetitive behaviors, and sensory abnormalities. Some of these behaviors can be identified in simplified form in mouse models of ASD. To explore the potential link with Eph proteins, Wurzman and colleagues72 performed a comprehensive series of behavioral tests on ephrin-A2/A3 double-knockout mice. In a three-chamber social interaction test, the knockout mice spent significantly less time than wild-types did in a chamber exposed to a novel mouse, indicating social aversion. Compared with wild-type mice, the ephrin-A2/A3 mice exhibited significantly greater repetitive and self-injurious grooming behavior. They also showed decreased acoustic startle response and increased prepulse inhibition of the startle reflex. These behavioral phenotypes are similar to those in other mouse models of ASD. This work, though still in its early stages, expands the relevance of developmental Eph-ephrin signaling in establishing normal sensory function and behavior.Apart from CFNS, an understanding of the impact of mutations affecting Eph-ephrin signaling on human brain conditions is in its very early stages. Genetic studies have linked these mutations with neurodevelopmental disorders, including autism spectrum disorder (ASD)A large body of work on the roles of Eph proteins and the mechanisms underlying their versatility has emerged. Recent work demonstrates the breadth of roles throughout development and the significance for assembly of sensory, motor, and cognitive neural systems. Determining how these multiple functions are coordinated remains a significant challenge. The recent studies highlighted here have begun to shed light on this complex issue, showing that specificity arises from differential clustering, forward and reverse signaling, and unique combinations of protein family members that engage distinct signaling pathways.L, lateral lateral motor column; LMCM, medial lateral motor column; MGE, medial ganglionic eminence; MMCM, medial motor column; POA, preoptic area.ASD, autism spectrum disorder; CFNS, craniofrontonasal syndrome; CR, Cajal-Retzius; FAK, focal adhesion kinase; LMC, lateral motor column; LMC


A proto-oncogene (hereafter simplified as oncogene) has the potential to initiate cancer. Oncogenes are often mutated and/or their products are expressed at high levels in various types of cancer. Several mechanisms and features of oncogenesis have been extensively discussed in the review of Hanahan and Weinberg “hallmarks of cancer: the next generation” . FundameSeveral ongoing preclinical studies and clinical trials propose to target the proliferation process ,3. SpeciHCC is the most frequent primary liver neoplasm which often arises in the predisposing liver disease states. HCC accounts for approximately 800,000 deaths each year and, making it the second most lethal cause of cancer worldwide GLOBOCAN 2008 v2.0). Various therapeutic approaches to the treatment of advanced HCC have been unsuccessfully implemented. One of the most beneficial HCC treatment is so far the well known kinase inhibitor Sorafenib that improves patient survival of a maximum of 2 to 3 months 008 v2.0.. Thus, lAlthough several pathways and molecular players were reported in HCC development, the lack of animal models that recapitulate the full spectrum of the human disease progression may impede the development of suitable therapies. Despite detailed etiological and clinical features, the pathogenesis of HCC is not well understood. The comprehension of the disease, identifying clinically relevant therapeutic targets and the generation of efficient medicines require powerful genetic tools that mimic the human clinical stages. In a recent study of our lab, we generated genetically engineered mouse models (GEMMs) of Unconventional prefoldin RPB5 interactor (URI) loss- and gain-of-function . DevelopAt the early stages, we demonstrate that DNA damage is the critical initiating event leading to dysplastic lesions and aggressive HCC. Interestingly, while apoptosis-induced compensatory proliferation is suggested to initiate liver tumors, in our model, abolishing apoptosis and increasing genotoxic stress by inactivating p53, accelerates tumor formation and death of mice. Thus, oncogenic URI-induced genotoxic stress, rather than excessive liver injury is essential to initiate the liver tumorigenic process. In support of this, chromosomal abnormalities represent the most reliable clinical aspect to determine precancerous stages of HCC .+ synthesis. The decrease in total NAD+ levels consequently provokes DNA damage  activity may be affected. We do not completely exclude that NAD+ depletion may affect Sirts activity. Furthermore, because NAD+ is a cofactor for inosine monophosphate dehydrogenase implicated in dNTPs synthesis, NAD+ deficits can also lead to insufficient dNTP production that may contribute to DNA damage during high replication. Replenishing the NAD+ levels by nicotinamide riboside (NR), a derivative of vitamin B3, prevented and abolished DNA damage and aggressive tumor formation [Next, using global quantitative transcriptomic and proteomic analysis, we demonstrate that prior to DNA damage, URI downregulates the L-tryptophan/kynurenine catabolism pathway and thus, leads to the inhibition of de novo NADA damage . Althougormation .+ depletion through a net reduction in tryptophan catabolism. Enzymes implicated in tryptophan degradation are downregulated by c-Myc over-expression. NAD+ depletion is apparently involved in the formation of pancreatic tumors. Importantly, these tumors could be tackled when NAD+ levels were enhanced by NR supplementation [+ depletion is a common molecular mechanistic basis for oncogene-induced DNA damage and tumor development.Based on previous observations and extending our work to other oncogenes known to induce tumors on the basis of DNA damage, we demonstrate that c-Myc expression in pancreas induced NADentation . We prop+ “boosters” and, understanding the mechanisms of NAD+ depletion-dependent DNA damage would offer a broad spectrum of new possibilities to decisively prevent and cure cancer in human beings. The development of drug discovery platform based on screening of new compounds that enable to abolish DNA damage by increasing NAD+ levels can thus be an exciting investment in the war against cancer.NR seems therefore to be an efficient therapy for the treatment of various cancers in which predictive and prognostic factors can be identified as oncogene-associated genotoxic stress. Clinical trials with NR for treatment of such cancers are under consideration. However, developing a methodological screen to find more efficient and stable NAD


The availability of fully sequenced genomes of many bacterial organisms has enabled mapping networks of binary protein interactions that form the basic building blocks of molecular pathways and dynamic assemblies defining all cellular activities. Few proteome-scale studies have been reported for pathogenic bacteria though, suggesting that a systems-wide network analysis of binary interaction partners could reveal groups of proteins that coordinate to achieve specific biological tasks important to pathogenesis and provide a functional map useful to the discovery of new antibiotics, vaccines, and diagnostic tools.Yersinia pestis and analytically identified more than 74,000 binary interactions. Using a library of biotinylated recombinant proteins to probe a planar microarray comprised of immobilized proteins that represented approximately 85%  of the Y. pestis proteome, we measured protein-protein interactions by fluorescence intensity of the laser-scanned microarrays. We obtained kinetic interaction data for >1,600 binary complexes by microarray-based, surface plasmon resonance imaging, and identified several high-affinity (D K~ nM) interactions. We applied a machine learning algorithm that used previously reported experimental protein-protein