Pseudomonas syringae produce the phytotoxin coronatine (COR). A major aspect of COR function is its ability to mimic a bioactive jasmonic acid (JA) conjugate and thus target the JA-receptor COR-insensitive 1 (COI1). Biological activities of COR include stimulation of JA-signaling and consequent suppression of SA-dependent defense through antagonistic crosstalk, antagonism of stomatal closure to allow bacterial entry into the interior of plant leaves, contribution to chlorotic symptoms in infected plants, and suppression of plant cell wall defense through perturbation of secondary metabolism. Here, we review the virulence function of COR, including updates on these established activities as well as more recent findings revealing COI1-independent activity of COR and shedding light on cooperative or redundant defense suppression between COR and type III effector proteins.Plant pathogens deploy an array of virulence factors to suppress host defense and promote pathogenicity. Numerous strains of  Pseudomonas syringae pathovars produce a repertoire of virulence effectors that are active inside plant cells, including numerous phytotoxins  and coronamic acid (CMA)  strain DC3000 and P. syringae pv. glycinea (Pgl) strain 4180 -7-iso-JA-Ile (JA-Ile)  has been noted -7-iso-JA-Ile  genes  or as-1-like promoter elements  is a key phytohormone in plant defense against a variety of biotrophic and hemibiotrophic pathogens, including bacterial strains producing COR Fig. . SA is aPR genes, SA-signaling also has an antagonistic effect on JA-signaling  bacteria induced less JA- and more SA-responsive gene expression, and the reduced growth of cor- strains was restored when the bacteria infected SA-signaling deficient plants  repress expression of genes controlling SA-biosynthesis, including an isochorismate synthase (SID2), and 2) induce expression of a benzoic/salicylic acid carboxyl methyltransferase (BSMT1) that reduces the pool of free  SA via methylation  is a plant counter-defense strategy to prevent bacterial entry  plants  plays a critical role in the contribution of COR to the induction of chlorotic disease symptoms by Pto DC3000  induced accelerated, coalescing necrotic lesions without apparent chlorosis , that work in a ‘multifunctional, cooperative, and redundant’ manner -deficient double mutant of Pto DC3000) revealed overlapping yet still distinct roles of COR and T3Es in reprogramming of the host transcriptome  gene, RAP2.6. Both a TTSS mutant and a cor- mutant of DC3000 failed to induce RAP2.6. Interestingly, AvrB complemented the ability of the cor- mutant strain to induce RAP2.6, indicating overlapping activities for the T3E and toxin. Further support for this overlap came from the observation that the ability of AvrB to induce RAP2.6 was dependent on COI1  strain 11528 destabilizes JAZ proteins without a requirement for COI1, likely via its cysteine protease activity that directly cleaves the central Zim domain of the JAZ proteins  to disrupt an SA-independent pathway supporting Arabidopsis defense  of PtoΔCEL (CEL deletion of Pto DC3000) elicits SA-signaling that overcomes the suppressive effect of COR. The new activity of COR was detected by examining defense responses against a PtoΔCEL cor- (CEL deletion and COR-deficient double mutant strain) in SA-signaling deficient mutant plants (sid2 and npr1). The PtoΔCEL cor- double mutant elicited more callose and grew less than the PtoΔCEL single mutant in SA-signaling mutant plants. Furthermore, those higher levels of callose elicited by PtoΔCEL cor- were suppressed by either exogenous application of COR or expression of hopM1 in the double mutant strain. Thus, COR and HopM1 carry out overlapping roles in suppressing cell wall-associated defense.The ability of COR to suppress cell wall-associated defense escaped detection until recently (Geng et al. PtoΔCEL cor- while COR could not. Thus, COR and HopM1 distinctly suppress signaling within the plant defense network by functioning in mechanistically distinct manners. The ability of COR to suppress callose deposition and promote bacterial growth in SA-signaling deficient mutants indicates that COR targets an SA-independent sector. Consistent with this idea, Geng et al. PtoΔCEL cor- and restores bacterial growth in both wild-type and SA-signaling deficient mutant plants. Thus, HopM1 suppresses both SA-dependent and SA-independent sectors, perhaps through downstream effects on defense-associated vesicle trafficking. The mode of action by which COR and HopM1 each target distinct sectors of a converged portion of the plant defense network remains to be elucidated.Although COR and T3Es of the CEL locus share the ability to suppress cell wall-associated defense, their mode of action differs. In SA-signaling competent plants, HopM1 suppressed the high levels of callose deposition induced by Plants consistently face environmental stresses, including biotic stresses, due to their sessile lifestyle. The key roles of hormone signaling and secondary metabolic pathways in the ability of plants to deal with these stresses make targeting of them an effective strategy deployed by plant bacterial pathogens to suppress host defense. Here, we reviewed the demonstrated ability of the phytotoxin COR to hijack JA-signaling and thus suppress SA-signaling. We also speculated about targeting of ET-signaling by COR and considered how one, or perhaps both, of these activities contributes to the various virulence activities of COR. The finding that COR promotes virulence independent of targeting COI1 opens a path to study this novel activity of COR separate from the confounding effects of COI1 activation. The perturbation of ET-dependent IGs metabolism provides an attractive system for this effort. In addition to producing hormones and/or hormone mimics, such as COR, bacteria also produce T3Es. COR cooperates with other T3Es to promote bacterial virulence and dampen the induced defense responses. The collaborative targeting of plant hosts by pathogen-produced virulence factors, for example by COR and T3Es that target plant hormone signaling pathways, is an area that, with further exploration, will reveal a better view of the elegant network comprising the plant immune system and how it is collaboratively defeated by pathogen-derived virulence factors.


We have previously shown that medication overuse is prevalent among individuals self-medicating regular headache.In this study we evaluated self-medicating headache patients from a broader perspective, exploring the interplay between headache and concomitant pain conditions, pain-related disability and pain medication use. Identification of subgroups of patients could be helpful to tailor intervention strategies.A hierarchical cluster analysis was used to group 1021 self-medicating headache patients according to their (1) sociodemographics, (2) pain characteristics, (3) pain-related disability and (4) pain medication use. Patients were recruited in 202 Belgian community pharmacies and fulfilled the following inclusion criteria: aged ≥18 years, purchasing an over-the-counter systemic analgesic, experiencing pain ≥1 full day/month and suffering from headache.Three subgroups were identified. Group 1 comprised patients with low socioeconomic status, low self-rated health, on average four concomitant pain conditions, high pain frequency, high disability, and high rates of medication overuse. Group 2 included older patients with a mean of two other pain syndromes, and low disability but high pain intensity. Group 3 comprised young highly-educated patients diagnosed with migraine, having on average one concomitant pain condition, low pain frequency, low disability but high pain intensity, and low rates of medication overuse.We have identified three subgroups in a large sample of individuals self-medicating headache. The marked differences across the three groups stress the importance of a holistic assessment of headache patients and the need for tailored strategies to reduce the risk of medication-overuse headache in primary care.No conflict of interest.


MECP2, which encodes the transcriptional regulator methyl-CpG-binding protein 2. Its core symptoms include problems with breathing control, which are most severe during wakefulness and are further exacerbated by behavioural arousal. Reduced levels of brain-derived neurotrophic factor (BDNF) are thought to be involved in the aetiology of respiratory dysfunction in RTT. Now, Katz et al. report that acute administration of LM22A-4, a partial BDNF mimetic, completely reverses spontaneous apnoeas  and respiratory dysregulation during behavioural arousal in Mecp2 mutant mice, an RTT model. Moreover, patch-clamp recordings in Mecp2-null brainstem slices indicate that LM22A-4 decreases synaptic hyperexcitability within specific cell groups in the brainstem’s respiratory network. These findings support the hypothesis that reduced BNDF signalling and respiratory dysfunction in RTT are linked and demonstrate the feasibility of improving neurological function in RTT by redressing imbalances in synaptic excitability. 1047Page Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in


PMN-MDSCs have previously been found to be enriched among low-density granulocytes (LDGs) in density gradient centrifuged blood.Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were originally identified in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity and even bacterial infections. Human MDSCs are commonly divided into monocytic (Mo-MDSCs) and granulocytic (PMN-MDSCs) subtypes. To what extent the In this study we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood as compared to density gradient centrifuged blood.+HLADRlow Mo-MDSCs in all sepsis patients, whereas the typical PMN-MDSCs as well as a prominent CD14low PMN-MDSC-like population appeared preferentially in Gram-positive cases (Figures low PMN-MDSC variant was demonstrated to suppress T-cell proliferation in vitro via a ROS-dependent mechanism, to display an increased IL-10:TNFα ratio, and to present with signs of immaturity: blast morphology and low cytokine levels (Figures We found a high frequency of typical CD14 Figures . The CD1 Figures .We conclude that a spectrum of cells with MDSC features are enriched in sepsis, and that microbial origin of sepsis contributes to the substantial interindividual patient variation in MDSC pattern.


