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Nod-like receptors (NLRs) are nucleotide-binding domain and leucine-rich repeats containing proteins that are important in plant resistance signaling. Many of the known pathogen resistance (R) genes in plants are NLRs and they can recognize pathogen molecules directly or indirectly. As such, divergence and copy number variants at these genes are found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild nonmodel species, it is necessary to identify the full range of NLRs and infer their evolutionary history. Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species Solanum pennellii. We use a combination of enrichment sequencing and pooling ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different mapping and single nucleotide polymorphism calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πS ratios and differing site frequency spectra. We compare our observed rate of heterozygosity with expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In nature, most plants are resistant to a wide range of phytopathogens. However, mechanisms contributing to this so-called nonhost resistance (NHR) are poorly understood. Besides constitutive defenses, plants have developed two layers of inducible defense systems. Plant innate immunity relies on recognition of conserved pathogen-associated molecular patterns (PAMPs). In compatible interactions, pathogenicity effector molecules secreted by the invader can suppress host defense responses and facilitate the infection process. Additionally, plants have evolved pathogen-specific resistance mechanisms based on recognition of these effectors, which causes secondary defense responses. The current effector-driven hypothesis is that NHR in plants that are distantly related to the host plant is triggered by PAMP recognition that cannot be efficiently suppressed by the pathogen, whereas in more closely related species, nonhost recognition of effectors would play a crucial role. In this review we give an overview of current knowledge of the role of effector molecules in host and NHR and place these findings in the context of the model. We focus on examples from filamentous pathogens (fungi and oomycetes), discuss their implications for the field of plant-pathogen interactions and relevance in plant breeding strategies for development of durable resistance in crops.

Some of the strongest electromagnetic fields (EMF) are found in the workplace. A European Directive sets limits to workers' exposure to EMF. This review summarizes its origin and contents and compares magnetic field exposure levels in high-risk workplaces with the limits set in the revised Directive. Pubmed, Scopus, grey literature databases, and websites of organizations involved in occupational exposure measurements were searched. The focus was on EMF with frequencies up to 10 MHz, which can cause stimulation of the nervous system. Selected studies had to provide individual maximum exposure levels at the workplace, either in terms of the external magnetic field strength or flux density or as induced electric field strength or current density. Indicative action levels and the corresponding exposure limit values for magnetic fields in the revised European Directive will be higher than those in the previous version. Nevertheless, magnetic flux densities in excess of the action levels for peripheral nerve stimulation are reported for workers involved in welding, induction heating, transcranial magnetic stimulation, and magnetic resonance imaging (MRI). The corresponding health effects exposure limit values for the electric fields in the worker's body can be exceeded for welding and MRI, but calculations for induction heating and transcranial magnetic stimulation are lacking. Since the revised European Directive conditionally exempts MRI-related activities from the exposure limits, measures to reduce exposure may be necessary for welding, induction heating, and transcranial nerve stimulation. Since such measures can be complicated, there is a clear need for exposure databases for different workplace scenarios with significant EMF exposure and guidance on good practices., (© The Author 2014. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Accelerated gene evolution is a hallmark of pathogen adaptation following a host jump. Here, we describe the biochemical basis of adaptation and specialization of a plant pathogen effector after its colonization of a new host. Orthologous protease inhibitor effectors from the Irish potato famine pathogen, Phytophthora infestans, and its sister species, Phytophthora mirabilis, which is responsible for infection of Mirabilis jalapa, are adapted to protease targets unique to their respective host plants. Amino acid polymorphisms in both the inhibitors and their target proteases underpin this biochemical specialization. Our results link effector specialization to diversification and speciation of this plant pathogen.

Aphids are phloem-feeding insects that, like other plant parasites, deliver effectors inside their host to manipulate host responses. The Myzus persicae (green peach aphid) candidate effectors Mp10 and Mp42 were previously found to reduce aphid fecundity upon intracellular transient overexpression in Nicotiana benthamiana. We performed functional analyses of these proteins to investigate whether they activate defenses through similar activities. We employed a range of functional characterization experiments based on intracellular transient overexpression in N. benthamiana to determine the subcellular localization of Mp10 and Mp42 and investigate their role in activating plant defense signaling. Mp10 and Mp42 showed distinct subcellular localization in planta, suggesting that they target different host compartments. Also, Mp10 reduced the levels of Agrobacterium-mediated overexpression of proteins. This reduction was not due to an effect on Agrobacterium viability. Transient overexpression of Mp10 but not Mp42 activated jasmonic acid and salicylic acid signaling pathways and decreased susceptibility to the hemibiotrophic plant pathogen Phytophthora capsici. We found that two candidate effectors from the broad-host-range aphid M. persicae can trigger aphid defenses through different mechanisms. Importantly, we found that some (candidate) effectors such as Mp10 interfere with Agrobacterium-based overexpression assays, an important tool to study effector activity and function.

Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

Background: Plant-microbe interactions feature complex signal interplay between pathogens and their hosts. Phytophthora species comprise a destructive group of fungus-like plant pathogens, collectively affecting a wide range of plants important to agriculture and natural ecosystems. Despite the availability of genome sequences of both hosts and microbes, little is known about the signal interplay between them during infection. In particular, accurate descriptions of coordinate relationships between host and microbe transcriptional programs are lacking., Results: Here, we explore the molecular interaction between the hemi-biotrophic broad host range pathogen Phytophthora capsici and tomato. Infection assays and use of a composite microarray allowed us to unveil distinct changes in both P. capsici and tomato transcriptomes, associated with biotrophy and the subsequent switch to necrotrophy. These included two distinct transcriptional changes associated with early infection and the biotrophy to necrotrophy transition that may contribute to infection and completion of the P. capsici lifecycle, Conclusions: Our results suggest dynamic but highly regulated transcriptional programming in both host and pathogen that underpin P. capsici disease and hemi-biotrophy. Dynamic expression changes of both effector-coding genes and host factors involved in immunity, suggests modulation of host immune signaling by both host and pathogen. With new unprecedented detail on transcriptional reprogramming, we can now explore the coordinate relationships that drive host-microbe interactions and the basic processes that underpin pathogen lifestyles. Deliberate alteration of lifestyle-associated transcriptional changes may allow prevention or perhaps disruption of hemi-biotrophic disease cycles and limit damage caused by epidemics.

MLL-rearranged infant acute lymphoblastic leukemia (ALL) (<1 year of age) are frequently resistant to glucocorticoids, like prednisone and dexamethasone. As poor glucocorticoid responses are strongly associated with therapy failure, overcoming glucocorticoid resistance may be a crucial step towards improving prognosis. Unfortunately, the mechanisms underlying glucocorticoid resistance in MLL-rearranged ALL largely remain obscure. We here defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples, demonstrating that, among other genes, high-level ANXA2 is associated with prednisolone resistance in this type of leukemia. Further investigation demonstrated that the underlying factor of this association was the presence of Src kinase-induced phosphorylation (activation) of annexin A2, a process requiring the adapter protein p11 (encoded by human S100A10). shRNA-mediated knockdown of either ANXA2, FYN, LCK or S100A10, all led to inhibition of annexin A2 phosphorylation and resulted in marked sensitization to prednisolone. Likewise, exposure of prednisolone-resistant MLL-rearranged ALL cells to different Src kinase inhibitors exerting high specificity towards FYN and/or LCK had similar effects. In conclusion, we here present a novel mechanism of prednisolone resistance in MLL-rearranged leukemias, and propose that inhibition of annexin A2 phosphorylation embodies a therapeutic strategy for overcoming resistance to glucocorticoids in this highly aggressive type of leukemia.

Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions.

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca(2+) fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca(2+) and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca(2+) levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca(2+) fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.

Unlabelled: Phytophthora capsici is a highly dynamic and destructive pathogen of vegetables. It attacks all cucurbits, pepper, tomato and eggplant, and, more recently, snap and lima beans. The disease incidence and severity have increased significantly in recent decades and the molecular resources to study this pathogen are growing and now include a reference genome. At the population level, the epidemiology varies according to the geographical location, with populations in South America dominated by clonal reproduction, and populations in the USA and South Africa composed of many unique genotypes in which sexual reproduction is common. Just as the impact of crop loss as a result of P. capsici has increased in recent decades, there has been a similar increase in the development of new tools and resources to study this devastating pathogen. Phytophthora capsici presents an attractive model for understanding broad-host-range oomycetes, the impact of sexual recombination in field populations and the basic mechanisms of Phytophthora virulence., Taxonomy: Kingdom Chromista; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Phytophthora; Species capsici., Disease Symptoms: Symptoms vary considerably according to the host, plant part infected and environmental conditions. For example, in dry areas (e.g. southwestern USA and southern France), infection on tomato and bell or chilli pepper is generally on the roots and crown, and the infected plants have a distinctive black/brown lesion visible at the soil line (Fig. 1). In areas in which rainfall is more common (e.g. eastern USA), all parts of the plant are infected, including the roots, crown, foliage and fruit (Fig. 1). Root infections cause damping off in seedlings, whereas, in older plants, it is common to see stunted growth, wilting and, eventually, death. For tomatoes, it is common to see significant adventitious root growth just above an infected tap root, and the stunted plants, although severely compromised, may not die. For many cucurbit fruit, the expanding lesions produce fresh sporangia over days (or even weeks depending on the size of the fruit) and the fruit often look as if they have been dipped in white powdered confectioner's sugar (Fig. 1). Generally, hyphae do not emerge from infected plants or fruit (common with Pythium infections) and all that is visible on the surface of an infected plant is sporangia., Importance: Phytophthora capsici presents an oomycete worst-case scenario to growers as it has a broad host range, often produces long-lived dormant sexual spores, has extensive genotypic diversity and has an explosive asexual disease cycle. It is becoming increasingly apparent that novel control strategies are needed to safeguard food production from P. capsici and other oomycetes. Considering that P. capsici is easy to grow, mate and manipulate in the laboratory and infects many plant species, this pathogen is a robust model for investigations, particularly those related to sexual reproduction, host range and virulence., Useful Websites: Phytophthora capsici genome database: http://genome.jgi-psf.org/Phyca11/Phyca11.home.html. Molecular tools to identify Phytophthora isolates: http://phytophthora-id.org., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