interactions from Escherichia coli as a training set in order to extract E. coli-like interactions from the Y. pestis dataset. The node degree distribution of the resulting network, comprised of 2344 interactions between 314 proteins, approximates a power-law distribution typical of scale-free networks. Functional annotation clustering of proteins within the network revealed statistically enriched complexes and pathways involved in diverse biological processes. Among the more notable protein assemblies identified were components of the RNA polymerase enzyme and ribosomes. Small modules of proteins related to various metabolic pathways, as well as previously reported interactions involved in homologous recombination and fatty acid biosynthesis, were also present in the network. Two highly interconnected network sub-regions contained a large percentage of proteins with functions linked to transcription and translation.We performed a comprehensive proteomics analysis of the pathogenic bacterium Y. pestis. This new benchmark data set will serve as a critical tool for the analysis of protein interaction networks functioning within an important human pathogen.We have systematically identified and analyzed thousands of direct binary protein interactions within


Uveal melanoma (UM) is recognized as the most common intraocular malignancy and the second most common form of melanoma. Nearly 50% of UM patients develop untreatable and fatal metastases. The 48-member nuclear receptor (NR) superfamily represents a therapeutically targetable group of transcription factors known for their regulation of key cancer pathways in numerous tumor types. Here, we profiled the expression of the 48 human NRs by qRT-PCR across a melanoma cell line panel including 5 UM lines, 9 cutaneous melanoma (CM) lines, and normal primary melanocytes. NR expression patterns identified a few key features. First, in agreement with our past studies identifying RXRg as a CM-specific marker, we found that UM cells also exhibit high levels of RXRg expression, making it a universal biomarker for melanoma tumors. Second, we found that LXRb is highly expressed in both UM and CM lines, suggesting that it may be a therapeutic target in a UM metastatic setting as it has been in CM models. Third, we found that RARg, PPARd, EAR2, RXRa, and TRa expressions could subdivide UM from CM. Previous studies of UM cancers identified key mutations in three genes: GNAQ, GNA11, and BRAF. We found unique NR expression profiles associated with each of these UM mutations. We then performed NR-to-NR and NR-to-genome expression correlation analyses to find potential NR-driven transcriptional programs activated in UM and CM. Specifically, RXRg controlled gene networks were identified that may drive melanoma-specific signaling and metabolism. ERRa was identified as a UM-defining NR and genes correlated with its expression confirm the role of ERRa in metabolic control. Given the plethora of available NR agonists, antagonists, and selective receptor modulators, pharmacologic manipulation of these NRs and their transcriptional outputs may lead to a more comprehensive understanding of key UM pathways and how we can leverage them for better therapeutic alternatives. Uveal melanoma (UM) is the most common form of intraocular cancer in adults and comprises about 5% of all malignant melanoma diagnoses . UM tumoV600E or NRAS mutations, only ~5% of UMs  exhibit BRAFV600E mutations, and NRAS mutations are typically not observed in UM tumors  to transform immortalized melanocytes . Some suThe nuclear receptor (NR) superfamily of transcription factors includes 48 members, most of which activate complex transcriptional programs via ligand binding . NRs regNuclear receptors have also been noted as having tumor suppressive functions including VDR’s protective function in colon cancer ; PPARg’sThe expression and activity of NRs in UMs have been, to date, completely unstudied. To address this issue, we have used high-throughput qRT-PCR to profile the expression of the 48 members of the NR superfamily in various UM cell lines derived from both primary and metastatic lesions and in a normal melanocyte cell line. Based on these results, we report UM-specific NR expression patterns as well as pharmacologically targetable NR-regulated gene networks that could be driving proliferative or oncogenic signaling in UM.All UM cell lines were a kind gift from Dr. Jerry Niederkorn at UT Southwestern and were grown as originally described . CM cellwww.nursa.org under the rapid release tab. Universal cDNA standards generated from human adult RNA  were used for analysis of all receptors except CAR, FXRb, PXR, SHP, DAX-1, ERb, LRH-1, PNR, SF-1, and TLX, which were too limited in expression to use the universal RNA set. For these receptors, commercially available tissue-specific total RNA standards derived from cell lines or adult organ donors were used from liver, ovary, eye, adrenal, and brain, as appropriate. qRT-PCR data were analyzed using ABI instrument software SDS2.1. Baseline values of amplification plots were set automatically, and threshold values were kept constant to obtain normalized cycle times and linear regression data. Because PCR efficiencies for each receptor primer set vary, individual receptor PCR efficiencies were determined to permit receptor-to-receptor comparisons. PCR efficiencies were calculated from the slope of the resulting standard curves as reported previously  was performed in triplicate using the TaqMan-based efficiency-corrected cycle threshold method with 12.5 ng cDNA per reaction for 50 cycles in an ABI 7900HT sequence detection system  as previously described . NR mRNAeviously . Normalihttp://www.ncbi.nlm.nih.gov/gds/) (All microarray data were obtained from published datasets available at the GEO Database (ov/gds/) –25. The p-Values for comparisons between groups of measurements were performed using Student’s t-tests. Bonferroni Corrections were used to account for multiple-hypothesis testing as appropriate.Dendrograms were generated using R Statistical Software. Analysis parameters included distance calculations using a Manhattan methodology and Ward’s method for cluster aggregation. Correlation coefficients for comparisons between data were calculated using Pearson Correlation. r > 0.6 was used). Culled lists were input into gene ontology (GO) analysis tool GOrilla and GO analyses were performed as described (Correlation coefficients (Pearson) were calculated for each gene and the NR in question (either ERRa or RXRg). Lists were then culled to retain the most significantly positively correlated genes (cutoff of escribed .n = 48) in UM, we performed high-throughput qRT-PCR expression analysis across a cell line panel consisting of five UM cell lines, nine CM cell lines from the NCI-60 , suggesting that the data presented here for UM lines are representative of findings in clinical UM samples.Data quality was assessed by comparison to 63 previously published microarray profiles generated from patient UM samples  . In thisTo visualize relationships within the dataset, unsupervised hierarchical clustering analyses were performed on the qRT-PCR dataset for both the 13 cell lines and the 48 NRs  were particularly distinct from the other NRs in the dataset due to their exceptionally high expression. Previously published work from our group noted that RXRg expression is very high in CM cell lines while essentially unexpressed in every other tissue type in the NCI-60 cancer cell line panel . High leWe next examined NRs differentially expressed between UM and CM Figure . First, To better understand the relationships between the NRs themselves within the melanoma panel, we calculated correlation coefficients (performed as before) for all possible pairwise combinations of the 48 NRs. The results of this unsupervised clustering analysis are shown as a heat