KRAS allele. While activating RAS mutations are relatively rare in human breast cancer, increased RAS-RAF-MEK pathwayBone is a common site for metastatic colonization in patients with breast cancer, hence the importance of identifying new treatments for this disease. Preclinical studies of bone metastases have commonly employed MDA-MB-231 cells that possess an activated in vitro. Our results suggest that MEK inhibition may be a strategy for triggering cell death within the hypoperfused, oxygen and nutrient poor regions of tumors with activated RAS alleles.activity is common in high-grade breast cancers. To study the consequences of MEK inhibition on bone metastases stemming from the intra-cardiac injection of luciferase-expressing MDA-MB-231 cells in mice, we used the MEK inhibitor AZD6244 (Selumetinib). We found that AZD6244 treatment caused decreased tumor bioluminescence that was associated with cavitation of the bone metastases, owing to apoptosis of cells specifically within the central region of the bone lesions. Hypothesizing that the latter effect was due to the increased sensitivity of poorly perfused regions to pro-apoptotic stimuli, we found that the combination of serum deprivation and AZD6244 led to dramatic induction pf MDA-MB-231 apoptosis  Bone is a frequent site of metastatic colonization in advanced breast cancer, occurring in up to 70% of individuals with this disease . Once tuRAS family genes, seen in a large percentage of all human malignancies [RAS mutations are most frequently found in cancers arising in the colon, lung, pancreas, and thyroid [RAS mutations in breast cancer has led to the notion that the RAS signal transduction pathway activity does not play an important pathogenic role in this disease. However, a large proportion of individuals with breast cancer show over-expression of EGFR, a kinase whose activation leads to RAS pathway activation. Indeed, 70-80% of breast carcinomas demonstrate evidence of EGFR over-expression [Mutational activation of gnancies , makes i thyroid , they ar thyroid . The relpression , and thipression . Furtherpression . TherefoKRAS.In this study we utilized a well-established model of breast cancer bone metastasis that mimics the processes involved in the metastatic colonization process, including extravasation, colonization of suitable marrow microenvironments -16, and In vivo bioluminescence imaging revealed that AZD6244 treatment dramatically inhibited the growth of luciferase-expressing MDA-MB-231 bone metastases and led to central cavitation of these lesions.in vitro data showing that MEK inhibition often resulted in a cytostatic effect.Injection of MDA-MB-231-Luc2 cells into the left ventricle of nude/beige mice invariably resulted in the development of skeletal metastasis, particularly affecting one or both distal femora and/or proximal tibiae. Following injection of MDA-MB-231-Luc2 cells, mice were screened for the development of knee tumor bioluminescence on days 7, 10, 14, 17 and 21 post-IC injection. Mice with nascent bone metastasis were subjected daily treatment of AZD6244, or vehicle control, from days 14 to 21 post-cell injection. AZD6244 treatment dramatically attenuated knee tumor photon emission rates throughout the treatment period Figure , exhibitIntracardiac injection of MDA-MB-231-Luc2 cells led to the development of homogenous osteolytic tumors that eventually filled the medullary cavity of the distal femur and/or proximal tibia. In contrast, bone metastases in mice that had been treated with AZD6244 exhibited large central cyst-like cavities  treatment regimen with AZD6244 was carried out on large late-stage knee metastases, and tumor viability was monitored via photon flux measurements and BLI Figure . Followiin vivo nutrient and/or trophic factor deprivation, MDA-MB-231 cells were grown in 10% FBS and 0.1% FBS. We found that MDA-MB-231-Luc2 cells grown in 10% FBS media and exposed to AZD6244 demonstrated no significant increase in Annexin V-FITC and PI staining  of bone metastases demonstrated that this agent was able to slow tumor growth as reflected by the decrease in bioluminescence signals as compared to controls. Unexpectedly, histological examination of the AZD6244-treated bone metastases revealed the presence of a single central fluid filled cyst-like space that occupied a large volume within these lesions. These cystic spaces were enveloped by a viable rim of proliferating cells that accounted for the residual bioluminescence that was detected in the AZD6244 treated tumors. Hypothesizing that cystic spaces were the result of MDA-MB-231-Luc2 cell death with subsequent clearance of cell corpses and other debris by macrophages , we alloRAS allele within MDA-MB-231-Luc2 cells was providing survival signals to tumor cells growing in regions that were being subjected to micro-environmental stress. For example, the abnormalities of the solid tumor microvasculature that result in central regions of nutrient and oxygen deprivation suggested an explanation for why AZD6244 had produced central cell death within the bone metastases. Perhaps consistent with this idea, we found that MDA-MB-231-Luc2 cells underwent apoptosis in response to serum deprivation when simultaneously exposed to AZD6244 in vitro. This suggested that MEK inhibition sensitized the cells to the pro-apoptotic stimulus of serum deprivation. Since serum contains a complex mixture of trophic factors, low concentrations of serum might plausibly mirror reduced nutrient availability within the central regions of rapidly growing tumors. Indeed, the RAS-MEK pathway may be important in helping tumor cells resist stressors [The development of central zones of apoptosis in response to AZD6244 was intriguing, and suggested that intra-tumoral heterogeneity was responsible for this phenomenon. It was plausible that the activated tressors . From a tressors . AlternaUnder low nutrient conditions, the mutant p53 in MDA-MB-231 cells was previously shown to be stabilized by phospholipase D (PLD), and this was proposed to be under control of the Ras-MAPK pathway . InteresBRAF allele [Although MEK inhibition may blunt the ability of tumor cells to respond to a variety of pro-apoptotic stimuli, a potential stumbling block lies in the finding that elevated levels of BCL-2 appear to have the capacity to counteract the increases in BIM brought about by MEK inhibition, thus inhibiting the pro-apoptotic function of BIM . TherefoF allele . These rMycoplasma spp. by PCR testing (Charles River Laboratories).AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen. A polyclonal population of MDA-MB-231 cells stably expressing an EGFP-Luciferase 2 (Luc2) fusion protein (designated herein as MDA-MB-231-Luc2) was generated and maintained as described previously . These c5 cells/well) seeded and incubated for 24 hrs, then media was replaced with fresh media containing either 10% or 0.1% FBS followed by 24 hrs of incubation. AZD6244 was added into respective wells and all the cells were incubated for a further 48 hrs. Non-adherent cells and adherent cells (using 1x Versene) were harvested, washed twice with PBS, and once with 1x binding buffer. Cells were stained with 5 μl of Annexin V-FITC in the dark for 15 min and PI was added to the cell suspension prior to being acquired and analyzed by flow cytometry.An annexin V-FITC apoptosis detection kit (BD Pharmingen) was used. MDA-MB-231-Luc2 .Ice-cold RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail  was used to lyse the cells. Following 30 min in RIPA buffer solution, lysates were centrifuged at 13,000 rpm for 15 minutes. Protein concentrations were determined using Bio-Rad protein dye reagent. Whole cell lysates (30 μg per lane) were then loaded, electrophoresed, and transferred to PVDF membrane (Millipore) using a semi-dry transfer method. Membranes were blocked with 5% skim milk in 1x TBS with 0.1% Tween-20, and probed with primary antibodies  in 1% skim milk in 1x TBS with 0.1% Tween-20 at 4 nu/nu; beige/beige) mice were purchased from Charles River Canada. All animal studies were performed in compliance with Canadian Council of Animal Care guidelines, and with ethics approval from the University of Calgary Animal Care Committee.Female 5-6 week old, NIH-III (5 per mouse) were injected into the left ventricle as previously described [To generate metastasis MDA-MB-231-Luc2 cells , or vehicle, in 5 ml/kg of 50 mM 2-(hydroxypropyl)-β-cyclodextrin, via daily oral gavage for 7 consecutive days. In contrast, the 3-day treatment regimen was commenced once total photon fluxes in one or both knees had reached an average of 10Upon sacrifice, hind limbs (femurs and tibiae) were harvested and fixed in 4% paraformaldehyde (PFA) for 24 hrs. The limbs were then placed into fresh 4% PFA for a period of 7 days, followed by 14 days of decalcification in 14% EDTA. Knees were sectioned at 6 μm for immunohistochemistry and histological staining with either Masson's trichrome or with haematoxylin and eosin (H&E). For TUNEL staining, the ApopTag Plus Apoptosis Detection Kit  was used.Data were plotted as mean ±SEM and statistical analyses were performed using one-way or two-way ANOVA, with Bonferonni post-tests to determine statistical significance, using Graphpad Prism 4.03 software.