MLL-rearranged infant acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia characterized by a unique gene-expression profile. We uncovered that the activation of particular (proto-onco)genes is mediated by promoter hypomethylation. In search for therapeutic agents capable of targeting these potential cancer-promoting genes, we applied connectivity mapping on a gene expression signature based on the genes most significantly hypomethylated in t(4;11)-positive infant ALL as compared with healthy bone marrows. This analysis revealed histone deacetylase (HDAC) inhibitors as suitable candidates to reverse the unfavorable gene signature. We show that HDAC inhibitors effectively induce leukemic cell death in t(4;11)-positive primary infant ALL cells, accompanied by downregulation of MYC, SET, RUNX1, RAN as well as the MLL-AF4 fusion product. Furthermore, DNA methylation was restored after HDAC inhibitor exposure. Our data underlines the essential role for epigenetic de-regulation in MLL-rearranged ALL. Furthermore, we show, for the first time, that connectivity mapping can indirectly be applied on DNA methylation patterns, providing a rationale for HDAC inhibition in t(4;11)-positive leukemias. Given the presented potential of HDAC inhibitors to target important proto-oncogenes including the leukemia-specific MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials.

Introduction: External fixation is the primary choice of temporary fracture stabilisation for specific polytrauma patients. Adequate initial fracture healing requires sufficient stability at the fracture site. The purpose of this study was to compare the rigidity of the Dynafix DFS(®) Standard Fixator (4 joints) with the Orthofix ProCallus Fixator(®) (2 joints), which differ in possibilities for adapting the configuration for clinical needs., Materials and Methods: Both devices were tested 10 times in a standardised model. In steps of 10N, loading was increased to a maximum of 160N in parallel, transversal and axial direction (distraction and compression). Translation resultant and rotation resultant were calculated., Results: With a force of 100N in parallel direction the mean translation resultant (Tr(mean)) of the Dynafix DFS(®) Standard Fixator (6.65±1.43mm) was significantly higher than the ProCallus Fixator(®) (3.29±0.83mm, p<0.001; Student's t-test). With a maximum load of 60N in transverse direction the Tr(mean) of the Dynafix DFS(®) Standard Fixator was significantly lower (8.14±1.20mm versus 9.83±0.63mm, p<0.005). Translation was significantly higher with the Dynafix DFS(®) Standard Fixator, for both distraction (2.13±0.32mm versus 1.69±0.44mm, p<0.05) and compression (1.55±1.08mm versus 0.15±0.33mm, p<0.005). The mean rotation resultant (Rr(mean)) at 160N distraction was lower for the Dynafix DFS(®) Standard Fixator (0.70±0.17° versus 0.97±0.21°, p<0.005)., Conclusions: Both fixators were most sensitive to transverse forces. The Dynafix DFS(®) Standard Fixator was less rigid with parallel and axial forces, whereas transverse forces and rotation at distraction forces favoured the Dynafix DFS(®) Standard Fixator. Repeated heavy loading did not influence the rigidity of both devices., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is the most aggressive type of childhood leukemia. To develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t(4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. In this study, we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression. We identified 11 miRNAs that were downregulated in t(4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, five of these miRNAs are associated either with MLL or MLL fusions, and for miR-152 we found both MLL and DNA methyltransferase 1 (DNMT1) as potential targeted genes. Finally, a high degree of methylation of the miR-152 CpG island was strongly correlated with a poor clinical outcome. Our data suggests that inhibitors of methylation have a potential beyond re-expression of hypermethylated protein-coding genes in t(4;11)-positive infant ALL. In this study, we provide additional evidence that they should be tested for their efficacy in MLL-rearranged infant ALL in in vivo models.

Background: ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity., Procedure: Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis., Results: Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC(50) values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways., Conclusions: We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials., (Copyright © 2010 Wiley-Liss, Inc.)