map in Figure Finally, we compared NR profiles across the different UM cell lines Figure . BecauseWithin the UM panel, three cell lines were derived from primary tumors  and two cell lines were derived from metastatic lesions (OMM1 and OMM2.3). Comparison between these two groups found that NGFIB was up-regulated in the metastatic lines versus the primary tumor lines and the melanocyte control line Figure B. There Examples of all three of the key UM mutations are represented in the cell line panel Table . Becauser = 0.82 for ERRa and 0.7 for RXRg). Lists of microarray probes positively correlated with either ERRa or RXRg expression patterns were generated, culled to the top 400 genes correlating with each receptor , and subjected to gene ontology (GO) analysis using publically available GO analysis tool Gorilla  and identified receptors  exhibiting mutation-specific expression patterns.In this study, we measured expression levels of the 48 human NRs by qRT-PCR across a panel of UM cell lines and a normal melanocyte control to expand on our original work across the NCI-60 panel. We demonstrated that UM (like CM) is distinguished from other cancer cell lines by high expression of RXRg, an NR that we previously reported separates melanomas from other cancers. Furthermore, UM and CM can be differentiated based solely on their NR expression profiles with several NRs differentially expressed between the two . Our results confirm the distinction of UM and CM as separate diseases in line with their differing mutational profiles. We also examined whether there are NRs preferentially expressed in the different mutational subtypes of UM  also showed generally higher expression in the metastasis-derived UM cell lines. The role of NR4 family members in cancer is controversial as it has been noted to be pro-proliferative in some contexts and tumor suppressive in others . One othFinally, we examined NR-to-NR correlation patterns as well as NR-to-genome correlation patterns to identify receptors that might be interacting with each other and to identify networks of genes that certain, key NRs might be controlling within the UM context. ERRa was one of the NRs that differentiated the UM cluster from the CM cluster, and is particularly important given its role as a therapeutic target in other cancers (particularly breast cancer) and the growing availability of ERRa-targeted therapeutics  includinAnalogous studies conducted previously by our group and others have successfully utilized NR expression profiles to subdivide between different cancer types. It has been demonstrated that expression profiles of the 48 NRs alone can properly distinguish between cancers of vastly different origins (within the NCI-60 panel) , betweenIt is of significant interest that several NRs are differentially expressed between UM and CM tumors. Particularly, the findings RARg, PPARd, EAR2, TRa, and LXRb that are retained in UM at levels comparable to the melanocyte while being reduced or lost in CM suggests that there may be opportunities for NR-directed therapeutic interventions in UM that are not available in CM due to CM-specific loss of these receptors. It is also worth noting that the UM-specific loss of RXRa expression. Because RXRa is a heterodimeric binding partner for many NRs, lower RXRa expression might suggest indirect downregulation of NR signaling in UM tumors.in vivo.It has recently been reported that metastatic CM can be inhibited by administration of LXRb agonists in pre-clinical models of CM . Here, wMelanoma is an aggressive, highly metastatic disease that is notoriously difficult to treat using standard cytotoxic agents . Mechaniin vitro growth of GNAQ/11 UM mutant cell lines  in more than 80% of UM cases. Although these mutations would seem obvious targets for therapeutic intervention, GNAQ/11 mutations have not been amenable to therapeutic development in UM and recent work has instead focused on inhibiting downstream events and gene networks driven by these mutations , 35. As ll lines , it is ill lines . Recent ll lines .As transcription factors, activated NRs are extremely effective in eliciting widespread physiologic changes in cells through alteration of the transcriptional output and architecture of the genome . One of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://journal.frontiersin.org/article/10.3389/fendo.2015.00093The Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


The incidence of acute aortic syndrome is low, but the spontaneous course is often life-threatening. Adequate ECG-gated imaging is fundamental within the diagnostic workup. We here report a case of a 53-year-old man presenting with atypical chest pain, slight increase of D dimers at admission, and extended diameter of the ascending aorta accompanied by mild aortic regurgitation. Interpretation of an initial contrast-enhanced computed tomography was false negative due to inadequate gating and motion artifacts, thereby judging a tiny contrast signal in the left anterior quadrant of the ascending aorta as a pseudointimal flap. By hazard, cardiac magnetic resonance imaging demonstrated an ulcer-like lesion superior to the aortic root, leading to aortic surgery at the last moment. As sensitivity of imaging is not 100%, this example underlines that second imaging studies might be necessary if the first imaging is negative, but the clinical suspicion still remains high. The incidence of acute aortic dissection is estimated with 2,9–3,5/100.000 annual lives . Severalμg/mL, reference <0.5 μg/mL). Immediately performed transthoracic echocardiography demonstrated a preserved left ventricular global function without significant wall motion abnormalities and no signs of right ventricular strain, though an extended diameter of the ascending aorta of 52 mm was accompanied by a mild aortic regurgitation with hemodynamically noncompromising pericardial effusion. Thus, under the working diagnosis of an acute aortic syndrome, a contrast-enhanced CT using a triple rule out protocol was initiated. Because of inadequate gating due to repeated premature ventricular contractions at the time of image acquisition despite administration of beta-blockers, finally a non-ECG-gated contrast-enhanced CT of the ascending aorta was completed in order to exclude aortic dissection. The CT showed an extended diameter of the ascending aorta with a maximum of 51 mm without hint for dissection membrane or false lumen. However, imaging of the aortic root as well as the supracoronary aorta was hampered by aortic motion artifacts, thereby judging a tiny contrast signal in the left anterior quadrant of the ascending aorta as a pseudointimal flap  shortly after initial presentation. Surprisingly, cine-CMR imaging demonstrated an ulcer-like lesion superior to the aortic root in the left anterior quadrant of the ascending aorta  but no tThe currently asymptomatic patient was directly admitted to our intensive care. Unfortunately, only a few minutes following the second CT the patient suffered a convulsive seizure with subsequent hemodynamic instability needing cardiopulmonary resuscitation for about 20 minutes until return of spontaneous circulation. The patient was brought immediately to the operation room for aortic surgery. At this time, the operative site demonstrated a supracoronary entry of an extensive classical type A aortic dissection reaching the aortic arch with an inversion of the intima flap that resulted in an occlusion of the supra-aortic limbs. A graft replacement of the ascending aorta under selective cerebral perfusion was done using a 30 mm artificial graft.Despite the worsening scenario the patient recovered well without relevant neurological residuals until discharge and during subsequent outpatient follow-up consultations.Type A dissection has a 40–60% risk of death within the first 48 hours. However, not only acute type A dissection but all types of type A acute aortic syndrome require timely diagnosis and early surgery . The claRetrospectively, a localized dissection membrane or ulcer-like lesion should have been assumed, but diagnosis failed by insufficient imaging quality and missing of immediate reevaluation. This case should be a reminder that sensitivity of the first performed imaging and particularly CT scan is high but not 100%. Vice versa, the rate of false positive activation remains low and justifiable in such life-threatening disorder [