Childhood obesity is an important early predictor of adult obesity and associated comorbidities. Common forms of obesity are underpinned by both environmental and genetic factors. However, the rising prevalence of obesity in genetically stable populations strongly suggests that contemporary lifestyle is a premier factor to the disease. In pediatric population, the current treatment/prevention options for obesity are lifestyle interventions such as caloric restriction (CR) and increase physical activity. In obese individuals, CR improves many metabolic parameters in peripheral tissues. Little is known about the effect of CR on the hypothalamus. This study aimed to assess the effect of CR on hypothalamic metabolic gene expression of young obese- and lean-prone animals.Male juvenile JCR:LA-cp obese-prone rats were freely fed (Obese-FF) or pair fed (Obese-FR) to lean-prone, free-feeding animals (Lean-FF). A group of lean-prone rats (Lean-FR) were matched for relative average degree of CR to Obese-FR rats.P<0.001) and showed greater increases in body weight, fat mass, plasma glucose, insulin and lipids (P<0.01). These metabolic differences were associated with alterations of feeding-related neuropeptides expression in the hypothalamus, as well as pro-inflammatory cytokines and oxidative stress markers. When submitted to the same degree of CR, the two genotypes responded differently; hypothalamic inflammatory and oxidative stress gene expression was improved in Obese-FR, while it was worsened in Lean-FR rats.In free-feeding conditions, obese-prone rats consumed more energy than lean-prone rats (We demonstrate in JCR rats that the metabolic and inflammatory response of the brain to CR is genotype dependent. Within the pediatric population, lifestyle modifications such as caloric restriction (CR) and increased physical activity are the first-line approach for the prevention or treatment of obesity.9Over the last few decades, the incidence of pediatric obesity has increased exponentially in developed countries and developing countries in economic transition.11 For the obese-prone phenotype, CR without malnutrition improves many metabolic and inflammatory parameters in peripheral tissues.13 However, the effects of CR on the hypothalamus, the ‘headquarters' in the regulation of many fundamental physiological activities, including metabolic and energy homeostasis14 are not well documented.Body weight is affected by biological, environmental and behavioral factors, all of which are influenced by genetic background.15 and CR.16In the hypothalamus (and mostly within the arcuate nucleus (ARC)), the mechanisms of energy-balance regulation involve the interaction of multiple neuropeptides including orexigenic peptides such as neuropeptide Y (NPY), agouti-related protein and hypocretin/orexin, as well as anorectic peptides such as pro-opiomelanocortin, cocaine–amphetamine-regulated transcript and corticotropin-releasing hormone. These orexigenic and anorxigenic peptides are altered in both obesity induced by leptin signaling deficiency18 In addition, intracellular inflammation and related oxidative stress generated by overfeeding are linked to metabolic syndrome via the hypothalamus20—impairing hypothalamic neurons involved in energy balance and feeding, and disrupting central adiposity sensitivity to leptin and insulin.20 On the other hand, chronic severe food restriction and depletion of fat stores also generate brain inflammation and oxidative stress. Owing to the role of central inflammation in metabolic syndrome initiation,21 it is possible that animals with chronic severe food restriction-induced brain inflammation can be well predisposed to metabolic syndrome under conditions of plenty, suggesting a vicious cycle between food availability, brain inflammation and metabolic syndrome. Therefore, the differential response of the brain to calorie availability may relate to genetic differences, duration of CR and/or the degree of caloric deprivation employed.22 The objective of the present study was to determine the levels of hypothalamic feeding-related neuropeptides, inflammation and oxidative stress gene expression of obese-prone and lean-prone juvenile JCR rats submitted to a similar degree of CR.Chronic overfeeding associated with accumulation of adipose tissue is known to trigger an atypical form of inflammation and oxidative stress, which lead to metabolic dysfunctions at cellular, organ and systemic levels.We hypothesized that young obese-prone animals under free-feeding conditions would display higher expression of hypothalamic appetite-regulatory, inflammatory and oxidative stress genes than their lean-prone counterparts. Furthermore, we hypothesized that obese- and lean-prone rats matched on CR would show differential expression of these genes in the hypothalamus.23 The JCR-LA-cp rats express a corpulent trait (cp) due to an autosomal recessive, nonsense Tyr763Stop mutation in the Ob-R gene, resulting in a total absence of functional leptin receptors.24 Thus, rats that are homozygous for the cp trait (cp/cp) are obese prone and display metabolic syndrome characteristics observed in human obesity, including hyperphagia, hyperinsulinemia and hyperlipidemia, which can be observed at 4 weeks of age;25 those that are heterozygous for the cp trait (+/cp) or wild type (+/+) are lean prone. As the wild type (+/+) and the heterozygous (cp/+) are both lean prone and are physically undistinguishable, they are represented as (+/?). The ‘+/?' means a 2:1 mix of cp/+ (heterozygote) and +/+ (homozygous wild type). The study was conducted in two separate batches (12 rats per batch) with 6 obese-prone (cp/cp) and 6 lean-prone (+/?) animals in each batch. The rats were housed individually in clear polycarbonate cages (47 cm × 27 cm × 20 cm) with sterile wood chip bedding in a temperature- (22±2 °C) and humidity-controlled environment and maintained on a 12 h light/dark cycle (lights off 1000–2200 h). Throughout the experiment, animals had free access to water and were fed as outlined in the experimental procedure. The care and use of animals was in accordance with the Guidelines of the Canadian Council of Animal Care and subject to prior review and approval by the University of Alberta Animal Care and Use Committee: Livestock.Twenty-four male JCR:LA-cp rats with an obese-prone phenotype (corpulent trait carriers: cp/cp) and 12 control rats with a lean-prone phenotype (non-expressing and non-corpulent trait carriers: +/?) 35–40 days of age (juveniles) were obtained from our established breeding colony at the University of Alberta.One day after arrival, rats were measured for lean and fat body mass using nuclear magnetic resonance . Obese-prone rats were matched for body weights and randomly assigned to free feeding (Obese-FF) or food-restricted (Obese-FR) groups (six rats per group). Lean-prone animals were randomly assigned to free-feeding (Lean-FF) or food-restricted (Lean-FR) groups (six rats per group). In obese-prone rats, CR consisted of pair feeding the obese-prone rats the daily mean amount of food consumed by the Lean-FF group from the previous day. The daily food intake of Lean-FR rats was a percentage of the food consumption of Lean-FF animals; we calculated this percentage from the difference in daily food intake of Obese-FF and Obese-FR groups (Lean-FR intake=Lean-FF intake*(Obese-FF intake−Obese-FR intake)/Obese-FF intake*100). Thus, Obese-FR and Lean-FR groups were matched on relative average amount of CR. Animals were fed standard laboratory chow . An electronic scale  was used to measure daily food and body weight to the nearest gram.g, 10 min). Plasma samples were stored at −80 °C until analysis. The rats were immediately perfused intracardially with ice-cold isotonic saline: the brain was removed and immediately frozen before sectioning, as described below. Lean body mass and white adipose tissue were expressed as a percentage of total body weight.After 10 days on the feeding schedule, rats were removed from their home cages, fat and lean body mass were measured again as at baseline and then animals were anesthetized with isoflorane in oxygen. Blood was taken by cardiac puncture, transferred to 10-ml polyethylene tubes containing EDTA and stored on ice until centrifugation  at an approximate operating temperature of −20 °C was used for coronary sections from frozen rat brains. We focused mostly on the ARC, owing to its close proximity to the peripheral blood stream and its strong response to peripheral hormonal and nutritional signals. Punches containing this targeted area of the brain were taken based on The Rat Brain Stereotaxic Coordinatesn=44) was measured by performing real-time quantitative PCR using a high-throughput method  with primers and corresponding universal probe library  probes designed with the Roche UPL design centre software as previously described27 (CT (2-ΔΔCt) method. All assays were performed in triplicate. With the 44 target genes assessed, we are only reporting the gene expression values mainly involved in appetite regulation, inflammation and oxidative stress; the remaining gene expression values are available in the An array of target genes . Plasma glucose was determined using a glucose oxidase method . Insulin and leptin levels were assessed by enzyme-linked immunosorbent assay kit for rodents .t-tests (paired or independent samples) and two-way analysis of variance to test genotype (obese-prone vs lean-prone) by feeding condition (free access vs CR) effects and one-way analysis of variance for comparison of the four groups with post-hoc Tukey's test for pairwise comparisons, and P<0.05 were considered statistically significant. For multiple comparisons of means tests, the least significance α-level for the several contrasts is reported in the text.Results are presented as means±s.e.m. and were plotted using GraphPad . We used P=0.03), but no time by genotype interaction. Not surprisingly, juvenile free-fed rats consumed more calories than CR animals over the 10-day period (P<0.01). Obese-prone rats also consumed more calories than lean-prone animals, yielding a genotype effect  in body weight (P<0.001) and a significant feeding condition effect. Free-fed juvenile rats had increased body weight compared with CR rats . After 10 days of feeding, Obese-FF rats weighted more than Obese-FR, Lean-FF or Lean-FR rats (P<0.001). Obese-FR and Lean-FF rats did not significantly differ in their absolute body weight, indicating that pair feeding of obese-prone rats to lean-prone animals normalized the body weight of juvenile animals. However, Lean-FR and Lean-FF rats did not significantly differ in their absolute body weight after 10 days of pair feeding despite that Lean-FR ate 64% of the intake of the Lean-FF rats. A trend analysis showed a difference in linear trend (P<0.01), indicating that juvenile Lean-FF increased body weight more over days than Lean-FR rats of the same age (There was a significant time by feeding interaction (P<0.001), while the opposite was found for the percentage of the lean body mass. CR induced by pair feeding significantly improved body composition. Caloric-restricted animals had significantly less fat mass than free-fed rats (P=0.02) and greater lean body mass than free-fed animals (P< 0.05).P<0.001), total cholesterol (P<0.001), glucose (P<0.001) and insulin (P<0.001) than the Obese-FF rats. Similar degree of CR significantly reduced triglyceride in both genotypes, while glucose and insulin levels were reduced significantly only in the obese-prone rats (P<0.05).P<0.05). This suggests that the leptin pathway may downregulate these neuropeptides in juvenile free-feeding animals. In lean-prone juvenile rats, CR had no significant effect on the expression of NPY and Orexin A. In obese-prone juvenile rats, CR significantly decreased NPY mRNA (P<0.05) and increased Orexin A mRNA (P<0.05) levels . Pair feeding significantly lowered these pro-inflammatory cytokine expression levels in juvenile obese-prone rats. As shown in P<0.05). In lean-prone juvenile rats, a similar degree of relative CR had no significant effect on tumor necrosis factor-α and nuclear factor-κB mRNA expression. Lean-FR rats had significantly increased expression of interleukin-6 compared with the Lean-FF rats . Interestingly, pair feeding of obese-prone to lean-prone rats significantly reduced the hypothalamic expression levels of superoxide dismutase-1, glutathione reductase, glutathione peroxidase and catalase mRNA in Obese-FR rats as compared with the Obese-FF group (P<0.05). In lean-prone rats, however, with the same degree of relative CR, the expression of superoxide dismutase-1 and catalase mRNA in the ARC was significantly increased compared with the Lean-FF rats , obese-prone rats, with defective leptin signaling, showed higher hypothalamic NPY and Orexin A mRNA expression than lean-prone animals of similar age (with intact leptin receptor), but no difference in pro-opiomelanocortin and cocaine–amphetamine-regulated transcript expression under the free-feeding condition. Leptin is known to regulate arcuate hypothalamic neurons directly by binding to the ObR receptor long isoform, activating Janus kinases (JAK) and (3) signal transducer and activator of transcription (STAT) signaling and suppressing the activation of NPY neurons.29 With free access to food, the energy intake of juvenile obese-prone rats  was consistently higher than that of lean-prone animals of similar age. This greater energy consumption of obese-prone juvenile animals, a consequence of non-functional leptin receptor, was associated with greater gains in body and fat mass weight, and increased plasma glucose, insulin and lipid concentrations. These findings are in agreement with previous studies.23 The lower fat depot found in the juvenile lean-prone rats compared with the obese-prone JCR counterparts is a result of leptin action on energy balance. Leptin is also known to induce expression of uncoupling proteins (UCP-1/-2/-3) in the mitochondria via stimulation of β3-adrenaergic receptors, thereby leading to increased thermogenesis30 and hence reduced fat deposition.The upregulation of orexigenic neuropeptides (NPY and Orexin A) in leptin receptor-deficient rodents is often accompanied by physiological and metabolic changes.31 In the present study, under free access to food, the increased hypothalamic inflammation observed in the juvenile obese-prone rats, with greater food consumption, could be a consequence of excessive nutrients presented to the hypothalamus, as previously reported.21 Moreover, De Souza et al.32 have found that high-fat feeding (lipidemic diet) induces a local pro-inflammatory status in the hypothalamus, which results in impaired anorexigenic insulin signaling. In the current study, our results show that under regular chow regime, the overeating of the obese-prone JCR juvenile rats promptly activates inflammatory and related oxidative stress pathways in the hypothalamus. Compared with lean-prone rats of similar age, obese-prone animals showed greater interleukin-6, tumor necrosis factor-α and nuclear factor-κB mRNA expression in the ARC, as well as increased expression of markers of oxidative stress . This finding is in line with a previous report that revealed increased microglial activation or neuroinflammation associated with obesity in JCR rats using positron emission tomography imaging.33 However, the underlying mechanism(s) of overfeeding-induced hypothalamic inflammation remain elusive.The link between overfeeding, hypothalamic inflammation and the control of body weight is fundamental to how insulin resistance develops when tissues are exposed to a supply of nutrients that exceeds their energy requirements.35, (ii) the Toll-like receptor pathway36 and (iii) the cytokine/chemokine receptor pathways.37 Compared with classical  inflammation, inflammation triggered by nutrient excess is termed ‘metabolic inflammation.'38 More importantly, unlike inflammation in peripheral tissues, hypothalamic inflammation induced by nutrient excess impairs neuronal sensitivity to leptin and insulin stimulation, which consequently leads to obesity development by altering feeding-related neuropeptides.21 In addition, this high inflammatory response induced by overnutrition in juvenile obese-prone JCR rats (before an overt obesity phenotype) associated with orexigenic neuropeptides upregulation could also participate in overt metabolic syndrome development during adulthood.Recent research, using genetic and molecular approaches, suggests that overnutrition-induced hypothalamic metabolic inflammation involves at least three levels of responses consisting of (i) membrane receptor-independent intracellular stresses29 However, owing to the experimental procedure, food availability is intermittent; hence, the catabolic pathway is also activated to produce energy, leading to the reduction of white adipose tissue in the CR animals.In addition to the negative outcome associated with obese-prone genotype under free-fed conditions, our study is also the first to illustrate that under a similar degree of CR, obese-prone and lean-prone JCR rats respond differently. Submitting juvenile obese-prone and lean-prone JCR rats to a similar degree of CR reduced plasma triglyceride concentrations and total fat mass, compared with freely fed controls. The lower plasma lipids and fat mass in the CR rats are associated with higher Orexin A expression rather than NPY expression in these animals. CR is known to increase NPY expression that triggers food intake.39 stimulating lipolysis and increasing β-oxidation.40 We also found that hypothalamic pro-inflammatory cytokines and oxidative stress markers were significantly reduced in the obese-prone juvenile rats after 10 days of CR, whereas a similar degree of CR significantly increased these parameters in the lean-prone juvenile rats for the same period. The high level of hypothalamic pro-inflammatory cytokines observed in lean-prone rats following the CR relative to the free-feeding animals is in agreement with recent report showing that food restriction significantly increases hypothalamic expression of tumor necrosis factor-α in Wistar rats,41 suggesting that in metabolically normal conditions, CR induces inflammatory stress, which might contribute to the onset of hypothalamic inflammation. Indeed, owing to the lower percentage of fat mass in the lean-prone rats, 36% of CR induced with the experimental protocol may be functionally defined as a severe CR that most likely induced physiological stress, which may contribute to the onset of hypothalamic inflammation as previously reported.43 Thus, taken on the account of the importance of central inflammation in the regulation of energy homeostasis, it cannot be ruled out that the increased hypothalamic inflammation observed in the Lean-FR rats may likely lead to the development of metabolic syndrome when food becomes freely available.This physiological adaptation explains the Orexin A upregulation in the food-restricted animals, as this neuropeptide is more likely to be involved in the control of energy metabolism rather than in caloric intakeIn conclusion, our findings report for the first time that under free-feeding conditions, the obese-prone genotype based on leptin receptor deficiency was detrimental for juvenile animals, inducing overexpression of hypothalamic orexigenic feeding-related neuropeptides and inflammatory genes associated with hyperphagia and body weight and fat mass gains. However, under a similar degree of CR the two genotypes (lean-prone vs obese-prone) respond differently; CR improves hypothalamic inflammation and oxidative stress in juvenile obese-prone rats, but aggravates these markers in lean-prone animals of similar age. Clinical implications of these findings lie in supporting the rationale for incorporating genotyping into obesity management based on CR.