The mammalian blood-brain barrier (BBB) consists of endothelial cells, linked by tight junctions, and the adjoining pericytes and extracellular matrix. It helps maintain a highly stable extracellular environment necessary for accurate synaptic transmission and protects nervous tissue from injury. An increase in its normally low permeability for hydrophilic and charged molecules could potentially be detrimental. Methods to assess the permeability of the BBB include histological staining for marker molecules in brain sections and measurement of the concentration of marker molecules in blood and brain tissue. Their advantages and disadvantages are discussed. Exposure to levels of radiofrequency electromagnetic fields (EMF) that increase brain temperature by more than 1°C can reversibly increase the permeability of the BBB for macromolecules. The balance of experimental evidence does not support an effect of 'non-thermal' radiofrequency fields with microwave and mobile phone frequencies on BBB permeability. Evidence for an effect of the EMF generated by magnetic resonance imaging on permeability is conflicting and conclusions are hampered by potential confounders and simultaneous exposure to different types and frequencies of EMF. The literature on effects of low frequency EMF, which do not cause tissue heating, is sparse and does not yet permit any conclusions on permeability changes. Studies on the potential effect of EMF exposure on permeability of the BBB in humans are virtually absent. Future permeability studies should focus on low frequency effects and effects in humans. Care should be taken to avoid the methodological limitations of earlier studies and to determine the pathophysiological relevance of any changes found., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A single session of footshocks in rats causes long-lasting sensitisation of behavioural, hormonal and autonomic responses to subsequent novel stressful challenges as well as altered pain sensitivity. These changes mimic aspects of post-traumatic stress disorder in humans. Our aim was to identify neuropeptide substrates in the central nervous system involved in stress sensitisation. Male Wistar rats were exposed to ten footshocks in 15 min (preshocked) or placed in the same cage without shocks (control). Two weeks later, rats were placed in a novel cage, subjected to 5 min of 85 dB noise, and returned to their home cage. Rats were killed either before or 1 h after noise and their brains processed for in situ hybridization for neuropeptide Y (NPY) and beta-preprotachykinin-I (PPT) mRNA. Additional groups of rats were killed under basal conditions and brains processed for NPY and neurokinin receptor binding with radiolabelled ligands. Two weeks after footshock treatment NPY mRNA expression was increased in the basolateral amygdala and showed preshockxnoise interaction in the locus coeruleus (down after noise in controls, lower basal and unchanged after noise in preshocked). PPT expression in the lateral parabrachial nucleus also showed preshockxnoise interaction (up after noise in controls, higher basal and down after noise in preshocked), and was increased after noise in the periaquaeductal grey. NK1 receptor binding in the agranular insular cortex and arcuate nucleus of the hypothalamus and NK2 receptor binding in the amygdala was lower in preshocked rats than in controls. Altered expression of NPY in the basolateral amygdala and locus coeruleus could contribute to or compensate for behavioural and autonomic sensitisation in preshocked rats. Altered PPT expression in the parabrachial nucleus may be involved in the altered pain processing seen in this model. Lower NK1 and NK2 receptor numbers in cortex, hypothalamus and amygdala may reflect secondary adaptations to altered neuropeptide release. These long-term changes in brain neuropeptide systems could offer novel leads for pharmacological modulation of long-term stress-induced sensitisation.

Conditioned taste aversion (CTA) is a behavioural response essential to the survival of an individual. The combination of taste and odour of most foods provides a strong conditioned stimulus (CS) for an animal to respond in an appropriate way to any harmful unconditioned stimuli (US) that follow. The most widely used conditioned stimuli are drinkable sweet solutions, such as saccharin and sucrose. CTA-like responses are also found for environmental unconditioned stimuli, but these usually take longer training. In the present study, the aversive nature of a duodenal distention with an implanted balloon catheter was studied in freely moving rats using either CTA against a sucrose solution, or a light-dark passive avoidance (PA) paradigm. In addition, the effect of spinal morphine on CTA and the cardiovascular response to duodenal distention were studied. CTA could be induced by a single, but long-lasting 20-minute duodenal distention, which did not induce PA behaviour in a light-dark box. Spinal infusion of morphine alone induced CTA, suggesting that the model is unsuitable to investigate spinal pharmacological modulation of visceral pain. Spinal morphine did reduce the cardiovascular response to duodenal distention, strengthening its validity as a visceral pain model. Since CTA is a complicating factor in the field of chemotherapy in cancer patients and spinal morphine causes nausea and vomiting in humans, CTA may also complicate spinal drug treatment or anaesthesia.