About half of these  also contain a mutation known as EGFRvIII,2 which is strongly tumorigenic. The signaling mechanisms underlying EGFRvIII’s tumorigenicity are not fully understood. Now, in a recent issue of Nature Neuroscience, Jahani-Asl et al.3 add to our knowledge of how EGFRvIII mediates its profound tumorigenicity.High-grade glioma (HGG) is the most common neoplasm of the brain and is almost universally lethal.4 This deletion leads to a ligand-independent receptor, although the constitutive activity of this receptor is lower than that of ligand-stimulated wtEGFR.4 Despite this sub-optimal activity, EGFRvIII imparts a significant growth advantage to HGG cells, especially in vivo. Moreover, because of this low level of activation, EGFRvIII is not downregulated, unlike wtEGFR following activation by ligand. This leads to sustained signaling through EGFRvIII,5 which is critical to its tumorigenicity.Previous studies have shown that EGFRvIII is a gain-of-function mutation that arises from genomic deletion of exons 2–7.4 There is some evidence of how EGFRvIII mediates its diverse range of biological effects. For example, we have shown that EGFRvIII signaling is amplified through interaction with a range of other receptor tyrosine kinases, especially c-Met.6 Indeed, EGFRvIII forms a heterodimer with c-Met, leading to its direct activation and enhancing its response to the c-Met ligand hepatocyte growth factor.6 As a consequence, the co-targeting of EGFRvIII and c-Met has synergistic anti-tumor activity in mouse xenograft models.6Nonetheless, this low level of sustained signaling through EGFRvIII is insufficient to explain its full tumorigenic activity, which promotes a diverse range of biological activities, including proliferation, survival and angiogenesis.7 These cytokines can activate signaling through gp130 in neighboring cells. Activated gp130, in turn, activates wtEGFR, even in the absence of EGFRvIII in these cells or of EGFR ligand, thereby enhancing HGG cell growth both in vitro and in xenograft models.7In addition, EGFRvIII has been shown to promote HGG growth through paracrine mechanisms. Cells expressing EGFRvIII secrete large amounts of interleukin-6 and leukemia inhibitory factor.et al.3 broadens our understanding of the tumorigenic capacity of EGFRvIII. The authors conducted global RNA sequencing (RNA-seq) on a series of HGG stem cell lines that express EGFRvIII. Using the knowledge that EGFRvIII activates the transcription factor STAT3, they simultaneously performed RNA-seq on mouse astrocytes  expressing EGFRvIII in the presence or absence of Stat3. Finally, they also conducted chromatin immunoprecipitation sequencing on mouse astrocytes expressing both EGFRvIII and STAT3. Using this wealth of data, the authors were able to identify genes whose expression is driven specifically by the EGFRvIII–STAT3 pathway.The recent study by Jahani-Asl Osmr gene promoter, and knockdown of Stat3 by lentivirus-mediated RNA interference resulted in a marked reduction in the level of Osmr mRNA, indicating that OSMR is probably a positive feed-forward signal in the EGFRvIII–STAT3 pathway  of EGFRvIII, as pharmacological inhibition of EGFRvIII with EGFR-directed tyrosine kinase inhibitors prevented the association of EGFRvIII and OSMR.8 Surprisingly, the authors show that the interaction of EGFRvIII and OSMR and the subsequent signaling of the EGFRvIII–OSMR complex is independent of gp130. They go on to demonstrate a similar direct interaction between OSMR and wtEGFR; however, the formation of this receptor complex was dependent on OSM. One important follow-up question from these studies is whether the EGFRvIII–OSMR complex also directly activates JAK signaling and, as a corollary, whether this activation is augmented by the addition of exogenous OSM. If this pathway is activated, there is a readily available path into the clinic using FDA-approved JAK inhibitors such as ruxolitinib.9The canonical signaling pathway for OSMR involves binding its cognate ligand (OSM), direct interaction with its co-receptor (gp130) and subsequent activation of the JAK–STAT pathway.6 Uncovering the role of this cysteine in the EGFRvIII–OSMR complex may provide new mechanistic insight into how this complex is formed and subsequently signals.EGFRvIII is known to contain a free cysteine that is not present in untruncated EGFR, and we have shown that this cysteine is critical for the activation of EGFRvIII and for its ability to transactivate other receptor tyrosine kinases such as c-Met.OSMR in the HGG stem cell lines reduced their tumorigenicity in an intracranial model, and knockdown of Osmr in EGFRvIII-expressing mouse astrocytes robustly reduced their in vivo tumor growth by attenuating cell proliferation. Knockdown of Osmr in EGFRvIII-expressing mouse astrocytes also had the surprising effect of significantly reducing EGFRvIII expression, which correlated with loss of phosphorylated STAT3. This raises the possibility that phospho-STAT3, driven by OSMR, may sustain EGFRvIII expression. This idea could be further explored in the human EGFRvIII-expressing HGG stem cell lines after OSMR knockdown. If replicated, the therapeutic targeting of OSMR could inhibit both EGFRvIII and STAT3 simultaneously. Taken together, these data suggest that targeting OSMR signaling in patients with EGFRvIII-positive HGG tumors would have therapeutic benefit.Importantly, the authors also analyzed two HGG databases and showed that high levels of OSMR were significantly associated with poorer survival. Consistent with this, knockdown of 10 even when the drug  reaches high enough concentrations within the tumor.11 This study raises the possibility that resistance to EGFR-targeted therapeutics could be mediated by EGFR- and/or EGFRvIII-independent OSMR signaling. This may involve OSM–OSMR autocrine loops or OSMR complexes with other receptor tyrosine kinases. Studies exploring this possibility are well warranted.This new study strongly supports the idea that EGFRvIII is a valid therapeutic target in HGG. Despite this, attempts to therapeutically target EGFR and EGFRvIII with single agents have largely failed in patients,12 contributes to the tumorigenicity of HGG cells and to identify OSMR as a therapeutic target. It also further highlights the promiscuous interactions between EGFRvIII and resident receptors in HGG cells. Understanding how these interactions function is vital for developing novel, efficacious therapies for HGG, a highly lethal cancer.This is the first study to show how the previously described EGFRvIII–STAT3 signaling pathway