We hypothesised that the proportion of refractory tumour tissue (Oxy-R) would be a robust biomarker of tumour hypoxia across multiple models with different vascular and hypoxic phenotypes.There is need for non-invasive methods to identify, quantify and map tumour hypoxia. In this study we used an emerging technology – R2 were evaluated in well vascularised renal cancer 786-O xenografts. Dynamic sensitivity of proportional Oxy-R to acute changes in hypoxia was evaluated using hydralazine challenge. Relationship of proportional Oxy-R to tissue immunohistochemistry and gadolinium DCE-MRI were explored in parental and drug-resistant 786-O models and in SW620 xenografts.Methods: OE-MRI signal precision, stability and relationship to tissue pOin vivo experiments demonstrated the accuracy, precision and stability of R1 measurement. The proportion of tumour Oxy-R increased significantly following hydralazine challenge (p=0.045) relative to control. The proportion of tumour with perfused Oxy-R voxels was correlated to chronic hypoxia in well perfused 786-O-R xenografts and in relatively necrotic SW620 xenografts .Phantom and The proportion of tumour perfused Oxy-R is a robust biomarker of tumour hypoxia. Voxel-wise analysis of dual oxygen and gadolinium challenge has potential to quantify and map tumour hypoxia as prognostic, predictive and pharmacodynamic biomarkers that could facilitate personalised healthcare.


However, patients require lifelong therapy and are at risk of developing non-AIDS complications . Despite T cells . WithoutCCR5 Δ32 mutation, which confers high resistance to most strains of HIV-1  and are subsequently killed by the immune system or viral cytopathic effects (“kill”) . cART ad results . However results –16. Alth results .ex vivo did not die from viral cytopathic effects and were not killed by autologous cytotoxic T lymphocytes (CTL), which may have been relatively quiescent in the presence of cART   , 20. AlsHLA-B57) . Still, (in vivo –24. Alth(in vivo ] that caWe postulate that HIV-1-specific antibodies might be able to mediate killing of reactivated latently infected cells through antibody-dependent cellular cytotoxicity (ADCC). HIV-1-specific ADCC involves the binding of antibodies to HIV-1 antigens expressed on the infected cell surface and the subsequent recruitment of innate effector cells, such as natural killer (NK) cells or monocytes . The croin vitro through the actions of the accessory proteins, Vpu and Nef  protein for the binding of ADCC antibodies. Even though results from recent clinical trials of LRAs seem promising, it is not known whether reactivated latently infected cells express sufficient Env antigens on the surface for efficient binding of ADCC antibodies. While the level of Env expressed on the surface is an important factor for anti-HIV-1 ADCC, the conformation of Env and consequent availability of epitopes for the binding of ADCC antibodies might be even more critical. As reviewed in Kramski et al. , several and Nef –38. Vpu  and Nef . In addi and Nef , 37. Ove and Nef .Another potential barrier for ADCC-mediated elimination of the latent HIV-1 reservoir is that levels of HIV-1-specific ADCC antibodies decline in HIV-1-infected individuals on long-term cART . Similarin vivo, and (2) if native Env trimer-binding, ADCC-competent bNabs can eliminate latently infected cells following administration of LRAs.Over the years, a series of potent and broadly neutralizing Env-specific monoclonal antibodies (bNabs) have been isolated from HIV-1-infected individuals –47. Manyin vivo cytokine therapy, or adoptive NK cell therapy may need to be implemented to boost NK cell functionality before latency reversal to unleash the full potential of ADCC antibodies in cure strategies.Even if ADCC antibodies can efficiently recognize HIV-1-infected cells, the demonstration that autologous CTLs from patients on cART were unable to kill reactivated latently infected cells without prior antigen stimulation raises concerns that NK cells in these patients may not be able to eliminate the reactivated reservoir either . While sDespite the increasing efforts funneled into HIV-1 cure research, an HIV-1 cure still remains elusive. The relapse of the “Boston patients” and the “Mississippi baby” after months of viral suppression emphasizes the difficulty of the task ahead. Recent studies show the potential utility of CTL immune responses in clearing reactivated latently infected cells. Moving forward, CTL functionality in cART-treated patients may need to be boosted and directed toward sub-dominant epitopes. In addition to CTL responses, ADCC represents another immune mechanism and further investigation to assess its potential to clear reactivated latently infected cells is warranted. Advances in HIV-1 cure strategies will require an improved understanding of not only how to reactivate HIV-1 from latently infected cells, but also how to clear them with the most effective immune responses.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.


Bacteria-infected cells are known to produce higher level of nitric oxide and reactive oxygen species. Although these reactive chemicals can contribute directly to kill pathogens, in this study, we focus on antibacterial autophagy regulation via downstream mediator. Autophagy is a cellular self-catabolic process in which organelles, biomolecules, and invading pathogens are sequestered in autophagosomes that fuse with lysosomes. However. signaling pathways leading to autophagy activation in innate immunity have not been fully clarified.A downstream mediator of nitric oxide signaling, 8-nitroguanosine 3’,5’-cyclic monophosphate (8-nitro-cGMP), has been recently identified in various mammalian cell lines ,2. This The results of our study suggested that 8-nitro-cGMP is an endogenous autophagy enhancer that defines targets for sequestration in autophagosomes by recruiting ubiquitin chains.