Rationale: A short session of repeated foot shocks in rats causes long-lasting sensitization of behavioural, hormonal and autonomic responses to novel stressful challenges. The behavioural sensitization can be reduced by anxiolytics and mimics aspects of stress-induced changes in patients with post-traumatic stress disorder., Objectives: The aim of this study was to test the efficacy of a group II metabotropic glutamate receptor (mGluR) agonist and assess altered brain mGluR receptor expression in shock-sensitized rats., Materials and Methods: Male Wistar rats were exposed to a 15-min session with ten 6-s foot shocks (preshocked). One and 2 weeks later, rats were intraperitoneally injected with the group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) or vehicle, and 30 min later exposed to 5 min of 85 dB noise. For in situ hybridization with probes for mGluR1, mGluR2, mGluR3 and mGluR5, preshocked and control rats were killed under basal conditions 2 weeks after foot shocks and their brains cryosectioned., Results: APDC had no clear effect in controls, but dose-dependently reduced high immobility and increased low locomotion and rearing seen in preshocked rats to the levels of controls. mGluR3 expression was increased in the basolateral nucleus of the amygdala, and mGluR2 expression was increased in the agranular insular cortex of preshocked rats compared to controls., Conclusions: Shock-induced behavioural sensitization in rats is reduced by acute treatment with a group II metabotropic glutamate receptor agonist. This effect may depend on the increased expression of amygdala mGluR3, which could be hypothesized as an endogenous mechanism to counteract stress-induced neuronal sensitization.

Post-traumatic stress disorder (PTSD) is a chronic, debilitating psychiatric disorder that can follow exposure to extreme stressful experiences. It is characterised by hyperarousal and increased startle responses, re-experiencing of the traumatic event, withdrawal or avoidance behaviour and emotional numbing. The focus of this review is on aspects that have received less attention. PTSD develops only in a substantial minority of people exposed to traumatic stress, and possible individual traits that increase vulnerability are discussed. An overview is given of the wide variety of physiological disturbances that accompany PTSD and may contribute to disability, including neuroendocrine, cardiovascular, gastrointestinal and immune function and pain sensitivity. Brain imaging and pharmacological studies have generated some insight into the circuitry that may be involved in the generation of PTSD symptoms. Major limitations of human studies so far are the issue of causality and our lack of understanding of the underlying molecular substrates in the brain, which are easier to address in relevant animal models and will be discussed in a companion paper.

Animal models that are characterised by long-lasting conditioned fear responses as well as generalised behavioural sensitisation to novel stimuli following short-lasting but intense stress have a phenomenology that resembles that of PTSD in humans. These models include brief sessions of shocks, social confrontations, and a short sequence of different stressors. Subgroups of animals with different behavioural traits or coping styles during stress exposure show a different degree or pattern of long-term sensitisation. Weeks to months after the trauma, treated animals on average also show a sensitisation to novel stressful stimuli of neuroendocrine, cardiovascular and gastrointestinal motility responses as well as altered pain sensitivity and immune function. Functional neuroanatomical and pharmacological studies in these animal models have provided evidence for involvement of amygdala and medial prefrontal cortex, and of brain stem areas regulating neuroendocrine and autonomic function and pain processing. They have also generated a number of neurotransmitter and neuropeptide targets that could provide novel avenues for treatment in PTSD.

Posttraumatic stress disorder (PTSD) is the fourth most common psychiatric disorder. It is associated with cardiovascular disorders and irritable bowel syndrome (IBS). Besides stressful life-events, a prior history of gastrointestinal infection is a predisposing factor for the development of IBS. Only a proportion of persons exposed to traumatic events develop PTSD. Several factors, like genetic predisposition, stressor intensity, cognitive appraisal mechanisms and coping processes influence the likelihood of developing PTSD after exposure to a trauma. We used a single session of footshocks in rats, an animal model with a high degree of validity for PTSD, to study whether transient colonic inflammation alters local and distal visceral sensitivity, and whether reactivity to the open-field (low (LA) or high (HA) active) predicts long-term stress-induced behavioural and cardiovascular sensitisation and altered visceral pain sensitivity. A distention series and noise challenge were given 2 weeks after foot-shocks, followed by a transient colonic inflammation period and a second distention series and noise challenge 4 weeks after foot-shocks. During exposure to noise, both before and after inflammation, footshocked rats showed increased immobility compared to controls, which was significantly greater in LA rats than in HA rats. LA preshocked rats also showed a greater blood pressure response to the noise test, but this only became evident in the second noise-test. Neither footshocks nor colonic inflammation affected duodenal pain sensitivity. The results provide additional evidence for long-lasting cardiovascular hyperresponsivity after a stressful event and indicate that its degree is predicted by personality traits or coping style.

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (<1 years of age). Since current chemotherapy fails in >50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (P< or =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL.

Luminal distention of the intestine can be aversive in humans and laboratory animals, and hypersensitivity to distention is found in functional gastrointestinal disorders. Current animal models either require anaesthesia or acute balloon intubation or use implanted balloons of irritant materials, for which the aversive quality of distention and physiological responses have not been well characterised. We report here that silicone balloon catheters implanted in the duodenum via the stomach have long patency without obvious tissue damage. Balloon inflation in freely moving rats caused passive avoidance learning and classic 'pain' postures, as well as graded cardiovascular responses which can be recorded telemetrically. The method should make long-lasting studies of pharmacological and environmental effects on visceral sensitivity more feasible.