Inflammation originating from the adipose tissue is considered to be one of the main driving forces for the development of insulin resistance and type 2 diabetes in obese individuals. Although a plethora of different immune cells shapes adipose tissue inflammation, this review is specifically focused on the contribution of macrophages that reside in adipose tissue in lean and obese conditions. Both conventional and tissue-specific functions of adipose tissue macrophages (ATMs) in lean and obese adipose tissue are discussed and linked with metabolic and inflammatory changes that occur during the development of obesity. Furthermore, we will address various circulating and adipose tissue-derived triggers that may be involved in shaping the ATM phenotype and underlie ATM function in lean and obese conditions. Finally, we will highlight how these changes affect adipose tissue inflammation and may be targeted for therapeutic interventions to improve insulin sensitivity in obese individuals. Historically, adipose tissue is known to perform a number of functions including storage of excess energy, cold insulation and protection of vital organs. Intense research efforts over the last decades have greatly furthered our understanding of adipose tissue biology and led to the identification of various additional functions. The adipose tissue—composed of adipocytes and non-adipocyte cells, including endothelial cells, pre-adipocytes and various types of immune cells —is knownThe central role of adipose tissue in homeostasis becomes apparent during the development of obesity, which is a major risk factor for the development of insulin resistance . During Macrophages are immune cells that have gained much attention as important contributors to adipose tissue functioning. Whereas macrophages in lean mice and humans make up around 5% of the cells in adipose tissue, during obesity they constitute up to 50% of all adipose tissue cells –8. MacroIn contrast to mice, inflammatory changes in human adipose tissue during obesity are somewhat less pronounced, although the presence of CLSs is positively correlated with a worsening in insulin resistance levels . Further+ T cells have recently gained considerable attention because of the observed early influx into adipose tissue upon HFD-feeding and their potential contribution either to attracting macrophages or affecting their phenotype [+ regulatory CD4+ T cells have been found to reduce in number prior to the accumulation of macrophages in obese adipose tissue [+ T cells in adipose tissue might explain the accumulation and/or proinflammatory signalling of ATMs during obesity. Overall, these findings suggest a strong interplay between adaptive and innate immunity that together determine the inflammatory characteristics of obese adipose tissue. An excellent overview of the contribution of various immune cells to adipose tissue biology can be found elsewhere [In addition to macrophages, numerous other types of immune cell populate the adipose tissue and affect its function , 13. Indhenotype , 19, 22.e tissue . In linelsewhere , 25. ThiMacrophages are unique in their capacity to quickly adapt to a changing environment, causing them to embrace a variety of phenotypes ranging from anti-inflammatory to proinflammatory. In virtually every tissue, macrophages are actively involved in maintaining tissue homeostasis by clearing cellular debris, participating in tissue immune surveillance and resolving inflammation . AlthougIn this review, we will touch upon both well established and recently identified functions of macrophages in lean and obese adipose tissue. The origin of macrophages residing in lean and obese adipose tissue will be addressed and several triggers that may account for an increase in number and a phenotypical switch of ATMs during the development of obesity will be discussed. Finally, this knowledge is used to pinpoint relevant future research directions to establish potential therapeutic targets for shifting macrophage phenotype and function in order to promote adipose tissue health.In 1908, Elie Metchnikoff received the Nobel Prize for his important discovery of the phagocytic activity of macrophages. Nowadays phagocytosis is still considered to be the most prominent function of macrophages. Although Metchnikoff specifically described the engulfment of microbes, phagocytosis also encompasses the engulfment of apoptotic endogenous cells in a process termed ‘efferocytosis’ . EfficieBuilding upon their unique role as professional phagocytes, recent data suggest that macrophages facilitate acute metabolic tasks executed by the adipose tissue. During lipolysis induced by either fasting or pharmacologic adrenergic activation, macrophages rapidly infiltrate the adipose tissue and adopt an anti-inflammatory phenotype . In adip+ adipocyte progenitors to differentiate [Interestingly, 7 days of continuous infusion of a β3-adrenergic receptor agonist to induce adipocyte lipolysis not only results in an influx of anti-inflammatory macrophages that adopt a lipid-laden cell appearance, it also increases the presence of dead adipocytes and the formation of CLSs. Within these CLSs, anti-inflammatory macrophages have been found to recruit and stimulate platelet-derived growth factor receptor (PDGFR)αrentiate . IntriguIn obese adipose tissue, macrophages surrounding dead adipocytes in CLSs also form multiple intracellular lipid droplets and activate transcriptional programs involved in lysosomal lipolysis . The preThe close correlation between adipocyte death and new cell formation through adipogenesis that has been observed in lean adipose tissue may not hold true during obesity. While macrophages in obese adipose tissue have been found to populate adipogenic clusters and facilitate angiogenesis and adipogenesis, adipogenic clusters are formed at sites away from CLSs , 43, 44.+ cells from obese adipose tissue blocks adipogenesis [In contrast to studies postulating a role for macrophages in adipogenesis, other studies have argued that ATMs inhibit adipogenesis in obese adipose tissue , 50. Forogenesis . A promiogenesis . Reducedogenesis , 52. Indogenesis , 54. How+CD11c+ or ‘classically activated’ M1 macrophages, characterised by increased expression levels of TNFα and inducible nitric oxide synthase (iNOS) [+CD206+CD301+CD11c− macrophages, resembling an alternatively activated M2 phenotype that is characterised by the expression of genes encoding anti-inflammatory proteins including Ym1, arginase 1 and IL-10 [+CD206−CD11c− phenotype has also been reported [Besides increasing in number, ATMs are known to dramatically change their phenotype during obesity. It has been suggested that the majority of the macrophage population in adipose tissue of obese mice consists of F4/80nd IL-10 . A balanreported , supportreported . Interesreported . Rather reported .+CD206+ macrophages has been associated with insulin resistance [In parallel with an increase in ATMs during obesity in mice, macrophage numbers have also been found to increase in human obese adipose tissue , 7. In csistance . Interessistance . As mentsistance –64. It iAdipose tissue is stored at various locations throughout the body, characterised by specific metabolic and inflammatory properties. Both subcutaneous (scAT) and visceral (vAT) storage depots exist, which are identified based on their anatomical location and differentially contribute to the development of metabolic abnormalities . EpidemiIn general, most studies are mainly focused on determining the inflammatory phenotypes of ATMs. However, in line with the metabolic functions executed by ATMs, inflammatory markers may not suffice to distinguish functional from dysfunctional ATMs. It would be interesting to unravel whether macrophages within human adipose tissue display a metabolically activated phenotype, as has been identified recently in mice . MoreoveContrasting with the prevalent idea that all tissue macrophages are derived from circulating monocytes, novel techniques that allow fate mapping have revealed that not all tissue-resident macrophages originate from monocyte precursors . Many reIt is well established that adipocytes are able to produce specific adipokines that function as chemoattractants for circulating monocytes. Monocyte chemoattractant protein-1 (MCP-1) is one such chemokine that is known to be produced in large amounts by adipocytes and is robustly increased in obesity . Indeed,+ tissue macrophages may have an increased inflammatory phenotype compared with CCR2− tissue macrophages, which is illustrated by an induction of genes involved in the regulating of the NLRP3 inflammasome, at least in the myocardium [In addition to increased secretion of MCP-1 by adipose tissue, obesity is characterised by a significantly increased number of CCR2 molecules on circulating monocytes . Moreoveocardium . The NLRocardium , 80. Simocardium . Howeverocardium . Indeed,ocardium .