Cancer cells require nutrients and energy to adapt to increased biosynthetic activity and depend on mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. Whereas they exhibit a pronounced Warburg effect, their TCA cycle remains intact and becomes more dependent on glutamine metabolism through glutaminolysis. BesidesE Coli L-ase (Kidrolase®) and E. Chrysanthemi L-ase (Erwiniase®) are not mediated by the asparaginase activity of the enzyme. L-ases have also a glutaminase activity and transform extracellular glutamine into glutamate. Both L-ases induce dose and time-dependent mTORC1-inhibition which correlates with extra-cellular glutamine depletion[35] methionine incorporation into newly synthesized proteins, indicative of global protein synthesis inhibition. Finally, we showed that L-ase treatment acts through inhibition of leucine entry into the cells by depleting intracellular glutamine, thus preventing mTORC1 activation by Rheb at the lysosomal surface[The dependence of acute myeloid leukemia (AML) cells to glutamine is little studied. In a recent work, we have tested the effects of glutamine depletion in AML cells: leukemidepletion. Downstrl surface.E. Coli L-ase in refractory/relapsed AML in adults revealed a significantly increased CR rate with the association (40% vs. 24%)[in vitro sensitivity to L-ase very close to that of acute lymphoblastic leukemia cells. Further preclinical studies are thus warranted to characterize new chemotherapeutic combinations in association to L-ase in AML.Overall, those data open the way for new therapies targeting glutamine uptake in AML. L-ase has been previously tested in AML. A randomized study that compared the effects of high dose cytarabine with or without  vs. 24%). Blast cWe also identified potential mechanisms of resistance to L-ase treatment in AML. L-ase upregulates glutamine synthase (GS) protein expression and this process may limit the L-ase-induced anti-leukemic activity in AML. GS inhibition by the L-methionine-sulfoximine (MSO) may increase the sensitivity of AML cells to L-ase as described in ALL. Moreover, L-ase triggers an autophagic process, probably resulting from mTORC1 inhibition (mTORC1 inhibits autophagy by controlling the activity of Atg1/ULK and ATG13 proteins). Knockdown of either ATG5 or Beclin, two proteins controlling autophagy, increase apoptosis in L-ase-treated AML cells. The future testing of a combination of autophagy inhibitors and L-ase is thus warranted in AML.Another therapeutic strategy to exploit AML glutamine dependence may be to target the glutamine transporters. We showed that the knockdown of the high affinity transporter SLC1A5 has strong anti-leukemic effects in vitro and inhibits tumor formation in an AML mouse xenotransplantation model. HoweverWe only address here the role of glutamine uptake by AML cells on the control of mTORC1 activation. Studies are needed to examine its role in mitochondrial anaplerosis through glutaminolysis. The key gatekeeper of glutaminolysis is the enzyme glutaminase (GLS) which catalyzes the hydrolysis of glutamine to glutamate, which is then converted to the TCA cycle intermediate α-ketoglutarate (α-KG). Two different forms of GLS exist: the liver-type glutaminase  and the glutaminase 1 (GLS gene) which exists as two splice variants: the longer form called kidney-type glutaminase (KGA) and the shorter called glutaminase C (GAC). The twoTo conclude, through inhibition of mTORC1 activity and/or through suppression of the mitochondrial TCA cycle, glutamine metabolism constitutes an appealing target for treating this particularly poor prognosis disease.


Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs.The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in  The plant hormone auxin , regulates a wide range of developmental and physiological processes in plants including for example, apical dominance, root development, vascular differentiation, shoot elongation, and embryo patterning , which underlie the functional properties of these proteins. Domain I mediates the transcriptional repression of the proteins, whereas domain II mediates protein degradation  there are opportunities for numerous combinatorial interactions among these proteins to mediate specific responses.Characterization of plant responses to IAA treatments led to the identification of various classes of early auxin-responsive genes. Members of the Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are important to define their unique biological functions  to generate pGBKT7-ARFs. Similarly, PCR products of Aux/IAAs were digested, purified and fused to the GAL4 DNA activation domain of pGADT7 vector (Clontech) to generate pGADT7-Aux/IAAs. All constructs were verified by sequencing.Full-length coding sequences for 19 Saccharomyces cerevisiae strain AH109 was co-transformed with 551 pairs of pGBKT7-ARF and pGADT7-Aux/IAA vectors and yeast cells containing both vectors were selected using SD/-Leu/-Trp medium. The interactions between ARFs and Aux/IAA were assayed by plating the transformed cells onto the stringent SD/-Ade-His-Leu-Trp selective medium using at least 10 independent colonies. Serial dilutions of yeast co-transformed cells were used to measure the strength of the interaction.ARF5, 6, and 19 were PCR amplified using forward and reverse primers containing restriction enzyme sites  epidermal cells using particle bombardment as previously described by Hewezi et al. . Quality assessment of sequence reads was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality filtering and trimming were conducted using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). After quality filtering and trimming, each sequence read was aligned to the TAIR10 Arabidopsis thaliana reference genome . Sample contribution scores were calculated by multiplying Z-score of ARFs and Aux/IAAs for each tissue as described in CoexViewer available at ATTED-II database   website. Sequences were aligned using ClustalX  assays , while Aux/IAA5, 6, 17, 32, and 34 were fused to the C-terminal half of a yellow fluorescent protein gene (cEYFP). Ten different combinations between nEYFP and cEYFP fusions were co-expressed in onion epidermal cells. All ARF-Aux/IAA interaction combinations, including those showing weak interaction in yeast (ARF6-Aux/IAA32 and ARF19-Aux/IAA34), reconstituted the fluorescent YFP in the nucleus of transformed cells  were cloned in a bait vector and full-length coding sequences of 29 Aux/IAAs (Aux/IAA1-20 and Aux/IAA26-34) were cloned in a prey vector. Yeast cells were co-transformed with 551 pairs of bait and prey vectors and potential interactions were visualized by differential growth on the non-selective synthetic dropout (SD) medium (SD/-Leu/-Trp) and on the selective medium (SD/-Leu/-Trp/-His/-Ade). An example of the interaction between ARF10 and Aux/IAAs is provided in Figure It is interesting to note that all ARFs that function as activators  were found to interact with all Aux/IAA proteins except ARF7 with Aux/IAA7 Figure . In contWe tested whether phylogenetically-related ARF and Aux/IAA proteins would have similar protein-protein interaction patterns. In general, we found that phylogenetically-related ARF and Aux/IAA proteins formed similar protein-protein interactomes. For example, ARF5-8 and ARF19, which are phylogenetically-related, interacted with almost all Aux/IAA proteins. Similarly, ARF10 and 16, which clustered together, were found to interact with the same Aux/IAA proteins including Aux/IAA5, and Aux/IAA32-34 .In order to map the co-expression events of the interacting ARF-Aux/IAA proteins to specific tissues or organs, the co-expression between pairs was determined using Z score as described in the Material and Methods. We set the “fragments per kilobase of transcript per million mapped reads” (FPKM) value of 2.0 as the threshold for expressed genes. As a result, cis regulatory element in the promoters of auxin-response genes, a middle region (MR) that functions as activator or repressor, and frequently a CTD involved in protein-protein interactions , interacted with almost all Aux/IAA proteins, a result that was also found in rice (Shen et al., ARF6 and 8 were found to co-express with various Aux/IAA genes only in flowers and floral buds. This finding is in agreement with the redundant function of ARF6 and 8 in regulating floral structures (Nagpal et al., While global protein-protein interaction networks provide an indication for all possible physical interaction combinations that can occur between proteins, the biological significance of such interaction requires the genes coding for these proteins to be co-expressed in particular cells. One approach to investigate the biological importance of such interactomes is to combine expression data with protein interaction data. This is because interacting proteins whose corresponding genes are co-expressed generally co-function in particular processes or pathways (Boruc et al., ARFs can mediate tissue-specific functions through their association with restrictedly expressed Aux/IAAs such as IAA33 and IAA34. It should be noted that several significant gene co-expression associations for the interacting proteins were identified in various tissues. This can be explained by the possibility that co-expression correlations may occur in various subsets of tissues or in specific cell types. In this context, analyzing the temporal and spatial gene expression patterns of the interacting protein pairs in the target tissues will precisely define their specific transcriptional signatures. On the other hand, we were unable to identify significant co-expression associations for about 30% of the interacting proteins in the tissues included in our analysis. The co-expression associations of these pairs may take place in specific tissues at particular developmental stages that are not included in our analysis. Alternatively, the co-functions of these pairs may be limited to particular physiological circumstances, including biotic and abiotic stresses.ARF4-8 and 19, which tend to interact with all Aux/IAAs were also found to be broadly expressed and hence constitute the main hubs in the interaction and co-expression networks Figure . Our datIn conclusion, our analysis confirmed most of the published protein-protein interactions between ARFs and Aux/IAAs and provided a new set of previously unknown interactions. In addition, the combination of co-expression data with protein-protein interaction data provided new leads to the site of the co-functions of ARF and Aux/IAA proteins. Taken together, these analyses set the stage for detailed functional analysis to reveal the biological significance of ARF-Aux/IAA interactions in the morphogenesis and development of various plant tissues and organs.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.


Most pre-workout supplements are based on the stimulant caffeine, containing anywhere from 100-300 mg of caffeine in a serving. While research has confirmed increased mental focus and acuity from the use of caffeine, stimulant sensitive individuals should assess their tolerance before using pre-workout supplements containing caffeine. Caffeine can have dose-dependent unwanted effects contributing to a nervous or anxious feeling that can keep athletes from staying focused and even sleeping well. Ingredients to increase the synthesis and release of neurotransmitters , and blood flow to the brain (Gingko Biloba), offer neuroprotection (blueberry extract), and improve mental regeneration and reduce mental stress (L-Theanine) might offer a stimulant-free alternative to improve pre-workout cognition. Therefore, the purpose of the current study was to investigate the effects of caffeine and a stimulant-free pre-workout formula on alertness (A), focus (F), calmness (CAL), motivation (MOT), cognition (COG), reaction (R), motor reaction time (MR), memory (MEM) and vertical jump power (VJP).Five college-aged males volunteered to participate in this study and were randomly assigned to consume MindSet , Caffeine, and a placebo (rice flour) in a double-blind, placebo-controlled, randomized, crossover design. After baseline testing, subjects consumed one of the assigned supplements 30 minutes prior to testing. Tests were separated by a 48 hour wash-out period. All subjects participated in a variety of mental aptitude tests, visual reaction tests, and power output measurements. Mental aptitude tests  were measured on an interval scale. COG was measured as serial subtraction test; accounting for improvement in scores from pre and post testing. RT and MRT were measured through the use of Dynavision, and VJP was measured through Vertical Jump Test via Tendo Unit. Consent to publish the results was obtained from all participants.Caffeine increased alertness (+19%), focus (+35%), cognition (+26%), memory (+11%), motivation (+10%) and vertical jump power (+1%), however, decreased calmness by 18%. MindSet increased alertness (56%), focus (58%), motivation (43%), cognition (26%), memory (+15%), vertical jump power (3%), and calmness by 6%.A stimulant-free multi-ingredient pre-workout formula can be as effective as caffeine in increasing cognitive functioning without the unwanted side-effects. The results of this pilot study should be confirmed in a larger scale study.


