Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator’s target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.     

Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25

Protein phosphatase 2C clade A members are major signaling components in the ABA-dependent signaling cascade that regulates seed germination. To elucidate the role of PP2CA genes in germination of rice seed, we selected OsPP2C51, which shows highly specific expression in the embryo compared with other protein phosphatases based on microarray data. GUS histochemical assay confirmed that OsPP2C51 is expressed in the seed embryo and that this expression pattern is unique compared with those of other OsPP2CA genes. Data obtained from germination assays and alpha-amylase assays of OsPP2C51 knockout and overexpression lines suggest that OsPP2C51 positively regulates seed germination in rice. The expression of alpha-amylase synthesizing genes was high in OsPP2C51 overexpressing plants, suggesting that elevated levels of OsPP2C51 might enhance gene expression related to higher rates of seed germination. Analysis of protein interactions between ABA signaling components showed that OsPP2C51 interacts with OsPYL/RCAR5 in an ABA-dependent manner. Furthermore, interactions were observed between OsPP2C51 and SAPK2, and between OsPP2C51 and OsbZIP10 and we found out that OsPP2C51 can dephosphorylates OsbZIP10. These findings suggest the existence of a new branch in ABA signaling pathway consisting of OsPYL/RCAR-OsPP2C-bZIP apart from the previously reported OsPYL/RCAR-OsPP2C-SAPK-bZIP. Overall, our result suggests that OsPP2C51 is a positive regulator of seed germination by directly suppressing active phosphorylated OsbZIP10.     

Differences in field‐scale N2O flux linked to crop residue removal under two tillage systems in cold climates

Residue removal for biofuel production may have unintended consequences for N₂O emissions from soils, and it is not clear how N₂O emissions are influenced by crop residue removal from different tillage systems. Thus, we measured field‐scale N₂O flux over 5 years (2005–2007, 2010–2011) from an annual crop rotation to evaluate how N₂O emissions are influenced by no‐till (NT) compared to conventional tillage (CV), and how crop residue removal (R?) rather than crop residue return to soil (R+) affects emissions from these two tillage systems. Data from all 5 years indicated no differences in N₂O flux between tillage practices at the onset of the growing season, but CT had 1.4–6.3 times higher N₂O flux than NT overwinter. Nitrous oxide emissions were higher due to R? compared to R+, but the effect was more marked under CT than NT and overwinter than during spring. Our results thus challenge the assumption based on IPCC methodology that crop residue removal will result in reduced N₂O emissions. The potential for higher N₂O emission with residue removal implies that the benefit of utilizing biomass as biofuels to mitigate greenhouse gas emission may be overestimated. Interestingly, prior to an overwinter thaw event, dissolved organic C (DOC) was negatively correlated to peak N₂O flux (r = ?0.93). This suggests that lower N₂O emissions with R+ vs. R? may reflect more complete stepwise denitrification to N₂ during winter and possibly relate to the heterotrophic microbial capacity for processing crop residue into more soluble C compounds and a shift in the preferential C source utilized by the microbial community overwinter.     

Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

Catabolism of 4-hydroxy-2-trans-nonenal by THP1 monocytes/macrophages and inactivation of carboxylesterases by this lipid electrophile

Oxidative stress in cells and tissues leads to the formation of an assortment of lipid electrophiles, such as the quantitatively important 4-hydroxy-2-trans-nonenal (HNE). Although this cytotoxic aldehyde is atherogenic the mechanisms involved are unclear. We hypothesize that elevated HNE levels can directly inactivate esterase and lipase activities in macrophages via protein adduction, thus generating a biochemical lesion that accelerates foam cell formation and subsequent atherosclerosis. In the present study we examined the effects of HNE treatment on esterase and lipase activities in human THP1 monocytes/macrophages at various physiological scales (i.e., pure recombinant enzymes, cell lysate, and intact living cells). The hydrolytic activities of bacterial and human carboxylesterase enzymes (pnbCE and CES1, respectively) were inactivated by HNE in vitro in a time- and concentration-dependent manner. In addition, so were the hydrolytic activities of THP1 cell lysates and intact THP1 monocytes and macrophages. A single lysine residue (Lys105) in recombinant CES1 was modified by HNE via a Michael addition reaction, whereas the lone reduced cysteine residue (Cys389) was found unmodified. The lipolytic activity of cell lysates and intact cells was more sensitive to the inhibitory effects of HNE than the esterolytic activity. Moreover, immunoblotting analysis using HNE antibodies confirmed that several cellular proteins were adducted by HNE following treatment of intact THP1 monocytes, albeit at relatively high HNE concentrations (>50 μM). Unexpectedly, in contrast to CES1, the treatment of a recombinant human CES2 with HNE enhanced its enzymatic activity ∼3-fold compared to untreated enzyme. In addition, THP1 monocytes/macrophages can efficiently metabolize HNE, and glutathione conjugation of HNE is responsible for ∼43% of its catabolism. The functional importance of HNE-mediated inactivation of cellular hydrolytic enzymes with respect to atherogenesis remains obscure, although this study has taken a first step toward addressing this important issue by examining the potential of HNE to inhibit this biochemical activity in a human monocyte/macrophage cell line.     

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.     

A Single Mutation at Residue 25 Populates the Folding Intermediate of E. coli RNase H and Reveals a Highly Dynamic Partially Folded Ensemble

Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H (RNase H) from Escherichia coli populates an on-pathway intermediate identified in both bulk studies and single-molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of heteronuclear single quantum coherences, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that they are well packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.