High-density mapping and marker development for the powdery mildew resistance gene PmAS846 derived from wild emmer wheat (Triticum turgidum var. dicoccoides)

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important foliar disease of wheat worldwide. The dominant powdery mildew resistance gene PmAS846 was transferred to the hexaploid wheat lines N9134 and N9738 from wild emmer wheat (Triticum dicoccoides) in 1995, and it is still one of the most effective resistance genes in China. A high resolution genetic map for PmAS846 locus was constructed using two F₂ populations and corresponding F_2:3 families developed from the crosses of N9134/Shaanyou 225 and N9738/Huixianhong. Synteny between wheat and Brachypodium distachyon and rice was used to develop closely linked molecular markers to reduce the genetic interval around PmAS846. Twenty-six expressed sequence tag-derived markers were mapped to the PmAS846 locus. Five markers co-segregated with PmAS846 in the F₂ population of N9134/Shaanyou 225. PmAS846 was physically located to wheat chromosome 5BL bin 0.75–0.76 within a gene-rich region. The markers order is conserved between wheat and Brachypodium distachyon, but rearrangements are present in rice. Two markers, BJ261635 and CJ840011 flanked PmAS846 and narrowed PmAS846 to a region that is collinear with 197 and 112 kb genomic regions on Brachypodium chromosome 4 and rice chromosome 9, respectively. The genes located on the corresponding homologous regions in Brachypodium, rice and barley could be considered for further marker saturation and identification of potential candidate genes for PmAS846. The markers co-segregating with PmAS846 provide a potential target site for positional cloning of PmAS846, and can be used for marker-assisted selection of this gene.     

CED-1, CED-7, and TTR-52 regulate surface phosphatidylserine expression on apoptotic and phagocytic cells

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Highlights? PS is expressed on the surface of apoptotic and phagocytic cells during apoptosis ? CED-7 and TTR-52 mediate time-dependent loss of PS from surface of apoptotic cells ? CED-7 and TTR-52 promote generation of extracellular PS vesicles ? CED-7/TTR-52/CED-1 promote phagocyte PS expression, important for corpse engulfment     

Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4⁺CD8⁺ double positive stage, were both upregulated in CD4⁺ or CD8⁺ single positive thymocytes, and both continued to be co-expressed in na?ve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8⁺ T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.     

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background

H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking.

Methodology/Principal Findings

Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8⁺ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8⁺ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8⁺ T cells.

Conclusions/Significance

Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.     

Association of natural intake of dietary plant sterols with carotid intima-media thickness and blood lipids in Chinese adults: a cross-section study

Background

Many studies showed a moderate cholesterol-lowering effect of plant sterols (PS), but increased circulating PS might be atherogenic. We evaluated the associations between natural dietary intake of PS and carotid intima–media thickness (IMT) and serum lipids.

Methodology/Principal Findings

This community-based cross-sectional study included 1160 men and 2780 women aged 31–75 years. Dietary intakes were assessed using a food-frequency questionnaire. The IMTs at the common, bifurcation and internal carotid artery segments, and fasting serum total (TC), LDL (LDLc) and HDL (HDLc) cholesterol, and triglycerides (TG) were determined. After adjusting for potential covariates, multivariate analysis showed a dose-dependent inverse association of total PS intake with serum TC, LDLc, non-HDLc in women (P<0.001) and in men (P<0.05). As compared to the lowest quartile of PS intake (<206 mg/d), the multivariate-adjusted means of TC, LDLc and non-HDLc in the highest quartile of PS intake (447 mg/d) decreased by 5.0%, 6.2% and 6.5% in women (P<0.005), and by 6.4%, 7.1% and 6.7% (P>0.05) in men. Although the IMTs tended to be lower with greater intake of dietary PS, only small differences in the left internal IMT between the highest and lowest groups were observed among men (−7.6%) and women (−5.1%) (P<0.05). The multivariate analysis showed no significant mean differences among the PS groups in HDLc, TG and IMTs at other studied sites among men and women (all P>0.05).

Conclusions

Greater PS consumption from natural diets is associated with lower serum total, LDL, non-HDL cholesterol and with thinner left internal IMT in women and men.     

Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

GPS-ARM: computational analysis of the APC/C recognition motif by predicting D-boxes and KEN-boxes

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org.