The most important means of identifying diseases before symptoms appear is through the discovery of disease-associated biomarkers. Recently, microRNAs (miRNAs) have become highly useful biomarkers of infectious, genetic and metabolic diseases in human but they have not been well studied in domestic animals. It is probable that many of the animal homologs of human disease-associated miRNAs may be involved in domestic animal diseases. Here we describe a computational biology study in which human disease miRNAs were utilized to predict orthologous miRNAs in cow, chicken, pig, horse, and dog.hsa-miR-143-3p which is associated with type 2 diabetes and targets AKT1 gene which is highly conserved in pig, horse and dog. Functional analysis of AKT1 gene using Gene Ontology (GO) showed that it is involved in glucose homeostasis, positive regulation of glucose import, positive regulation of glycogen biosynthetic process, glucose transport and response to food.We identified 287 human disease-associated miRNAs which had at least one 100% identical animal homolog. The 287 miRNAs were associated with 359 human diseases referenced in 2,863 Pubmed articles. Multiple sequence analysis indicated that over 60% of known horse mature miRNAs found perfect matches in human disease-associated miRNAs, followed by dog (50%). As expected, chicken had the least number of perfect matches (5%). Phylogenetic analysis of miRNA precursors indicated that 85% of human disease pre-miRNAs were highly conserved in animals, showing less than 5% nucleotide substitution rates over evolutionary time. As an example we demonstrated conservation of human http://agbase.hpc.msstate.edu/cgi-bin/animal_mirna.cgi.This data provides the animal and veterinary research community with a resource to assist in generating hypothesis-driven research for discovering animal disease-related miRNA from their datasets and expedite development of prophylactic and disease-treatment strategies and also influence research efforts to identify novel disease models in large animals. Integrated data is available for download at  Caenorhabditis elegans[C. elegans in 2000 [MicroRNAs (miRNAs) are naturally occurring single-stranded small RNA molecules that play important roles in post-transcriptional regulation of gene expression . Studiess elegans,12. Lin- in 2000 , with ho in 2000 -16. Cons in 2000 -26, and  in 2000 -32 inclu in 2000 -43 and c in 2000 ,44-53, i in 2000 -57, and  in 2000 -60.While databases for human disease-associated miRNAs are publicly available -70, therThe main objective of our study was to identify animal miRNA homologs of published human disease-associated miRNAs in cow, chicken, pig, horse and dog using phylogenetic techniques. Using the current available human and animal miRNA resources, we identified potential disease-related miRNAs in domestic animals based on integrated computational and manual approaches including assessing the sequence similarities and evolutionary relationships between human disease miRNAs and their animal orthologs. These predictions will serve as a resource to facilitate hypothesis-driven research in domestic animals, which upon verification in animals could suggest animal models for human diseases and strategies for developing therapeutic measures.We compared the sizes of all known mature human miRNAs with the sizes of all known cow, chicken, pig, horse, and dog to establish their length diversity.http://202.38.126.151/hmdd/html/tools/hmdd_req.html to request all human disease-associated miRNA data from Dr. Qinghua Cui of department of Biomedical Informatics, Peking University Health Science Center. From this data we retrieved the miRNA IDs (identifiers) that were named according to miRBase [Briefly, we surveyed publicly available databases that link human miRNAs with diseases ,67,71,72 miRBase  nomenclaWe used the IDs of human disease miRNAs (from HMDD) to extract corresponding mature  miRNA nucleotide sequences from miRBase version 20 . We alsoThe precursor miRNA sequences (pre-miRNAs) of HDMACs were compared using a multiple alignment and phylogenetic approach to detect conservation profiles and rapid sequence divergence in human and domestic animals. Briefly, we used Clustal Omega  tool forWe manually annotated the genes targeted by the human disease-associate miRNAs from the associated Pubmed articles and predict their animal orthologs using the Ensembl Biomart  tool. AlWe integrated the information gathered from this study to form a computationally predicted animal disease miRNA resource which contained miRNA information including standardized miRBase identifiers of mature miRNAs, pre-miRNAs, and miRNA families linked to the associated human disease. Whenever applicable we used OMIM (Online Mendelian Inheritance in Man) , OMIA  version 2.0  updated. This daWe identified 694 domestic animal mature miRNA sequences that showed 100% sequence identity with the 287 human disease miRNAs  of its pre-miRNA is highly conserved in pig, horse and dog. The hsa-miR-143-3p target gene is AKT1 [We have demonstrated how experimentally confirmed s Figure , Table 2 is AKT1  which ha is AKT1 ,86 indicOne means of identifying diseases before symptoms appear is through the discovery and utilization of disease-associated molecular biomarkers. Many biomarker techniques that have been widely applied in human and model organism studies have not been adequately implemented in the study of domestic animal diseases. It is now clear that miRNA play major regulatory role in various disease processes but financial investment is more committed to investigate miRNA involvement in human disease more than any other species Figure . There ahsa-miR-143-3p which has been associated with diabetes type 2 [In this study we used homology approach to generate a resource that integrates animal miRNA data with human disease-associated miRNAs. As demonstrated using s type 2   or the Institutional Animal Care and Use Committee (IACUC).BRENDA: BRaunschweig ENzyme Database; BTO: BRENDA Tissue ontology; DO: Disease ontology; GO: Gene ontology; HDMACs: Human disease miRNAs with animal counterparts; HMDD: Human miRNA disease database; IGBB: Institute for genomics, biocomputing and biotechnology; MFE: Minimum free energy; miRNAs: MicroRNAs; NJ: Neighbor-joining; OMIA: Online mendelian inheritance in animal; OMIM: Online mendelian inheritance in man; pre-miRNAs: Precursor miRNAs; UTRs: Un-translated regions.The authors declare that they have no competing interests.TB generated data for this manuscript and performed data analysis and interpretation, MA provided computational data integration assistance, HW generated standardized disease phenotype names for the integrated resource, TB and DGP wrote the manuscript. All authors read, critiqued, edited and approved the final manuscript.Distribution of human disease microRNAs across animal counterparts. Multiple human diseases and locations are separated by a pipe (column 3). The (+) or (−) in columns 2–6 indicates presence or absence of homologous human disease miRNA (column 1) in the selected animal species.Click here for filePhylogenetic distances of pre-miRNAs of HDMACs. The data in column 1 & 3 are the pre-miRNA ID for animals and human, respectively prefixed with abbreviation of species scientific name i.e. bta (Bos Taurus - cow), gga , ssc (Sus scrofa - pig), eca (Equine canibus - horse), cfa (Canis familiaris - dog), and hsa (Homo sapiens - human). Column 2 & 4 show the phylogenetic distances calculated based on nucleotide substitution rate within similar clusters.Click here for fileSample of disease miRNAs targets and their animal orthologs. Experimentally verified genes targeted by the disease miRNAs are manually curated from Pubmed articles and their % identity with an animal ortholog is generated via Ensembl Biomart [ Biomart .Click here for file


Recent findings suggest these microRNAs (miRNAs) have a critical role that affects the stroma rather than epithelial cells. Understanding the part played by the miR-143/miR-145 cluster in the tumor microenvironment is essential for the development of future miRNA-related therapies. The contribution of the microenvironment to promoting tumor growth has been recognized for decades . Immune MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level, inhibiting mRNA translation or degrading mRNA. They are crucial players in tumorigenesis, and can act as oncogenes or tumor suppressors . One setG12D/+, p53–/–) in which tumors resemble human lung tumors. However, no differences in tumor burden or survival rate were found. In the same model, mice infected with a conditional lentivirus vector for forced expression of either miR-143 or miR-145 showed no difference in tumor burden when compared with controls. These results contradict earlier suggestions that the miR-143/miR-145 cluster has a tumor suppressor role [G12D/+, p53–/– cells with miR-143/miR-145 null alleles. Altogether, these results suggested that systemic miR-143/miR-145 expression could support tumor growth and launched the hypothesis that these miRNAs affected the stroma rather than epithelial cells [To explore the endogenous role of the miR-143/miR-145 cluster, Dimitrova, Gocheva and colleagues initially used mice with a conditional knockout for miR-143/miR-145, but these animals failed to develop tumors, and differences in survival between these mice and controls were not detected [To further explore the role of the tumor microenvironment, stromal cells were isolated and sorted, and the researchers found that endothelial cells, but not epithelial cells, were enriched in miR-143/miR-145. In miR-143/miR-145-deficient mice, active neoangiogenesis was reduced, and this effect could be partially explained by miRNA targeting of calcium/calmodulin dependent protein kinase ID (Camk1d), a protein involved in angiogenesis [This is the second in vivo study arguing against a tumor-suppressor function for the miR-143/miR-145 cluster [In the majority of gene expression studies in cancer, RNA is isolated from heterogeneous tumor samples. This approach can generate misleading conclusions, as expression of a miRNA exclusively in one specific cell type can appear as bulk expression . TherefoDelivery of exogenous miRNAs is considered as one of the most promising novel strategies for treatment of human cancer . However


