Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

Introduction

Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.

Methods

A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.

Results

A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites.

Conclusion

Chinese XLHED carriers often have a hypermethylated EDA promoter.     

P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms.

Methodology/Principal Findings

Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death.

Conclusions

Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.     

The Potential Biomarkers to Identify the Development of Steatosis in Hyperuricemia

Hyperuricemia (HU) often progresses to combine with non-alcoholic fatty liver disease (NAFLD) in the clinical scenario, which further exacerbates metabolic disorders; early detection of biomarkers, if obtained during the HU progression, may be beneficial for preventing its combination with NAFLD. This study aimed to decipher the biomarkers and mechanisms of the development of steatosis in HU. Four groups of subjects undergoing health screening, including healthy subjects, subjects with HU, subjects with HU combined with NAFLD (HU+NAFLD) and subjects with HU initially and then with HU+NAFLD one year later (HU→HU+NAFLD), were recruited in this study. The metabolic profiles of all subjects’ serum were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry. The metabolomic data from subjects with HU and HU+NAFLD were compared, and the biomarkers for the progression from HU to HU+NAFLD were predicted. The metabolomic data from HU→HU+NAFLD subjects were collected for further verification. The results showed that the progression was associated with disturbances of phospholipase metabolism, purine nucleotide degradation and Liver X receptor/retinoic X receptor activation as characterized by up-regulated phosphatidic acid, cholesterol ester (18:0) and down-regulated inosine. These metabolic alterations may be at least partially responsible for the development of steatosis in HU. This study provides a new paradigm for better understanding and further prevention of disease progression.     

Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.     

The Genetics of Leaf Flecking in Maize and Its Relationship to Plant Defense and Disease Resistance