Infants with acute lymphoblastic leukemia (ALL) are more resistant to chemotherapeutic drugs than older children with ALL, except for Ara-C. Drug resistance mechanisms in infant ALL, however, remain unknown. Possibly, multidrug resistance (MDR) proteins like P-glycoprotein, MDR-associated protein (MRP1), lung resistance-related protein (LRP/MVP) and the breast cancer resistance protein (BCRP) play a role. Accordingly, we measured the mRNA levels of these proteins in infants (n=13) and non-infants (n=13) with ALL, using quantitative RT-PCR. Infants expressed 2.4-fold less BCRP mRNA (P=0.009) than non-infants with ALL. MDR1, MRP1 and LRP/MVP expression did not differ between both groups. MDR gene expression levels did not correlate to prednisolone, vincristine, daunorubicin or Ara-C cytotoxicity, except for BCRP expression, which correlated with resistance to Ara-C (Rs=0.53, P=0.012), suggesting that Ara-C might be a BCRP substrate. However, culturing patients ALL cells in the presence of the BCRP inhibitor Ko143 had no effect on Ara-C sensitivity. Inhibiting Bcrp1 in the Mdr1a-, Mdr1b- and Mrp1-deficient and Bcrp1-overexpressing mouse cell line Mef3.8/T6400, also did not modulate Ara-C cytotoxicity. Therefore, we conclude that Ara-C is not a substrate for BCRP and that MDR proteins do not play a significant role in drug resistance in infant ALL.

Background & Aims: The highly prevalent functional gastrointestinal disorders involve visceral pain and disturbed bowel habit and are associated with preceding stressful experiences, although causality and biological mechanisms remain unclear. The aim of the present study was to establish whether stress can directly and lastingly alter central nervous system responsivity to colonic distention in the rat as well as which neural pathways are likely to be involved., Methods: Rats were treated with a brief session of stressful foot shocks known to induce long-term behavioral and autonomic sensitization. Two weeks later, after induction of inhalation anesthesia, a balloon catheter was inserted in the distal colon and repeatedly inflated with brief, constant-pressure air pulses., Results: Reflex decreases in blood pressure and heart rate indicative of visceral afferent activation were greater in previously shocked rats than in controls. Colonic distention increased the expression of Fos, a marker of neuronal activation, in the sacral spinal cord and caudal brain stem. In the central amygdala and several cortical areas (prelimbic, infralimbic, agranular insular, cingulate), previously shocked rats showed reduced Fos expression following colonic distention compared with relevant controls., Conclusions: The results indicate that a brief but intense stressful experience causes long-lasting alterations in higher-order central nervous system responsivity to colonic distention even in the absence of conscious affective responses, pointing to basic alterations in the neural pathways involved.

Rationale: Traumatic life events can induce long-term alterations in neuronal substrates, which may ultimately lead to the development of anxiety disorders. It has been postulated that corticotropin-releasing hormone (CRH) plays an important role in anxiety-like behavior., Objectives: (1) To study the long-term effects of a single foot-shock experience on defensive withdrawal (DW) behavior in rats. (2) To examine the effects of the benzodiazepine anxiolytic drug chlordiazepoxide on the behavior of preshocked and control rats in the DW test. (3) To study the role of endogenous CRH in the long-term stress-induced increase in DW behavior., Methods: (1) Rats were exposed to a single session of foot shocks or exposed to the grid cage without receiving any shocks. Two, six and ten weeks later, rats were tested in the DW tests (2, 3). In subsequent experiments, rats were exposed to foot shocks or exposed to the grid cage without receiving any shocks, and 2 weeks later the effect of pharmacological treatments on the behavioral response in the DW test was investigated. Chlordiazepoxide (1, 5, 10 mg/kg BW, i.p.) and the CRH antagonists D-Phe CRH(12-41) (0.2, 1, 5 microg per rat, i.c.v.) and alpha-helical CRH(9-41) (5 microg per rat, i.c.v.) were injected 30 min before the test., Results: A single session of foot shocks induced a long-term increase in DW behavior, which persisted after repeated testing for at least 10 weeks. Chlordiazepoxide decreased the latency but did not affect time spent in light, distance moved, or the number of entries in the open field. D-Phe CRH(12-41) had no behavioral effects. alpha-Helical CRH(9-41) increased the time spent outside the box, primarily as a result of effects of alpha-helical CRH(9-41) in controls., Conclusion: These data demonstrate that preshocked rats display long-term increased anxiety-like behavior in the DW test but that CRH is unlikely to be involved in its expression.

