P2Y6 receptors and ADAM17 mediate low-dose gamma-ray-induced focus formation (activation) of EGF receptor

The EGF receptor (EGFR) is frequently expressed in tumors of epithelial origin. Although it is well known that ionizing radiation induces activation of EGFR, the mechanism remains unknown. Recently, we reported that activation of P2Y receptors is involved in γ-radiation-induced activation of extracellular signal-regulated kinase1/2 (ERK1/2), which is dependent on activation of EGFR. Here we focused on the mechanism of activation of EGFR in response to low-dose γ radiation, mainly in terms of the activation-associated formation of EGFR foci in A549 cells. Irradiation of cells with 0.1 Gy γ rays induced biphasic phosphorylation of EGFR and ERK1/2 as well as biphasic formation of EGFR foci. The radiation-induced focus formation of EGFR was abolished by ecto-nucleotidase, P2Y receptor antagonists and knockdown of P2Y6 receptor, suggesting the involvement of extracellular nucleotides and activation of P2Y6 receptors in this process. Further, a disintegrin and metalloprotease 17 (ADAM17) is expressed in A549 cells and an ADAM17 inhibitor significantly blocked the radiation-induced focus formation of EGFR. We conclude that activation of both P2Y6 receptors and ADAM17 mediates the low-dose γ-radiation-induced activation of EGFR, as evaluated in terms of focus formation, in A549 cells.     

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Molecular Characterization and Expression Profiling of the Protein Disulfide Isomerase Gene Family in Brachypodium distachyon L

Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H₂O₂) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.     

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3⁺ T-cells than photo-damaged skin, however, the abundance of CD3⁻CD56⁺ Natural Killer (NK) cells, CD11c⁺HLA-DR⁺ conventional Dendritic Cells (cDC), BDCA-2⁺HLA-DR⁺ plasmacytoid DC (pDC), FoxP3⁺ Regulatory T-cells (T-reg), Vα24⁺Vβ11⁺ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4⁺ T-cells were associated with SCC, yet CD8⁺ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8⁺ T-cells than IEC. We propose that differences in CD8⁺ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4⁺ T-cells to CD8⁺ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.     

Impact of Groundwater Table and Plateau Zokors (Myospalax baileyi) on Ecosystem Respiration in the Zoige Peatlands of China

Peatlands contain large amount of carbon stock that is vulnerable to release into the atmosphere. Mostly because of human impact, the peatlands at Zoige Wetlands face severe degradation, and the groundwater table is now lower than before, which has increased the population of the plateau zokor, a burrowing rodent. However, the impact of these changes on ecosystem carbon flows has not been studied. To investigate how the plateau zokor and the groundwater level alter the ecosystem respiration of the Zoige peatlands, we sampled the CO₂ flux of hummocks shaped by the zokors and compared it with the CO₂ flux of undisturbed sites with different groundwater table levels. The soil organic carbon (SOC), soil water content (SWC) and soil temperature at 5 cm (T₅) were measured. SOC showed no significant difference among the four sampling sites and did not correlate with the CO₂ flux, while SWC was found to partly determine the CO₂ flux. A linear equation could adequately describe the relationship between the natural logarithm of the ecosystem respiration and the soil temperature. It is demonstrated that descending groundwater table might accelerate ecosystem respiration and the CO₂ flux from hummocks was higher than the CO₂ flux from the control site in the non-growing season. With rising temperature, the CO₂ flux from the control site accelerated faster than that from the hummocks. Our results show that ecosystem respiration was significantly lower from hummocks than at the control site in the growing season. The results on the impact of zokors on greenhouse gas emissions presented in this paper provide a useful reference to help properly manage not only this, but other litter-burrowing mammals at peatland sites.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

基于GIS和地统计学的低山茶园土壤肥力质量评价

亚热带低山区是我国茶园的主要分布区域,研究其土壤肥力质量状况是茶园精准化和信息化管理的基础。利用地统计学、GIS以及多元统计分析相结合的方法,对蒙顶山茶园土壤肥力质量进行了定量化综合评价研究。结果表明：（1）蒙顶山茶园土壤肥力质量沿海拔高度垂直变化明显,大部分土壤肥力区沿蒙顶山阳坡面水平方向呈带状分布。土壤肥力较高的区域分布在山体的中上部,随着海拔高度的降低土壤肥力质量水平也逐渐降低。（2）蒙顶山茶园土壤肥力质量总体水平不高。区域内土壤各项肥力指标与优良茶园相比还存在差距,同时肥力质量偏低的区域所占面积较大：第四、五两级肥力区所占面积最大,占总面积的42．35％。肥力较低的后5级占总面积的近70％,而肥力最高的3级不到10％。（3）采用该方法进行土壤肥力质量评价能较客观地反映土壤肥力状况,为地统计研究成果实际运用提供了思路。     

基于GIS的滨海盐渍化农田土壤空间变异及其分区管理

针对滨海盐渍化农田盐碱瘠薄、土壤属性空间变异大、粗放管理效益低的现实,研究管理分区精准划分方法,采取差异化措施,提升盐渍化土地利用水平。该文以无棣县农田为研究区,采用网格法结合土地利用现状定点野外采样、室内化验分析获取土壤属性数据,运用ArcGIS 10.2地统计方法分析土壤属性的空间变异特征;在MATLAB R2016a中采用模糊c-均值聚类法（FCM）计算各样点的模糊隶属度,通过插值预测模糊隶属度的空间分布,基于最大隶属度原则进行分区;通过变异性分析和最小极差法（LSR）差异显著性检验,对分区结果进行精度验证。结果表明：无棣县农田土壤总体呈轻中度盐渍化,有效氮含量偏低,有机质、有效磷含量中等,速效钾含量较高;有机质、有效氮、有效磷、速效钾和含盐量呈中等变异性（变异系数25.0%-52.3%）,空间变异性较大,应分区调控;速效钾、含盐量和pH的块金效应值小于25%,主要受土壤质地、地下水矿化度等结构因素影响,有机质、有效氮和有效磷的块金效应值在50%-75%之间,受耕作方式、施肥等人为因素影响较大。将全县农田划分为3类管理区,估算面积分别为2.56万hm²、1.76万hm²、3.24万hm²;各分区土壤养分变异系数分别为23.9%-51.5%、15.9%-50.3%、14.7%-33.0%,检验结果表明各分区间差异显著,而各分区内部变异性明显低于未分区。管理分区与土壤属性空间分布特征具有较高的拟合度,分区结果可以作为差异化管理的作业单元。研究结果为各分区内部统一、不同分区间差异化管理提供了依据,研究有助于推进滨海盐渍化农田精准化管理水平的提高。