Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-β

BACKGROUND: XIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and – independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-β. METHODS: We used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-β. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-β-mediated sensitization to TRAIL-induced cell death. RESULTS: We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-β across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-β-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-β induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-β-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-β-mediated TRAIL sensitivity. CONCLUSION: Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-β treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer

Techniques for Arbuscular Mycorrhiza Inoculum Reduction

It is well established that arbuscular mycorrhizal (AM) fungi can play a significant role in sustainable crop production and environmental conservation. With the increasing awareness of the ecological significance of mycorrhizas and their diversity, research needs to be directed away from simple records of their occurrence or casual speculation of their function (Smith and Read 1997). Rather, the need is for empirical studies and investigations of the quantitative aspects of the distribution of different types and their contribution to the function of ecosystems. There is no such thing as a fungal effect or a plant effect, but there is an interaction between both symbionts. This results from the AM fungi and plant community size and structure, soil and climatic conditions, and the interplay between all these factors (Kahiluoto et al. 2000). Consequently, it is readily understood that it is the problems associated with methodology that limit our understanding of the functioning and effects of AM fungi within field communities. Given the ubiquous presence of AM fungi, a major constraint to the evaluation of the activity of AM colonisation has been the need to account for the indigenous soil native inoculum. This has to be controlled (i.e. reduced or eliminated) if we are to obtain a true control treatment for analysis of arbuscular mycorrhizas in natural substrates. There are various procedures possible for achieving such an objective, and the purpose of this chapter is to provide details of a number of techniques and present some evaluation of their advantages and disadvantages. Although there have been a large number of experiments to investigated the effectiveness of different sterilization procedures for reducing pathogenic soil fungi, little information is available on their impact on beneficial organisms such as AM fungi. Furthermore, some of the techniques have been shown to affect physical and chemical soil characteristics as well as eliminate soil microorganisms that can interfere with the development of mycorrhizas, and this creates difficulties in the interpretation of results simply in terms of possible mycorrhizal activity. An important subject is the differentiation of methods that involve sterilization from those focussed on indigenous inoculum reduction. Soil sterilization aims to destroy or eliminate microbial cells while maintaining the existing chemical and physical characteristics of the soil (Wolf and Skipper 1994). Consequently, it is often used for experiments focussed on specific AM fungi, or to establish a negative control in some other types of study. In contrast, the purpose of inoculum reduction techniques is to create a perturbation that will interfere with mycorrhizal formation, although not necessarily eliminating any component group within the inoculum. Such an approach allows the establishment of different degrees of mycorrhizal formation between treatments and the study of relative effects. Frequently the basic techniques used to achieve complete sterilization or just an inoculum reduction may be similar but the desired outcome is accomplished by adjustments of the dosage or intensity of the treatment. The ultimate choice of methodology for establishing an adequate non-mycorrhizal control depends on the design of the particular experiments, the facilities available and the amount of soil requiring treatment

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Genetic analysis of the interaction between Allium species and arbuscular mycorrhizal fungi

The response of Alliumcepa, A. roylei, A. fistulosum, and the hybrid A. fistulosum × A. roylei to the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied. The genetic basis for response to AMF was analyzed in a tri-hybrid A. cepa × (A. roylei × A. fistulosum) population. Plant response to mycorrhizal symbiosis was expressed as relative mycorrhizal responsiveness (R′) and absolute responsiveness (R). In addition, the average performance (AP) of genotypes under mycorrhizal and non-mycorrhizal conditions was determined. Experiments were executed in 2 years, and comprised clonally propagated plants of each genotype grown in sterile soil, inoculated with G. intraradices or non-inoculated. Results were significantly correlated between both years. Biomass of non-mycorrhizal and mycorrhizal plants was significantly positively correlated. R′ was negatively correlated with biomass of non-mycorrhizal plants and hence unsuitable as a breeding criterion. R and AP were positively correlated with biomass of mycorrhizal and non-mycorrhizal plants. QTLs contributing to mycorrhizal response were located on a linkage map of the A. roylei × A. fistulosum parental genotype. Two QTLs from A. roylei were detected on chromosomes 2 and 3 for R, AP, and biomass of mycorrhizal plants. A QTL from A. fistulosum was detected on linkage group 9 for AP (but not R), biomass of mycorrhizal and non-mycorrhizal plants, and the number of stem-borne roots. Co-segregating QTLs for plant biomass, R and AP indicate that selection for plant biomass also selects for enhanced R and AP. Moreover, our findings suggest that modern onion breeding did not select against the response to AMF, as was suggested before for other cultivated species. Positive correlation between high number of roots, biomass and large response to AMF in close relatives of onion opens prospects to combine these traits for the development of more robust onion cultivars

Can Arbuscular Mycorrhizal Fungi Reduce the Growth of Agricultural Weeds?