There is evidence that the experience of traumatic events may play a role in the pathogenesis of somatic diseases, including cardiovascular disorders. In this study, telemetry was used to investigate the long-term effects of a single stressful experience on cardiovascular and behavioral responses to novel challenges 2 weeks later. Rats were exposed to footshocks and tested for sensitization using the following challenges: novel cylinder (Day 14); shock prod acquisition test (Day 15); and shock prod retention test (Day 16). No difference in basal somatomotor activity (SA), heart rate (HR) and blood pressure between preshocked rats and control rats was found. However, preshocked rats displayed an enhanced blood pressure response compared to controls during the shock prod acquisition test and the shock prod retention test. No differential increase in HR response between both groups was found. During the novel cylinder test, the preshocked rats displayed less SA while no behavioral differences were found in the shock prod acquisition test and the shock prod retention test. We conclude that a single stressful experience induces long-term sensitization of blood pressure responses to novel challenges that are not necessarily linked to sensitized behavioral responses. The footshock model may be a useful model to study autonomic hyperresponsivity found in posttraumatic stress disorder (PTSD).

Purpose: The purpose of this study was to determine the primary factors causing the differential oxygen uptake (VO2) response at submaximal intensities between one-legged and two-legged exercise, and whether peak oxygen uptake (VO2peak) increases in proportion to the increase in active muscle mass., Methods: Two different types of exercise were used for this experiment, each requiring a different movement, a different method of stabilizing posture, and, finally, a different limiting VO2peak. In experiment 1, nine male subjects performed one-legged cycling (OLC) and two-legged cycling exercise (TLC) at a pedaling rate of 80 rpm. The exercise intensity was first set at 80 W and was increased by 40 W every 3 min until exhaustion. In experiment 2, six healthy male subjects performed one-legged knee-extension (OKE) and two-legged knee-extension (TKE) exercise at a rate of 50 contractions per minute. The knee-extension exercise was done at constant work rates for a 3-min session in OKE or a 4-min session in TKE. The exercise bouts were performed intermittently at four to seven different submaximal intensities and VO2 was determined at each intensity in all exercises., Results: At submaximal intensities, VO2 in relation to work rate of one-legged exercise was more steep than those of two-legged exercise, and the mean values of VO2 were significantly higher in one-legged exercise than those in two-legged exercise in both knee extension and cycling exercise. Mean values of VO2peak for two-legged exercise were significantly higher than that for one-legged exercise (P < 0.01); however, it was much lower than two times of that for one-legged exercise even in knee extension exercise where the VO2peak would be limited peripherally., Conclusion: The findings of this study suggest that the differential VO2 response between one-legged and two-legged exercise would be attributed not only to the difference in force application throughout the exercise movement and to the effect of a postural component but also to the inhibited circulatory response caused by the multiple limb exercise. In addition, it was supposed that VO2peak does not increase in proportion to the exercising muscle mass even during smaller muscle activity where the cardiac pumping capacity has not reached its upper limit.

The involvement of the corticotropin-releasing hormone (CRH) type 1 receptor in CRH-induced cardiac responses was studied in freely moving rats. Intracerebroventricular (icv) infusion of 2 microg CRH under resting conditions resulted in a significant increase in heart rate (HR), but did not significantly affect the PQ interval of the electrocardiogram. This effect involves sympathetic nervous system (SNS) activation, since CRH-treatment resulted in a marked increase in plasma norepinephrine (NE) and epinephrine (E), and sympathetic blockade by subcutaneously injected atenolol (1 mg/kg), a beta1-selective adrenergic antagonist, completely prevented the CRH-induced tachycardia. CRH infusion after sympathetic blockade resulted in an elongation of the PQ interval, indicating CRH-induced vagal activation. Gross locomotor activity (GA) was determined to study its possible indirect effects on cardiac activity. Although CRH induced a marked increase in GA, this effect followed the tachycardiac response, indicating that the HR response was not a consequence of increased locomotor activity, but was a direct effect of icv CRH. Treatment with CP-154,526 (icv, 10 or 25 microg), a selective CRH type 1 receptor antagonist, did not affect baseline HR, plasma NE and E, whereas it partially blocked the CRH-induced increase in HR, plasma NE and E levels. CP-154,526 treatment had no significant effects on baseline or CRH-induced changes in GA. These results indicate that CRH activates the sympathetic nervous system at least in part via the CRH type 1 receptor.

The role of the endogenous corticotropin-releasing hormone (CRH) system in the regulation of heart rate, PQ interval (a measure of vagal activity), gross activity and release of adrenocorticotropic hormone (ACTH), noradrenaline and adrenaline into the blood during conditioned fear was studied in freely moving rats. Intracerebroventricular (i.c.v.) infusion of alpha-helical CRH-(9-41) (10 microgram/3 microliter), a non-selective CRH receptor antagonist, under resting conditions had no significant effect on gross activity, heart rate and PQ interval, indicating that alpha-helical CRH at this dose was devoid of agonist effects. Conditioned fear was induced by 10 min forced exposure to a cage in which the rat had experienced footshocks (5x0.5 mAx3 s) 1 day before. Conditioned-fear rats showed freezing behaviour, associated with an increase in heart rate, PQ interval, noradrenaline and adrenaline, indicating that the conditioned-fear-induced cardiac effects were the result of coactivation of the sympathetic and parasympathetic nervous system. The i.c.v. pre-treatment of rats with alpha-helical CRH significantly reduced the conditioned-fear-induced tachycardiac and ACTH response, and enhanced the increase in PQ interval, without affecting the noradrenaline and adrenaline response. These results suggest that endogenous CRH reduces the vagal response to conditioned-fear stress in rats. To test this, rats were pre-treated with atropine methyl nitrate (0.3 mg/kg, subcutaneously; s.c.), a peripherally acting cholinergic receptor antagonist. This resulted in a complete blockade of the alpha-helical CRH-induced decrease in heart rate response and increase in PQ interval. From these findings, it is concluded that endogenous CRH in the brain inhibits vagal outflow induced by emotional stress.

There is considerable evidence for a role of stressful experiences in psychosomatic disorders in humans, but the mechanisms leading to altered responsivity and the relative contributions of central and peripheral neuronal changes, however, are still under debate. To investigate the contribution of specific brain areas to sensitized responsivity, rats were exposed to a single brief session of inescapable footshocks (preshocked) or no shocks (control) in a gridcage. Two weeks later, an electrified prod was inserted in the home cage for 15 min and the behaviour recorded. One hour later rats were perfused and brain sections were stained for Fos protein immunoreactivity. The number of Fos positive neurons was quantified in 27 brain areas. No significant difference in behaviour was found between the groups during the shock prod challenge. A significantly higher number of Fos positive neurons was found in preshocked rats compared to controls in the following brain areas: agranular insular cortex, frontal cortex, nucleus accumbens, bed nucleus of the stria terminalis, basolateral amygdala, CA1 area of the hippocampus, paraventricular hypothalamic nucleus, dorsolateral central grey, locus coeruleus, nucleus of the solitary tract and lateral paragigantocellular nucleus. We conclude that altered reactivity to stressful challenges in brain areas involved in neuroendocrine and autonomic control may play a role in long-term sensitization of neuroendocrine and autonomic responses in preshocked rats under conditions where behavioural sensitization is not expressed., (Copyright 1999 Elsevier Science B.V.)

Functional bowel disorders are more prevalent in women than in men, but the reason for this is unclear. Stressful experiences can increase the risk for or precipitate intestinal dysfunction. Using a model for long-term stress-induced sensitisation in rats, it was investigated whether male and female rats differ in susceptibility for long-term colonic, behavioural and hormonal disturbances following brief but intense stress. Male and female Wistar rats were fitted with chronic electrodes on proximal colon and given either a 15-minute session of foot shocks or no shocks. Two weeks later, rats were exposed to two different novel stressful challenges in the home cage: an electrified prod (day 14) and an 85 dB noise stressor (day 15). Digitalised colonic myoelectric spike burst activity was quantified automatically. Behaviour during prod and noise exposure was scored blindly from videotape. Resting plasma hormone concentrations at the end of the study were determined by radio-immuno assay. Following prod stress on day 14, both male and female preshocked rats showed a greater increase in colonic spike burst frequency than controls, but similar behaviour, and the dynamics of colonic motility differed between sexes. Following noise stress on day 15, only a small change in burst frequency was seen in all rats, but preshocked rats showed less self-grooming behaviour and there was a tendency for preshocked females to show increased noise-induced immobility. Preshocked rats also had lower levels of plasma free thyroxine. While both male and female rats show long-term stress-induced colonic sensitisation and hormonal changes, females show a different activation pattern of colonic motility, and may be more vulnerable for altered behavioural reactivity, following stress.

Myoelectric activity of the small intestine was recorded digitally in fasted, freely moving rats fitted with multiple pairs of electrodes in the antimesenterial smooth muscle. The electrodes were implanted under strict aseptic conditions to safeguard the animal's health and thereby benefit experimental results. As in many other mammalian species, the fasted electrical and mechanical activity of the small intestine in the rat consists of alternating periods of activity and quiescence jointly called the Migrating Motility Complex (MMC). Existing methods for the analysis of small bowel myoelectric activity either integrate spike activity over longer periods of time or describe characteristics of single bursts. We have developed a computer program for fast, automated analysis that distinguishes the three characteristic phases of the MMC. The computer program recognizes periods of varying spike burst frequency and then produces a graphical and numerical output of the length and mean burst frequency of the different phases of each MMC that correlates well with the results of visual evaluation. The techniques used are discussed in relation to existing recording and analysis methods, and specific decisions in the program are motivated. The system will be a useful tool in assessing the effects of drugs, peptides, or stress on small bowel motility.