+ ATMs after bone marrow transplantation [Recent evidence has shed light on mechanisms and signals that have a vital role in controlling the recruitment of monocytes during obesity and go beyond MCP-1 and other chemokine-signalling modules. Indeed, the bone marrow has been identified as an important contributor to the ATM pool , 83. Theantation . TogetheIn addition to recruitment during obesity, multiple lines of evidence suggest that adipose tissue is equipped with tools allowing for local regulation and proliferation of ATM pools independently of the influx of blood precursors. vAT that is part of human omentum displays enhanced myelopoiesis during acute inflammatory conditions, which is suggestive for in situ production of cell populations in the abdomen , 91. In Besides the contribution of local precursors, local proliferation has been held responsible for increasing ATM numbers during obesity . MoreoveTo summarise, the origin of ATMs in lean and obese adipose tissue is still a matter of debate. The presence of specific cell surface markers on ATMs might help us learn more about their origin. Moreover, lineage tracing of ATMs using time-lapse microscopy, cell labelling and/or genetic markers will help us understand the origin of ATMs. On the basis of the currently available data, ATMs appear to be mainly derived from bone marrow-controlled pathways involving adipose-derived signals. Other lines of evidence suggest that local mechanisms may determine macrophage numbers in adipose tissue, including prolonged survival or increased retention of ATMs. Probably, various pathways at multiple levels are at work to set ATM numbers in adipose tissue. Whether the number of ATMs is primarily regulated at the level of the bone marrow or is determined by local factors in the adipose tissue will need further study.Both during myelopoiesis and in the circulation, monocytes can be primed by different factors that will eventually contribute to their migration and inflammatory fate after infiltration into the adipose tissue. It is likely that adipose tissue-derived factors will further determine the final ATM phenotype, which may differ between lean and obese adipose tissue. Some important triggers that shape the metabolic, inflammatory and functional traits of ATMs will be discussed below.As explained earlier, other immune cells residing in the adipose tissue probably shape ATM phenotype as well, but these will not be discussed in this review. The same also applies to various adipokines that are known to be differentially secreted by lean rather than obese adipose tissue. Their role in affecting the phenotype of macrophages has been reviewed elsewhere , 99.HIF1α [Both in humans and mice, rapid adipose tissue mass expansion during the development of obesity occurs without a concurrent increase in blood flow towards the tissue . Indeed,HIF1α , 101–103HIF1α , 105, unHIF1α  in obeseAs a result of their profound increase in size upon the development of obesity, adipocytes are generally thought to suffer from hypoxia in obese adipose tissue. Hypoxic conditions in adipocytes promote angiogenesis and fibrotic remodelling and have often been associated with the development of adipose tissue insulin resistance , 107.+ cells from human obese vAT are exposed to hypoxic conditions, the secretion of proinflammatory cytokines is enhanced in comparison with culturing under normoxic conditions [Hif1α [Only recently has attention been directed to the effects of hypoxic conditions on macrophages in the adipose tissue. In line with adipocytes, ATMs from obese adipose tissue have increased expression levels of hypoxia-related genes , 108. Itnditions . Moreovenditions . Interess [Hif1α . TogetheImportantly, the response of macrophages to hypoxia might depend on their initial inflammatory state. Indeed, it has been proposed that stimulation of bone marrow-derived macrophages with IL-4 prior to hypoxia exposure decreases the expression of proinflammatory genes, while leaving hypoxia-related gene expression unaltered . AlternaHif2α has been linked to adipose tissue inflammation, glucose intolerance and insulin resistance [The isoform HIF2α is also upregulated under low oxygen levels. While HIF1α increases the glycolytic flux and proinflammatory signalling, HIF2α is associated with a more anti-inflammatory macrophage phenotype , 113. Insistance  but so fOverall, the inflammatory state of macrophages might be linked to the relative presence of HIF isoforms that may influence the metabolic and inflammatory response of ATMs under hypoxic conditions. Importantly, in addition to hypoxia, one should not ignore the fact that HIF-dependent gene expression can also be regulated independently of oxygen availability, through the involvement of inflammatory-related signals. In addition to direct effects of hypoxia on macrophages, signals originating from hypoxic adipocytes may also shape the phenotype of ATMs in obese adipose tissue. Indeed, in vitro experiments have revealed that adipocytes exposed to hypoxic conditions release potential stressors that affect macrophage function. Examples include the proinflammatory adipokines IL-6, visfatin, leptin and macrophage migration inhibitory factor (MIF) . ImportaIn animal studies, cell death in obese adipose tissue has been well established. Dead adipocytes that have lost membrane integrity are surrounded by macrophages that cluster in CLSs and together form proinflammatory, fibrotic lesions . SignalsThe type of adipocyte cell death occurring in adipose tissue may determine the inflammatory phenotype of macrophages as well. Apoptotic cells induce a predominant anti-inflammatory response, whereas necrotic cells stimulate proinflammatory cytokine secretion . Dead ceImportantly, adipocyte cell death is unique in the fact that it leads to the release of substantial amounts of lipids and one might hypothesise that prolonged exposure to excess lipids affects ATM phenotype.n-3 FAs, are recognised for their anti-inflammatory properties [A lipid-rich environment during the development of obesity may represent an important metabolic stressor for ATMs. In general, there are three ways through which ATMs are exposed to lipids; via chylomicrons or VLDLs, by adipocyte lipolysis, or after adipocyte cell death. Hyperlipidaemia seems not to lead to macrophage accumulation in obese adipose tissue , indicatoperties , 144. Heoperties  may trigoperties , 145–147Obesity-induced adipose tissue inflammation is associated with systemic insulin resistance and hyperinsulinaemia in obese rodents and humans. Multiple lines of evidence suggest that insulin itself directly affects inflammatory processes. In fact, insulin has been assigned with both anti- and proinflammatory properties. In circulating mononuclear cells of obese individuals, insulin inhibits NF-kB activity, suggesting an anti-inflammatory effect . By contMCP-1 gene expression levels aggravating the influx of monocytes [It is difficult to pinpoint the sole effects of insulin in vivo during the development of obesity, as changes in many other circulating factors accompany hyperinsulinaemia. However, several lines of evidence suggest that insulin directly contributes to ATM homing. The start of insulin therapy in patients diagnosed with type 2 diabetes has been shown to promote the appearance of macrophages in scAT . Intriguonocytes .ob/ob mice have a defect in efferocytosis that was associated with defective phosphoinositide (PI)3 kinase activity, whereas cells lacking the insulin receptor were protected [Insulin resistance has also been shown to negatively affect macrophage function