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are known for their beneficial effects on plants. However, there is increasing evidence that some ruderal plants, including several agricultural weeds, respond negatively to AMF colonization. Here, we investigated the effect of AMF on the growth of individual weed species and on weed-crop interactions. METHODOLOGY/PRINCIPAL FINDINGS: First, under controlled glasshouse conditions, we screened growth responses of nine weed species and three crops to a widespread AMF, Glomus intraradices. None of the weeds screened showed a significant positive mycorrhizal growth response and four weed species were significantly reduced by the AMF (growth responses between -22 and -35%). In a subsequent experiment, we selected three of the negatively responding weed species--Echinochloa crus-galli, Setaria viridis and Solanum nigrum--and analyzed their responses to a combination of three AMF (Glomus intraradices, Glomus mosseae and Glomus claroideum). Finally, we tested whether the presence of a crop (maize) enhanced the suppressive effect of AMF on weeds. We found that the growth of the three selected weed species was also reduced by a combination of AMF and that the presence of maize amplified the negative effect of AMF on the growth of E. crus-galli. CONCLUSIONS/SIGNIFICANCE: Our results show that AMF can negatively influence the growth of some weed species indicating that AMF have the potential to act as determinants of weed community structure. Furthermore, mycorrhizal weed growth reductions can be amplified in the presence of a crop. Previous studies have shown that AMF provide a number of beneficial ecosystem services. Taken together with our current results, the maintenance and promotion of AMF activity may thereby contribute to sustainable management of agroecosystems. However, in order to further the practical and ecological relevance of our findings, additional experiments should be performed under field conditions

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>Similarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.</p> <p>Methods</p> <p>Relative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.</p> <p>Results</p> <p>Survivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.</p> <p>The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.</p> <p>Conclusion</p> <p>Our study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.</p

Inoculations with Arbuscular Mycorrhizal Fungi Increase Vegetable Yields and Decrease Phoxim Concentrations in Carrot and Green Onion and Their Soils

Background As one of the most widely used organophosphate insecticides in vegetable production, phoxim (C12H15N2O3PS) is often found as residues in crops and soils and thus poses a potential threat to public health and environment. Arbuscular mycorrhizal (AM) fungi may make a contribution to the decrease of organophosphate residues in crops and/or the degradation in soils, but such effects remain unknown. Methodology/Principal Findings A greenhouse pot experiment studied the influence of AM fungi and phoxim application on the growth of carrot and green onion, and phoxim concentrations in the two vegetables and their soil media. Treatments included three AM fungal inoculations with Glomus intraradices BEG 141, G. mosseae BEG 167, and a nonmycorrhizal control, and four phoxim application rates (0, 200, 400, 800 mg l−1, while 400 mg l−1 rate is the recommended dose in the vegetable production system). Carrot and green onion were grown in a greenhouse for 130 d and 150 d. Phoxim solution (100 ml) was poured into each pot around the roots 14d before plant harvest. Results showed that mycorrhizal colonization was higher than 70%, and phoxim application inhibited AM colonization on carrot but not on green onion. Compared with the nonmycorrhizal controls, both shoot and root fresh weights of these two vegetables were significantly increased by AM inoculations irrespective of phoxim application rates. Phoxim concentrations in shoots, roots and soils were increased with the increase of phoxim application rate, but significantly decreased by the AM inoculations. Soil phosphatase activity was enhanced by both AM inocula, but not affected by phoxim application rate. In general, G. intraradices BEG 141 had more pronounced effects than G. mosseae BEG 167 on the increase of fresh weight production in both carrot and green onion, and the decrease of phoxim concentrations in plants and soils. Conclusions/Significance Our results indicate a promising potential of AM fungi for enhancing vegetable production and reducing organophosphorus pesticide residues in plant tissues and their growth media, as well as for the phytoremediation of organophosphorus pesticide-contaminated soils

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.This work was supported by Universidad de Granada, Plan Propio 2015;#P3B: FAM, VMC (http://investigacion.ugr.es/pages/planpropio/2015/ resoluciones/p3b_def_28072015); Universidad de Granada CEI BioTic;#CAEP2-84: VMC (http:// biotic.ugr.es/pages/resolucionprovisional enseaanzapractica22demayo/!); and Canadian nstitutes of Health Research;#231421, #318176, #361847: STB, ECL, RK (http://www.cihr-irsc.gc. ca/e/193.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript