Stellar Evolutionary Effects on the Abundances of PAH and SN-Condensed Dust in Galaxies

Spectral and photometric observations of nearby galaxies show a correlation between the strength of their mid-IR aromatic features, attributed to PAH molecules, and their metal abundance, leading to a deficiency of these features in low-metallicity galaxies. In this paper, we suggest that the observed correlation represents a trend of PAH abundance with galactic age, reflecting the delayed injection of carbon dust into the ISM by AGB stars in the final post-AGB phase of their evolution. AGB stars are the primary sources of PAHs and carbon dust in galaxies, and recycle their ejecta back to the interstellar medium only after a few hundred million years of evolution on the main sequence. In contrast, more massive stars that explode as Type II supernovae inject their metals and dust almost instantaneously after their formation. We first determined the PAH abundance in galaxies by constructing detailed models of UV-to-radio SED of galaxies that estimate the contribution of dust in PAH-free HII regions, and PAHs and dust from photodissociation regions, to the IR emission. All model components: the galaxies' stellar content, properties of their HII regions, and their ionizing and non-ionizing radiation fields and dust abundances, are constrained by their observed multiwavelength spectrum. After determining the PAH and dust abundances in 35 nearby galaxies using our SED model, we use a chemical evolution model to show that the delayed injection of carbon dust by AGB stars provides a natural explanation to the dependence of the PAH content in galaxies with metallicity. We also show that larger dust particles giving rise to the far-IR emission follow a distinct evolutionary trend closely related to the injection of dust by massive stars into the ISM.Comment: ApJ, 69 pages, 46 figures, Accepte

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material

The ERCC6 Gene and Age-Related Macular Degeneration

Background: Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the developed countries and is caused by both environmental and genetic factors. A recent study (Tuo et al., PNAS) reported an association between AMD and a single nucleotide polymorphism (SNP) (rs3793784) in the ERCC6 (NM_000124) gene. The risk allele also increased ERCC6 expression. ERCC6 is involved in DNA repair and mutations in ERCC6 cause Cockayne syndrome (CS). Amongst others, photosensitivity and pigmentary retinopathy are hallmarks of CS. Methodology/Principal Findings: Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study) were used to analyse the genetic association between ERCC6 and AMD (2682 AMD cases and 3152 controls). We also measured ERCC6 mRNA levels in retinal pigment epithelium (RPE) cells of healthy and early AMD affected human donor eyes. Rs3793784 conferred a small increase in risk for late AMD in the Dutch population (The Rotterdam and AMRO-NL study), but this was not replicated in two non-European studies (AREDS, Columbia University). In addition, the AMRO-NL study revealed no significant association for 9 other variants spanning ERCC6. Finally, we determined that ERCC6 expression in the human RPE did not depend on rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (P = 0.018). Conclusions/Significance: Our meta analysis of four Caucasian cohorts does not replicate the reported association between SNPs in ERCC6 and AMD. Nevertheless, our findings on ERCC6 expression in the RPE suggest that ERCC6 may be functionally involved in AMD. Combining our data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 may be caused by diverse, small and heterogeneous genetic and/or environmental determinants

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

The fungal pathogen Magnaporthe oryzae can cause rice blast and wheat blast diseases, which threatens worldwide food production. During infection, M. oryzae follows a sequence of distinct developmental stages adapted to survival and invasion of the host environment. M. oryzae attaches onto the host by the conidium, and then develops an appressorium to breach the host cuticle. After penetrating, it forms invasive hyphae to quickly spread in the host cells. Numerous genetic studies have focused on the mechanisms underlying each step in the infection process, but systemic approaches are needed for a broader, integrated understanding of regulatory events during M. oryzae pathogenesis. Many infection-related signaling events are regulated through post-translational protein modifications within the pathogen. N-linked glycosylation, in which a glycan moiety is added to the amide group of an asparagine residue, is an abundant modification known to be essential for M. oryzae infection. In this study, we employed a quantitative proteomics analysis to unravel the overall regulatory mechanisms of N-glycosylation at different developmental stages of M. oryzae. We detected changes in N-glycosylation levels at 559 glycosylated residues (N-glycosites) in 355 proteins during different stages, and determined that the ER quality control system is elaborately regulated by N-glycosylation. The insights gained will help us to better understand the regulatory mechanisms of infection in pathogenic fungi. These findings may be also important for developing novel strategies for fungal disease control. Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

ABSTRACT: BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. METHODS: Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. RESULTS: Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. CONCLUSIONS: Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression