Identification of an Epi-metabolic dependency on EHMT2/G9a in T-cell acute lymphoblastic leukemia

Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.Subject terms: Translational research, Cancer metabolism, Acute lymphocytic leukaemia     

Characterization of a new class of potent inhibitors of the voltage-gated sodium channel Nav1.7

Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.     

Visfatin, an adipocytokine with proinflammatory and immunomodulating properties

Adipocytokines are mainly adipocyte-derived cytokines regulating metabolism and as such are key regulators of insulin resistance. Some adipocytokines such as adiponectin and leptin affect immune and inflammatory functions. Visfatin (pre-B cell colony-enhancing factor) has recently been identified as a new adipocytokine affecting insulin resistance by binding to the insulin receptor. In this study, we show that recombinant visfatin activates human leukocytes and induces cytokine production. In CD14(+) monocytes, visfatin induces the production of IL-1beta, TNF-alpha, and especially IL-6. Moreover, it increases the surface expression of costimulatory molecules CD54, CD40, and CD80. Visfatin-stimulated monocytes show augmented FITC-dextran uptake and an enhanced capacity to induce alloproliferative responses in human lymphocytes. Visfatin-induced effects involve p38 as well as MEK1 pathways as determined by inhibition with MAPK inhibitors and we observed activation of NF-kappaB. In vivo, visfatin induces circulating IL-6 in BALB/c mice. In patients with inflammatory bowel disease, plasma levels of visfatin are elevated and its mRNA expression is significantly increased in colonic tissue of Crohn's and ulcerative colitis patients compared with healthy controls. Macrophages, dendritic cells, and colonic epithelial cells might be additional sources of visfatin as determined by confocal microscopy. Visfatin can be considered a new proinflammatory adipocytokine.     

Survival of Conidia of Clonostachys rosea on Stored Barley Seeds and Their Biocontrol Efficacy Against Seed-borne Bipolaris sorokiniana

Survival and biocontrol activity of Clonostachys rosea (isolate IK726) conidia during storage on barley seeds were investigated. The initial density of colony forming conidia on seed was 4 &#50 10 3 to 9 &#50 10 4 colony forming units (cfu)/seed. After 5 months storage at 4°C, the density decreased by less than one order of magnitude and the biocontrol efficacy against seedling blight caused by seed-borne Bipolaris sorokiniana was maintained at a significantly high level ( > 80% disease reduction) for > 5 months. Conidial survival on seeds stored at 20°C declined more rapidly than at 4°C, and biocontrol efficacy was significantly reduced after 3-5 months. However, conidia produced on solid media over 20 days survived better than conidia produced in liquid culture and conidia from solid media produced over 12 days. In contrast, when seeds treated with conidia were packed with silica gel and stored at 20°C, the cfu density decreased by less than one order of magnitude after 5 months and the biocontrol efficacy was still high after 6 months. A dose-response curve revealed that 103 cfu/seed were needed for 80% control of seedling blight. Similar control was obtained in storage experiments when approximately 103 cfu/seed were recovered from seed, indicating that conidia which survived also retained a high ability to control disease.     

Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinerea and strawberry

Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for beta-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated while no change in expression of two endochitinases was measured. In strawberry leaves, the chitinase genes were upregulated 2-12-fold, except one of the endochitinases, whereas no change in expression of the two endoglucanases was measured. The results suggest that three out of four chitinase genes of IK726 are involved in biocontrol on leaves. This is the first example of monitoring of expression of chitinolytic genes in interactions between biocontrol agents and pathogens in plant material.     

Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities

To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach. The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities. However, endophytic populations correlated to a certain extent with plant growth performance. Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes. A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations. In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris. The proportion of isolates with antagonistic activity was highest against Streptomyces sp. (43%) followed by those against Xanthomonas sp. (29%). As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists. The antifungal activity of the isolates was generally low. The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high. Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.     