itself, especially in the context of atherosclerotic disease. Although glucose uptake primarily takes place via insulin-independent pathways involving GLUT1, circulating monocytes express the insulin receptor machinery and develop resistance to the effects of insulin. Interestingly, insulin receptor deficiency on macrophages reduces their influx into the adipose tissue and alleviates the development of low-grade inflammation and systemic insulin resistance in mice on an HFD . Functiorotected .It remains to be determined whether insulin resistance develops in ATMs in obese adipose tissue. Moreover, the functional consequences and possible contributions to adipose tissue inflammation are currently unknown. Another interesting aspect involves the development of insulin resistance in circulating monocytes . It is nChronic hyperglycaemia is known to induce insulin resistance by mechanisms that may partly be conveyed by an enhanced inflammatory state. Indeed, there is a substantial amount of evidence demonstrating proinflammatory effects of prolonged periods of hyperglycaemia. In humans, hyperglycaemia is associated with inflammation in individuals with diabetes . SimilarTxnip using small interfering (si)RNA approaches prevents hyperglycaemia-induced ROS generation and the induction of inflammation [IL-1β mRNA expression levels and intracellular pro-IL-1β accumulation [In both adipocytes and macrophages, harmful effects of prolonged exposure to high levels of glucose have been reported. In vitro approaches in which adipocytes are cultured in medium with 25 mmol/l glucose for prolonged periods of time revealed increased production of various proinflammatory cytokines including IL-6 , and simammation . Moreoveammation , a pathwammation , 164. Inmulation .The consequences of hyperglycaemia may also be conveyed via changes in cellular metabolism leading to alterations in intermediate metabolites. It has been documented that cultured adipocytes exposed to high levels of glucose produce and secrete enhanced quantities of lactate . InteresHigh levels of glucose may thus represent an important trigger that activates proinflammatory changes in macrophages, either directly or indirectly. Interestingly, both shorter periods of hyperglycaemia, either during a clamp procedure or postprandially, and chronic periods in the presence of obesity and insulin resistance, appear to induce proinflammatory macrophage characteristics.Macrophages can be identified via specific transcription factors that control tissue-tailored transcriptional programs and allow the cells to adopt extremely tissue-specific functions. An elegant example is the identification of GATA-6 as master regulator of the peritoneal-specific gene transcriptional program. By comparing the transcriptome of various tissue macrophages including those from lung, liver, adipose tissue and peritoneum , the autBecause of the high versatility of macrophages, which have a phenotype largely dependent on their environment, conclusions solely based on in vitro studies should be taken with caution. Rather, in vivo profiling of macrophage phenotype via several techniques, including metabolomics and transcriptomics, and identification of key regulators of distinct macrophage phenotypes, residing in different adipose tissue depots and/or during the development of obesity and insulin resistance in both humans and mice, will advance our knowledge on adipose tissue biology. Determining a causal relationship between triggers, a switch in the metabolic and inflammatory status of ATMs and ATM function will be an important challenge. This information will be crucial for our understanding of initial triggers that ultimately link obesity to adipose tissue inflammation and the development of insulin resistance, and might provide us with future therapeutic targets. An overview of stressors that may determine the phenotype of ATMs in lean vs obese conditions and the subsequent consequences for adipose tissue function are shown in Fig. The presence of adipose tissue inflammation translates the development of obesity into insulin resistance and type 2 diabetes. Macrophages play a key role in maintaining adipose tissue homeostasis, yet fuel adipose tissue inflammation upon the development of obesity.Current efforts to unravel adipose tissue-specific functions of macrophages have revealed an essential role in lipid buffering for ATMs, alongside their more conventional functions such as clearing cellular debris and participating in tissue immune surveillance to maintain homeostasis. Importantly, both in lean and obese adipose tissue, lipid storage by macrophages prevents spillover into the circulation. However, the metabolically activated macrophage that resides in obese adipose tissue appears to be continuously exposed to an overabundance of lipids. Chronic lipid overloading of ATMs translates into a proinflammatory phenotype and may drive dysfunction of ATMs, ultimately fuelling the inflammatory traits of obese adipose tissue.This new knowledge might shift intervention approaches away from targeting the inflammatory traits of ATMs, towards targeting their metabolic programming. Interventions aimed at increasing metabolic capacity might be used to reprogram macrophage metabolism, allowing macrophages to cope with metabolic challenges during obesity, in order to maintain adipose tissue homeostasis.


Complex profiles of deregulated miRNAs are of high interest, whereas the importance of non-expressed miRNAs was ignored. Since miRNAs regulate gene expression rather negatively, absent miRNAs could indicate genes with unaltered expression that therefore are normally expressed in specific compartments or under specific disease situations. For the first time, non-detectable miRNAs in different tissues and body fluids from patients with different diseases  were analyzed and compared in this study. miRNA expression data were generated by microarray or TaqMan PCR-based platforms. Lists of absent miRNAs of primarily cardiac patients  were clustered and analyzed for potentially involved pathways using two prediction platforms, i.e., miRNA enrichment analysis and annotation tool (miEAA) and DIANA miRPath. Extensive search in biomedical publication databases for the relevance of non-expressed miRNAs in predicted pathways revealed no evidence for their involvement in heart-related pathways as indicated by software tools, confirming proposed approach.MicroRNAs (miRNAs) can be found in a wide range of tissues and body fluids, and their specific signatures can be used to determine diseases or predict clinical courses. The miRNA profiles in biological samples (tissue,  Cardiovascular diseases as life-threatening diseases are the most common cause of death in Western European countries Improvements in human genetic studies and the continuously-expanding field of biomarker discovery revealed the potential of physiological biomarkers such as microRNAs (miRNAs) or gene expression profiles for diagnosis of complex diseases such as cardiomyopathies and for applications in personalized medicine miRNAs are 20–22 nucleotides in length and highly-conserved non-coding RNAs. They have been demonstrated to play multiple roles in negative or positive regulation of gene expression including transcript degradation, translational suppression, or transcriptional and translational activation. miRNAs are present in a wide range of tissues Up to now, about 800 expressed miRNAs have been experimentally detected in EMBs Analyses of expression data by bioinformatic software  and two TaqMan PCR-based high-throughput systems including low density array (LDA) and OpenArray . Based on the analysis of deregulated miRNAs, we presented lists and pathways of non-detectable miRNAs in different tissues of primarily cardiac patients. All data were generated in the same laboratory to facilitate comparative data analysis.n = 284), PBMCs (n = 67), or serum (n = 287) including corresponding controls  and one for PBMCs (50 others), respectively. For the 650 miRNAs exclusively absent in serum samples, miEAA analysis revealed 14 pathways other than heart-relates ones. Since these patients suffer from cardiac diseases, the missing heart-related pathways are in concordance with the absence of these 650 miRNAs in serum. DIANA miRPath analysis for these miRNAs could not be performed due to limited miRNA input possibility.To validate experimental findings for cardiac patients and minimize methodological bias, panels of absent RNAs were evaluated with data from large cohorts of patients with different diseases We compared the aforementioned 1107 miRNAs absent in any one or more sample groups of EMBs, serum, and PBMCs taken from cardiac patients to those absent in spinal fluid (Alzheimer’s disease patients), urine (bladder cancer patients) or ocular fluid (ocular cancer patients) samples. There are totally 432, 217, and 187 miRNAs absent in spinal fluid, ocular fluid, and urine samples, respectively. Venn diagram showed that 24 absent miRNAs were found to be common among all different tissue types tested. These 24 absent miRNAs were listed in Next, different pathway prediction tools were employed to analyze the pathways involving the 24 absent miRNAs shared by all samples examined . miEAA aThe importance of differentially-expressed miRNAs for characterization of various disease situations has been shown impressively The current study described, for the first time, the set of non-expressed miRNAs of the largest published cohort of patients  with inflammatory or virally-induced cardiomyopathies that were diagnosed using EMBs in silico prediction tools for the involvement of the remaining 3 miRNAs in any disease or pathway.Bioinformatic evaluation of identified absent miRNAs was performed by application of two freely-available pathway prediction tools (miEAA and DIANA miRPath) to confirm experimental findings. For cardiac patients, 6 heart-related pathways were recovered using miEAA. For the 6 miRNAs commonly not expressed in EMBs, serum, and PBMCs of cardiac patients, the software predicted association with myocarditis and DCM. Intensive search of biomedical publication databases provided no hint for their involvement in heart muscle diseases. Instead, hsa-miR-16-1-3p is related to chronic lymphocytic leukemia DIANA analysis revealed one DCM-related pathway based on the 24 common miRNAs that are never detected in any of EMB, serum, PBMC, spinal fluid, ocular fluid or urine samples. Literature screening in PubMed retrieved no publications related to DCM or other cardiomyopathies for all 24 common absent miRNAs, therefore no experimental proof as well , Table 7Both examples of detailed search (6 miRNAs and 24 miRNAs) for the relevance of miRNAs in distinct pathways revealed no evidence for their involvement in heart-related pathways as stated in DIANA tool. Pathway prediction tools could generate a broad amount and variety of potential networks which might only exist in theory but not in reality. In addition, these prediction tools have their limits in terms of amount of miRNAs that can be uploaded for analysis , literature evidence of theirs predicted pathways, and comparability between different prediction tools. The best way to overcome this deficiency in pathway prediction is the evaluation of larger sample cohorts or multiple data sources. The involvement of sets of non-expressed miRNAs for more diseases, as presented in this study, will sharpen the predictive power of bioinformatic analyses. These data are easily available but often not requested for publication. In future, predicted pathways should be double checked against list of absent miRNAs. The theoretical output of prediction tools shows high divergence from experimental validation, at least for our study. Therefore, users of prediction tools should take caution and assess the output critically.The spectrum of non-expressed miRNAs in body fluids for defined diseases such as serum of patients suffering from cardiomyopathies is of keen interest. Today circulating miRNAs have the most important scientific and diagnostic impact Further studies have to confirm especially which of these absent serum miRNAs in cardiomyopathies are not versatile. Circulating miRNAs will be the novel diagnostic biomarkers, also for heart muscle diseases There are some limitations in the current study. Three analytical platforms were used in generating data for overlapping sample sets to infer miRNAs absent alone or in different combinations. EMBs and PBMCs were measured with microarray-based technology for former sets of available miRNAs (miRBase v14), whereas Taqman PCR-based analysis were performed later and used to measure miRNAs in serum , EMBs (LDA and OpenArrays) The bioinformatic and translational perspective of presented approach is manifold. This first preliminary study on non-detectable miRNAs should sensitize scientific community to present not only data of deregulated candidates, but also data of completely absent miRNAs EMB, PBMC, and serum samples were obtained from healthy controls and patients suffering from inflammatory or virally induced myocarditis as shown in Spinal fluid samples were received from healthy controls and patients suffering from Alzheimer’s disease, with the ethical statement described previously miRNAs were obtained from patients, using mirVana™ miRNA Isolation Kit  resp. mirVana™ PARIS™ RNA and Native Protein Purification Kit  for low content samples such as serum, urine, ocular fluid, and spinal fluid according to manufacturer’s instructions. All presented expression studies were performed in the same laboratory.Total RNA including miRNA fraction was reversely transcribed to cDNA using Megaplex stem-loop RT primer  for Human Pool A and B in combination with the TaqMan MicroRNA Reverse Transcription Kit . This allowed simultaneous cDNA synthesis of 377 unique miRNAs for Pool A and B each. Except for biopsy materials, a pre-amplification protocol was performed for all low content samples to increase the detection rate. The entire procedure for quantification using TaqMan® OpenArray® ∗ sequences as annotated in Sanger miRBase version 14.0 was performed with the Geniom Real Time Analyzer  and the Geniom biochip MPEA hsapiens V14. Sample labeling with biotin was carried out by using the ULS labeling Kit from Kreatech . All essential steps such as hybridization, washing, as well as signal amplification and measurement, were done automatically by Geniom Real Time Analyzer. The resulting detection images were evaluated using the Geniom Wizard Software for background correction and normalization of generated data. miRNA expression analyses were carried out using the normalized and background-subtracted intensity values.The expression analysis of all 906 miRNA and miRNAhttp://bioinfogp.cnnb.csic.es/tools/venny/index.html). miEAA (http://www.ccb.uni saarland.de/mieaa_tool) and DIANA miRPath v.2.0 miRNAs not detectable in all samples of corresponding biological material were regarded as absent for this material and disease. All following bioinformatics analyses by pathway prediction tools were based on the list of these candidates. Venn diagrams of intersecting sets of miRNAs between different tissues and platforms are generated using Venny v2.0 (CS conducted the bioinformatic algorithms and miRNA target identification, and drafted the manuscript. CS and MR carried out miRNA expression studies. DL conceived the study, and participated in study design and coordination. UK, FE, and HPS had primary responsibility for patient characterization and management. All authors discussed the results, read, and approved the final manuscript.The authors declare no competing financial interests or relationships relevant to the content of this paper to disclose.