Physiological leaf spotting, or flecking, is a mild-lesion phenotype observed on the leaves of several commonly used maize (Zea mays) inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4,998 recombinant inbred lines from 25 biparental families, and in an association population, comprising 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7,386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million single-nucleotide polymorphisms (SNPs) in the NAM population and with 246,497 SNPs in the association population, resulting in the identification of 18 and three loci associated with variation in flecking, respectively. Many of the candidate genes colocalizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid- and jasmonic acid-dependent pathways, redox homeostasis, stress response, and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response, increased disease resistance, and increased pest resistance. A nonlinear relationship with total kernel weight also was observed whereby lines with relatively high levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.The plant hypersensitive response (HR) is a form of programmed cell death (PCD) characterized by rapid, localized cell death at the point of attempted pathogen penetration, usually resulting in disease resistance (Coll et al., 2011). It is often associated with other responses, including ion fluxes, an oxidative burst, lipid peroxidation, and cell wall fortification (Hammond-Kosack and Jones, 1996). van Doorn et al. (2011) suggested that HR is a type of PCD sharing features with, but distinct from, both vacuolar cell death and necrosis.HR has been associated with resistance to almost every class of pathogen and pest, including bacteria, viruses, fungi, nematodes, insects, and parasitic plants (Wu and Baldwin, 2010), and generally is most effective against biotrophic pathogens, since biotrophs require a long-term feeding relationship with living host cells. It is generally mediated by dominant resistance (R) genes whose activation is triggered by the direct or indirect detection of specific pathogen-derived effector proteins (Bent and Mackey, 2007). R proteins are maintained in their inactive state if their corresponding effector is not present. Mutants in which HR is constitutively active have been identified in many plant species, including maize/corn (Zea mays; Walbot et al., 1983; Johal, 2007), Arabidopsis (Arabidopsis thaliana; Lorrain et al., 2003), barley (Hordeum vulgare; Wolter et al., 1993), and rice (Oryza sativa; Yin et al., 2000).One well-known class of plant mutants spontaneously form lesions (patches of dead or chlorotic cells) in the absence of any obvious injury, stress, or infection to the plant. Since these lesions in some cases resemble HR, they have been termed disease-lesion mimics (Neuffer and Calvert, 1975). These mutants, which we will here collectively term Les mutants, have been studied extensively, especially in maize (Walbot et al., 1983; Johal et al., 1995; Johal, 2007) and Arabidopsis (Coll et al., 2011). While some of these lesion phenotypes are indeed caused by perturbations in the plant defense response (Hu et al., 1996; Rustérucci et al., 2001), some of the genes underlying this mutant class affect various other pathways that cause cell death if their function is perturbed (Johal, 2007). For instance, the Arabidopsis gene acd2 and the maize gene lls1 are defective in chlorophyll degradation (Gray et al., 1997; Mach et al., 2001).We have defined leaf flecking as the mild, genetically determined spotting observed on many maize inbred cultivars (Vontimitta et al., 2015; Fig. 1). The trait is qualitatively and visually similar to, but quantitatively less severe than, Les mutant phenotypes. The distinction between what constitutes a flecking versus a mild Les trait is necessarily somewhat arbitrary, but for our purposes, we have defined any nonproliferating and distinct leaf-spotting phenotype as flecking.Open in a separate windowFigure 1.A, Examples of variation in the flecking phenotype among inbred lines, with severity increasing from left to right (flecking scores in parentheses, from 0 to 4, scored on a scale of 1–10). B, Leaves of the lines nearly isogenic to inbred Mo20W, into which specific indicated dominant Les mutant genes have been introgressed (Rp1-D21 mutation in an H95 inbred background). Photographs were taken in Clayton, North Carolina, 12 weeks after planting. This figure is adapted from Figure 1 of Vontimitta et al. (2015).Leaf flecking is familiar to most corn breeders, appearing in such well-known and widely used lines such as Mo17 (Zehr et al., 1994) and in several other species such as barley (Makepeace et al., 2007), wheat (Triticum aestivum; Nair and Tomar, 2001), and oat (Avena sativa; Ferdinandsen and Winge, 1930). Flecking tends to be more noticeable in inbreds compared with their derived hybrids (M. Goodman and W. Dolezal, personal communication). Anecdotally, it is often thought to be indicative of a constitutive low-level defense response and as a marker for increased disease resistance.In previous work, we and others have defined the genetic architectures associated with resistance to several maize diseases, including southern leaf blight (SLB; causal agent, Cochliobolus heterostrophus), northern leaf blight (NLB; causal agent, Exserohilum turcicum), and gray leaf spot (GLS; causal agent, Cercospora zeae-maydis; Kump et al., 2011; Poland et al., 2011; Wisser et al., 2011; Benson et al., 2015), and with the control of the maize HR (Chintamanani et al., 2010; Chaikam et al., 2011; Olukolu et al., 2013). For much of this work, we used two powerful mapping populations: the maize association population (Flint-Garcia et al., 2005), a collection of 302 diverse inbred lines with low linkage disequilibrium, and the 5,000-line nested association mapping (NAM) population (McMullen et al., 2009), which is made up of 25 200-line recombinant inbred line (RIL) subpopulations derived from crosses between the common parent B73 and 25 diverse inbreds. Using these populations, it is possible to both sample a diverse array of germplasm and map quantitative trait loci (QTLs) precisely, in some cases to the gene level (Tian et al., 2011; Cook et al., 2012; Hung et al., 2012; Larsson et al., 2013; Olukolu et al., 2013; Wang and Balint-Kurti, 2016).A recent study using 300 lines from the maize intermated B73 × Mo17 population advanced intercross line mapping population identified low but moderately significant positive correlations between increased flecking and increased disease resistance and defense response (Vontimitta et al., 2015). Loci associated with variation in flecking were mapped, although these loci did not colocalize with QTLs identified previously for disease resistance and defense response traits (Balint-Kurti et al., 2007, 2008, 2010; Olukolu et al., 2013). In this study, we have extended this work to examine the genetic basis of leaf flecking over a much more diverse set of maize germplasm using a substantially larger population. We mapped loci associated with variation in leaf flecking and identified candidate genes and pathways that may be involved in this phenotype. Additionally, we have examined the correlations between leaf flecking and disease resistance, the hypersensitive defense response, and total kernel weight.     

A N‐(2‐hydroxyethyl)piperazine dangled 2,5‐diphenyl‐1,3,4‐oxadiazole‐based fluorescent sensor for selective relay recognition of Cu2+ and sulfide in water

A new 2,5‐diphenyl‐1,3,4‐oxadiazole‐based derivative (L) was synthesized and applied as a highly selective and sensitive fluorescent sensor for relay recognition of Cu²⁺ and S^2? in water (Tris–HCl 10 mM, pH = 7.0) solution. L exhibits an excellent selectivity to Cu²⁺ over other examined metal ions with a prominent fluorescence ‘turn‐off’ at 392 nm. L interacts with Cu²⁺ through a 1:2 binding stoichiometry with a detection limit of 4.8 × 10^–7 M. The on‐site formed L–2Cu²⁺ complex exhibits excellent selectivity to S^2? with a fluorescence ‘off–on’ response via a Cu²⁺ displacement approach. Copyright © 2016 John Wiley & Sons, Ltd.