Regionalizing Indicator Values for Soil Reaction in the Bavarian Alps – from Averages to Multivariate Spectra

We present an approach to produce maps of Ellenberg values for soil reaction (R-value) in the Bavarian Alps. Eleven meaningful environmental predictors covering GIS-derived information on climatic, topographic and soil conditions were used to predict R-values. As dependent variables, Ellenberg indicator values for soil reaction were queried from plot records in the vegetation database WINALPecobase. We used an additive georegression model, which combines complex prediction models and the increased prediction accuracy of a boosting algorithm. In addition to environmental predictors we included spatial effects into the model to account for spatial autocorrelation. As we were particularly interested in the usefulness of averaged R-values for spatial prediction, we applied two different models: (1) a geo-additive regression model that estimates mean R-values and (2) a proportional odds model predicting the probability distribution over R-values 1 to 9. We found meaningful dependencies between the R-value and our predictors. Both models produced the same spatial pattern of predictions. Spatial effects had an impact only in the first model. The main drawback of mean R-values is the oversimplification of complex conditions of soil reaction, which is entailed by averaging and regression to mean values. Therefore, regionalized average indicator values provide only limited information on site-ecological characteristics. Model 1 failed to predict the range and shapes of original indicator spectra precisely. In contrast, the second model provided a more sophisticated picture of soil reaction. To make the multivariate output of model 2 comparable to that of model 1, we propose to plot the distribution in a three-dimensional color-space. In addition, comparison of both models based on a multiple linear regression model resulted in a R ² of 0.93. The proportional odds model is a promising approach also for other indicator values and different regions as well as for other ordinal-scaled ecological parameters.     

M13 DNA fingerprinting can be used in studies on phenotypic reversions of forest tree mutants

 Solitary revertants which have been observed on single mutant tree individuals have up to now been believed to be grow-through cells belonging to the rootstock on which they are commonly grafted. In this study three different phenotypically visible mutants revealing revertant shoots on the same tree were chosen for genetic analysis. The mutant Quercus robur L. ‘argenteomarginata’ was grafted on a normal rootstock, an individual of Carpinus betulus L. var. quercifolia Desf. as well as an individual of Picea glauca (Moench) Voss. ‘conica’ are supposed to have grown from seeds. By means of a highly specific M13 PCR fingerprinting technique the mutant and revertant tissues were analysed in comparison to different individuals of each of the species. With the grafted mutant, cambium tissue of the rootstock was also investigated. Whereas conspecific individuals could be clearly distinguished from each other, mutant and revertant tissues revealed the same banding patterns for each of the three trees. In case of the grafted mutant, the fingerprint obtained from cambium tissue of the rootstock was clearly different from the pattern of mutant and revertant tissue. Results demonstrate the potential of the tool for genetic differentiation between individuals of three tree species hence in the case of the grafted mutant, the hypothesis that the observed reversion is caused by a grow-through of the rootstock is rejected. Furthermore, identical fingerprints of mutant and revertant tissue support identical genetic background of the tissues excluding the gene(s) responsible of the mutation. Possible causes of mutations and reversions regarding the three mutant trees are discussed. Received: 15 September 1997 / Accepted: 24 November 1997     

Soil warming alters microbial substrate use in alpine soils

Will warming lead to an increased use of older soil organic carbon (SOC) by microbial communities, thereby inducing C losses from C‐rich alpine soils? We studied soil microbial community composition, activity, and substrate use after 3 and 4 years of soil warming (+4 °C, 2007–2010) at the alpine treeline in Switzerland. The warming experiment was nested in a free air CO₂ enrichment experiment using depleted ¹³CO₂ (δ¹³C = ?30‰, 2001–2009). We traced this depleted ¹³C label in phospholipid fatty acids (PLFA) of the organic layer (0–5 cm soil depth) and in C mineralized from root‐free soils to distinguish substrate ages used by soil microorganisms: fixed before 2001 (‘old’), from 2001 to 2009 (‘new’) or in 2010 (‘recent’). Warming induced a sustained stimulation of soil respiration (+38%) without decline in mineralizable SOC. PLFA concentrations did not reveal changes in microbial community composition due to soil warming, but soil microbial metabolic activity was stimulated (+66%). Warming decreased the amount of new and recent C in the fungal biomarker 18:2ω6,9 and the amount of new C mineralized from root‐free soils, implying a shift in microbial substrate use toward a greater use of old SOC. This shift in substrate use could indicate an imbalance between C inputs and outputs, which could eventually decrease SOC storage in this alpine ecosystem.