There is a number of drugs demonstrating specific activity towards hereditary cancers. For example, tumors in BRCA1/2 mutation carriers usually arise via somatic inactivation of the remaining BRCA allele, which makes them particularly sensitive to platinum-based drugs, PARP inhibitors (PARPi), mitomycin C, liposomal doxorubicin, etc. There are several molecular assays for BRCA-ness, which permit to reveal BRCA-like phenocopies among sporadic tumors and thus extend clinical indications for the use of BRCA-specific therapies. Retrospective data on high-dose chemotherapy deserve consideration given some unexpected instances of cure from metastatic disease among BRCA1/2-mutated patients. Hereditary non-polyposis colorectal cancer (HNPCC) is characterized by high-level microsatellite instability (MSI-H), increased antigenicity and elevated expression of immunosuppressive molecules. Recent clinical trial demonstrated tumor responses in HNPCC patients treated by the immune checkpoint inhibitor pembrolizumab. There are successful clinical trials on the use of novel targeted agents for the treatment or rare cancer syndromes, e.g. RET inhibitors for hereditary medullary thyroid cancer, mTOR inhibitors for tumors arising in patients with tuberous sclerosis (TSC), and SMO inhibitors for basal-cell nevus syndrome. Germ-line mutation tests will be increasingly used in the future for the choice of the optimal therapy, therefore turnaround time for these laboratory procedures needs to be significantly reduced to ensure proper treatment planning. First tumor-predisposing germ-line mutations were discovered a quarter of century ago and were immediately translated into appropriate diagnostic tests –5. IdentBreast-ovarian hereditary cancer syndrome is by far more common than other categories of familial cancers, with BRCA1 and BRCA2 being among the most studied genes. BRCA1/2-driven tumors usually arise via 2-hit mechanism: while the involved gene is present in heterozygous but still proficient state in the normal cells of the carrier, cancer cells are characterized by somatic loss of the remaining BRCA allele and therefore demonstrate deficiency in DNA repair by homologous recombination (HR). This opens an elegant therapeutic window by making tumor cells specifically vulnerable to DNA damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors  study involving mainly patients with small tumors . Given tData on the use of platinum compounds in metastatic BC are limited. Byrski et al.  recruitePlatinum-based therapy forms a backbone for the standard systemic treatment of ovarian cancer (OC). Multiple studies indicate that BRCA1/2-driven OC are characterized by increased sensitivity to platinating agents as compared to sporadic carcinomas –30. InteThere is a number of PARP inhibitors, including olaparib , veliparib , rucaparib , niraparib , talazoparib , etc. . InipariOlaparib is the only PARP inhibitor already approved for the clinical use. Its initial trial as monotherapy agent demonstrated its preferential effect towards cancers arising in BRCA1/2 germ-line mutation carriers . SubsequGiven that neither olaparib nor conventional cytotoxic compounds are capable to achieve high rate of prolonged complete responses while administered separately, there is some interest to the use of these drugs in combination. Preclinical experiments support this concept –45. CombOnly a few studies assessed the efficacy of olaparib against standard cytotoxic treatment. Comparison of olaparib vs. pegylated liposomal doxorubicin (PLD) in recurrent BRCA1/2-associated ovarian cancer produced similar results for both drugs . It has Data on the use of other PARP inhibitors are less extensive. Veliparib was evaluated in heavily pretreated ovarian cancer patients with BRCA1/2 mutation, and induced tumor responses in 13/50 (26%) women; similarly to experience with olaparib, there was a clear difference in response rates between platinum-resistant and platinum-sensitive disease (20% vs. 35%) . NiraparIt is important to acknowledge, that all published clinical trials on PARP inhibitors involved pretreated BRCA1/2-mutated patients. This could be a critical limitation, as at least some BRCA1/2-driven tumors demonstrate restoration of intratumoral BRCA1/2 function during cytotoxic therapy , 29. LowIn addition to cisplatin, there are several other non-expensive cytotoxic drugs showing BRCA-specific activity in preclinical experiments . Based oTrabectedin  is a novel DNA damaging cytotoxic drug approved by FDA and EMEA for the therapy of inoperable soft tissue sarcomas . In addiEribulin  is a novel microtubule inhibitor, which demonstrated improvement of overall survival in patients with metastatic breast cancer after failure of multiple lines of systemic therapy. It was evaluated in combination with carboplatin in neoadjuvant trial involving triple-negative breast cancer patients. The study included 3 patients with BRCA1/2 mutation; clinical response was observed in all these women, with 2 of them achieving pathologic complete response .BRCA1 is required for the execution of taxane-induced apoptosis, and at least some data indicate that taxane-containing regimens show limited efficacy towards BRCA1-associated breast cancers , 17, 60.Anthracyclines appear to exert substantial activity against BRCA1/2-driven tumors , 16, 17,There are some data on the activation of PTEN/PI3K/AKT/mTOR pathway in cancers arising in BRCA mutation carriers. Two mTOR inhibitors, temsirolimus and everolimus, are already available for the clinical use, however their performance in BRCA-driven cancers has not been assessed yet in preclinical or clinical settings. Recent studies demonstrate that mTOR down-regulation may sensitize cancer cells to PARP inhibitors , 71. TheBRCA1/2-deficient tumors are characterized by increased mutational load and therefore appear to be more antigenic than sporadic cancers. In accordance with this, these cancers demonstrate increased lymphocyte infiltration and show distinct pattern of expression of immune-related molecules , 76. TheCarriers of BRCA1 and BRCA2 germ-line mutations usually develop breast and/or ovarian cancers, however there is also some association with other cancer types. Inherited BRCA2 heterozygosity is associated with elevated prostate cancer risk. BRCA2-driven prostate cancers demonstrate good response to platinum-containing therapy and PARP inhibitors , 77–79. Many sporadic tumors share biological characteristics with BRCA1/2-associated hereditary cancers , 83. ForWhile some “BRCA-ness assays” rely on the identification of genetic causes of this phenotype, i.e. the detection of biallelic inactivation of BRCA or similar genes, novel generation of diagnostic tests utilizes characteristic mutational pattern of BRCA1/2-driven (HR-deficient) tumors –89. For In addition to characteristic mutational signatures, HR-deficient tumors demonstrate specific expression profiles , 96. CelClinical data on the role of BRCA-ness demonstrate high level of consistency. BRCA-ness phenotype is associated with higher tumor sensitivity to platinum-containing agents and PARP inhibitors , 98, 99 It is essential to acknowledge that the participation of BRCA1 in DNA damage repair is not limited to HR, therefore the definitions of BRCA-ness and HR-ness are not necessarily identical. Hill et al.  studied Most of disease-causing BRCA1/2 mutations are represented by relatively small alterations in DNA sequence, which cause frame-shift or generation of premature stop-codon. There is a series of fascinating reports, which demonstrate a somatic restoration of the open reading frame of BRCA genes in the therapy-resistant tumor cells; this is achieved by the occurrence of the second mutation in the proximity to the first one. As expected, second mutations are observed both in platinum- and in PARPi-treated tumors , 102–108While the above mechanisms represent mutational evolution of tumor genome under the pressure of BRCA1/2-specific therapy, we observed a distinct root of acquiring platinum resistance . We analThere are some other proposed mechanisms of the resistance of BRCA1/2-mutated cells to the specific therapy, such as inactivation of TP53BP1 protein, efflux of platinum drugs or PARP inhibitors, or some other molecular events , 112–115High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation is used for the treatment of germ-cell tumors, several hematological malignancies, childhood cancers etc. It was a popular experimental approach for the treatment of breast cancer in mid 1990s, but failed to demonstrate improved long-term outcomes in unselected BC patients –118. HowAlthough normal cells in BRCA1/2 mutation carriers retain a wild-type copy of the involved gene and therefore are able to cope with DNA damage, some experiments suggest that the loss of even single BRCA1 allele results in some decrease of BRCA1 functional capacity . If the While BRCA1/2-related cancers have been studied with a high level of comprehension, the available information on clinical behavior of novel categories of hereditary breast cancer remains very limited. Importantly, BC arising in CHEK2, NBN/NBS1 and BLM heterozygotes usually demonstrates retention of the wild-type allele in the tumor, therefore there is no ground to expect selective chemosensitivity in these tumor types . In agreThere are multiple types of hereditary colon cancer , with thMSI-H carcinomas combine two distinct categories of malignancies. First, there are hereditary cancers obtained from the germ-line mutation carriers; they tend to be associated with younger age of the patients. Second, there are sporadic MSI-H tumors; they often arise in elderly subjects and frequently carry an actionable activating mutation in BRAF oncogene. In addition to genuine biological diversity of MSI-H tumors, there are some debates regarding technical aspects of MSI status determinations –136. TheHigh antigenicity of MSI-H tumors makes them recognizable by the immune system. In order to overcome host defense mechanisms, MSI-H carcinomas express some immune checkpoint molecules thus creating local immunosuppressive environment . Le et aMSI-H tumors may have distinct spectrum of chemosensitivity, which is critically influenced by the individual pattern of somatically mutated genes , 139. ThThere are studies aimed to develop systemic therapy for the familial adenomatous polyposis (FAP). Sulindac, a non-steroidal anti-inflammatory drug, was shown to be effective against colorectal adenomatous polyps. However, it has limited efficacy against duodenal carcinogenesis; duodenal carcinomas are the main cause of death in those FAP patients, who have already undergone colectomy. Synergistic interaction between COX2 and EGFR inhibitors was observed in murine FAP models. Combination of sulindac with erlotinib was assessed in a clinical trial involving 46 FAP patients in the experimental group and 46 subjects treated by placebo. Use of the above drugs resulted in clinically significant reduction of the number and size of duodenal polyps .Medullary thyroid cancer is often driven by inherited activating mutation in RET oncogene. There are RET kinase inhibitors, vandetanib  and cabozantinib , which demonstrate clinical activity against both hereditary and sporadic medullary thyroid carcinomas –145.Everolimus , an inhibitor of mTOR kinase, has been approved for the treatment of several tumor types. mTOR pathway is specifically activated in tumors arising in patients with tuberous sclerosis (TSC). Everolimus was systematically evaluated in TSC-related giant-cell astrocytomas and angiomyolipomas, and showed remarkable efficacy in these malignancies –148.Vismodegib  inhibits Hedgehog pathway via interaction with SMO protein. Its antitumor activity was examined in patients with basal-cell nevus (Gorlin) syndrome . This drThe number of known hereditary cancer syndromes will rapidly grow within next several years, thanks to the invention of whole exome sequencing . Many ofaCGH, array comparative genomic hybridization; BC, breast cancer; EMEA, European Medicines Agency; FAP, familial adenomatous polyposis; FDA, Food and Drug Administration; HNPCC, hereditary non-polyposis colorectal cancer; HR, homologous recombination; LOH, loss of heterozygosity; MSI-H, high-level microsatellite instability; OC, ovarian cancer; PARP, poly(ADP-ribose) polymerase; PARPi, PARP inhibitors; pCR, pathologic complete response; PFS, progression-free survival; PLD, pegylated liposomal doxorubicin; TSC, tuberous sclerosis


CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter, and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that  Arabidopsis, DRM2 catalyses de novo methylation in all sequence contexts and CMT2 is involved in non-symmetrical methylation while MET1, CMT3 and DRM2 participate in methylation maintenance at the CG, CHG and CHH sites, respectively15Spontaneous epimutations can result from heritable changes in DNA methylation without alteration in the underlying sequence, but these changes can influence gene expression and associated phenotypes1234678910111213123415Colourless non-ripening (Cnr) is one of the best characterized naturally occurring epimutantsCnr differs from structural epi-variants such as CmWIP, FWA, FOLT1 and SP11171819Arabidopsis, melon and Brassica, of which the epigenetic changes are either induced by transposon or trans-acting small RNAs, or genetic non-ripening mutants such as tomato rin, ripening-inhibitorCnr contains eighteen hypermethylated cytosines in a 286-bp region of the LeSPL-CNR promoter at the Cnr locus and the Cnr epimutation and phenotype are very stableCnr fruits with revertant sectors showing red stripes out of thousands of fruits grown over more than twenty years. In this paper, using the spontaneous Cnr epimutant together with VIGS-based gene functional screening, targeted and whole-genome DNA methylation profiling and qRT-PCR assay, we investigate the mechanism responsible for somatic inheritance of Cnr. We unravel that SlCMT3 silencing results in reduction of DNA methylation and leads to Cnr-to-ripening reversion in tomato. Our results demonstrate that SlCMT3, possibly along with other key components including SlCMT2, SlDRM7 and SlMET1 in the RdDM and methylation maintenance pathways, is required to maintain the Cnr epi-allele, and CMT3 possesses an important role in epigenetic regulation of structural genes such as transcription factors in addition to its role in maintaining the methylation of repetitive DNA and transposon-related sequences.The tomato Cnr phenotype could be recreated in normal fruits by repression of LeSPL-CNR3LeSPL-CNR promoter are thought to be responsible for the non-ripening phenotype -based VIGS3SlDRM7, SlMET1, SlCMT2, SlCMT3 and SlCMT4SlDMT genes were cloned into the PVX-based VIGS vector  approximately 4 days earlier than Cnr fruits mock-inoculated with TE buffer or injected with PVX  should rescue Cnr ripening. It is also intriguing that SlCMT3 silencing had a greater effect on reverting the Cnr phenotype than silencing of SlDRM7 (a homologue of the Arabidopsis de novo methyltransferase DRM2) or other SlDMTs  genes including LeMADS-RIN, LeHB1, SlAP2a and SlTAGL1222526SlCMT3 RNA was detected , and confirmed the loss of methylation at the eight specific cytosines in the 286-bp promoter region  genes including LeMADS-RIN, SlAP2a and SlTAGL1 form a regulatory network that controls tomato development and fruit ripening. These TFs can regulate each other and they are involved in possible feedback loops in the genetic regulation of ripening252627252627323mC) is under dynamic changes during tomato development and fruit ripening, and chemical-mediated demethylation can facilitate early premature ripening24283132Cnr, DNA methylation maintenance is critical for maintaining epigenetic stability of the naturally occurring epimutation. Silencing of key SlDMTs in RdDM and 5mC maintenance pathways can destabilise epigenetic status which is required to down-regulate LeSPL-CNR. Such negative epigenetic control may also play a direct or indirect role in modulation of key ripening-associated TFs, and ethylene biosynthetic and signalling genes. Furthermore, microRNAs may be also involved in the fine-tuning of LeSPL-CNR expression in modulation of tomato fruit ripeningSlCMT3 involve tomato development and fruit ripening  vectorSlCMT3 was also cloned into PVX to produce PVX/SlCMT3UTR. The full-length cDNA sequences of the nine tomato DMT genes and the sequences of the short non-translatable fragments that were used for construction of the PVX-based VIGS constructs are included in LeSPL-CNR gene and the 286-bp region of the LeSPL-CNR promoter were cloned into the PVX/GFP vectorNon-translatable 300–525-bp fragments corresponding to the 5′ ends of each gene were PCR-amplified and cloned into the Cnr, rin and wild-type tomato (Solanum lycopersicum cv. Ailsa Craig) fruits were performed as described3PVX-based VIGS and Virus-induced transcriptional gene silencing in −ΔΔCt and normalized to the amount of 18S rRNA detected in the same sample as describedTotal RNA was extracted from tomato tissues using RNeasy Plant Mini Kit (Qiagen). cDNA was synthesized using a FastQuant RT Kit (Tiangen). qRT-PCR was performed on a Bio-Rad CFX96 Real-Time system (Bio-Rad) using an UltraSYBR Mixture Kit (CoWin Bioscience). At least three technical replicates for each of three biological replicates for each sample were analyzed. The relative level of specific gene expression was calculated using the formula 2Total DNA was isolated from tomato tissues using DNeasy Plant Mini Kit (Qiagen). Bisulfite conversion, PCR amplification and sequencing were performed using the EZ DNA Gold Methylation Kit (Zymo Research), Blue MegaMix Double PCR mixture (Microzone) and BigDye Terminator Reaction Mixture (Applied Biosystems) as describedW.C., J.K., C.Q. and Y.H. designed and performed experiments; J.T., C.W., H.W., Y.S., C.L., B.L., P.Z., Y.W., T.L. Z.Y., X.Z. and N.S. performed experiments; Y.C., S.Y. and S.Z. performed the WGBS and analysed the data; H.-z.W., T.O., Y.L., K.M., S.J., D.R., S.Z. and G.B.S. were involved in discussions and helped writing the paper; P.G. analysed data and helped writing the paper; Y.H. initiated the project, analysed data and wrote the paper.Supplementary Information


P > 0.05); however, there was a trend toward significance with regard to bevacizumab therapy and the risk of pulmonary embolism (P = 0.07). As there was a trend toward significance with regard to bevacizumab therapy and the risk of pulmonary embolism, anticoagulation may be advisable in certain newly diagnosed adult GBM patients who display a history of thromboembolism and/or more serious risk factors for thromboembolic events.Previous evidence suggests that the humanized anti-VEGF antibody bevacizumab increases thrombosis risk in glioma patients. Here, we comprehensively assessed the risk of adverse vascular events in adult glioma patients receiving bevacizumab therapy. Systematic searches of MEDLINE, EMBASE, and the Cochrane Library were conducted to find prospective phase II/III clinical trials on adult bevacizumab-treated glioma patients and non-bevacizumab-treated controls that reported data on adverse vascular events. Four high-quality trials were finally included in the systematic review, scoring greater than or equal to 7/8 on the Newcastle-Ottawa Scale. Three trials provided sufficient data for four meta-analytical comparisons between bevacizumab-treated and control groups of newly diagnosed glioblastoma multiforme (GBM) patients: all-cause discontinuation, thrombocytopenia, deep vein thrombosis (DVT), and pulmonary embolism. None of these adverse outcomes were found to be significantly different between bevacizumab-treated and control groups ( Therefore, the aim of this systematic review and meta-analysis will be to comprehensively assess the risk of cerebrovascular and other adverse vascular events in adult glioma patients receiving bevacizumab therapy.Despite this evidence of increased VTE risk in bevacizumab-treated glioma patients from Simonetti A systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelinesThe purpose of this study was to determine whether bevacizumab increases the risk of cerebrovascular and other adverse vascular events in adult patients with glioma. Therefore, we only selected (i) prospective phase II/III clinical trials including (ii) adult patients with glioma treated with bevacizumab and (iii) non-bevacizumab-treated controls that (iv) reported data on cerebrovascular and other adverse vascular events , pulmonary embolism, etc.).Reports limited to non-general populations  and studies failing to report adverse vascular events were excluded. If single-center reports were compiled into larger, multicenter series, single-site data were not included unless unique patients could be identified. Non-English studies, non-Chinese studies, Phase I studies, retrospective studies, animal studies, pediatric studies, case reports, reviews, editorials/commentaries, conference abstracts/summaries, and technical reports were all excluded.Data extraction was conducted independently by two co-authors, and any discrepancies between these co-authors were resolved by discussion and consensus. For each study, we extracted the following information: first author’s name, year of publication, country of publication, number of included patients, study arms, bevacizumab dose (mg/kg), additional therapies and dose (if any) , radiation therapy and total dose (Gy), median follow-up period, proportion of protocol violations (%), rates of all-cause discontinuation (%), and rates of adverse vascular events (%), including thrombocytopenia, angina pectoris, myocardial infraction (MI), transient ischemic attacks (TIA), ischemic stroke, hemorrhage stroke, DVT, pulmonary embolism (PE), other vascular events , and vascular-related mortality.We assessed the methodological quality of all included studies by the Newcastle-Ottawa Scale (NOS)P-value < 0.05 was considered statistically significant. For the calculation of incidence, the number of patients with each adverse vascular event and the number of bevacizumab-treated patients were extracted from the safety profiles of the included studies. The proportion of patients with each adverse vascular event and the 95% CIs were then derived from each trial. Statistical heterogeneity among the included trials was quantified with the I2 statistic (100%*[Q-df]/Q), which estimates the percentage of total variation across studies attributable to heterogeneity over chance2 value was greater than 50%, the assumption of homogeneity was deemed invalid, and the random-effects model was used; otherwise, the fixed-effects model was usedSTATA 12.0  and the Cochrane Collaboration Review Manager 5 (RevMan 5) were used for the statistical analysisThe flowchart for study selection is provided in 111213P > 0.05; P = 0.07). There was significant heterogeneity (I2 > 50%) in the comparisons of all-cause discontinuation  using the World Health Organization grading scheme (WHO I-IV): WHO grade II (diffuse infiltrating low-grade astrocytomas/oligodendrogliomas), WHO grade III (anaplastic astrocytomas/oligodendrogliomas), or WHO grade IV (high-grade GBM)15Two large clinical studies , FVIII activity, and D-dimeret al. showed that elevated D-dimer and F 1 + 2 could stratify adult glioma patients prone to developing VTEet al. has suggested a prediction model that combines circulating D-dimer, F 1 + 2, VEGF or plasminogen activator inhibitor-1 (PAI-1) levels, as well as tumoral tissue factor (TF) expression to risk stratify for VTE in adult glioma patientsIn order to assess this risk of thromboembolic events in adult glioma patients, several studies have provided guidance with respect to prognostic biomarkers for thromboembolic risk in this patient population. For example, a recent prospective study by Thaler 2 > 50%) was found in the comparisons of all-cause discontinuation , or vascular-related mortality. Second, the included studies did not distinguish distal from proximal DVT when reporting DVT outcomes. Third, the ability to accurately detect the reported outcomes may have varied among the study centers, resulting in bias of the reported incidence rates. Fourth, although all the included studies applied conventional temozolomide and radiotherapy in conjunction with bevacizumab treatment, the Chauffert 2014 study also used irinotecan in both the experimental and control groups, which may contributed to the heterogeneity in some of the comparisons. Fifth, significant heterogeneity .