Predicting DNA methylation level across human tissues

Differences in methylation across tissues are critical to cell differentiation and are key to understanding the role of epigenetics in complex diseases. In this investigation, we found that locus-specific methylation differences between tissues are highly consistent across individuals. We developed a novel statistical model to predict locus-specific methylation in target tissue based on methylation in surrogate tissue. The method was evaluated in publicly available data and in two studies using the latest IlluminaBeadChips: a childhood asthma study with methylation measured in both peripheral blood leukocytes (PBL) and lymphoblastoid cell lines; and a study of postoperative atrial fibrillation with methylation in PBL, atrium and artery. We found that our method can greatly improve accuracy of cross-tissue prediction at CpG sites that are variable in the target tissue [R² increases from 0.38 (original R² between tissues) to 0.89 for PBL-to-artery prediction; from 0.39 to 0.95 for PBL-to-atrium; and from 0.81 to 0.98 for lymphoblastoid cell line-to-PBL based on cross-validation, and confirmed using cross-study prediction]. An extended model with multiple CpGs further improved performance. Our results suggest that large-scale epidemiology studies using easy-to-access surrogate tissues (e.g. blood) could be recalibrated to improve understanding of epigenetics in hard-to-access tissues (e.g. atrium) and might enable non-invasive disease screening using epigenetic profiles.     

Gene transfer in Leptolyngbya sp. strain BL0902, a cyanobacterium suitable for production of biomass and bioproducts

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira ("pirulina" strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a "elper"plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain.     

Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.     

The stimulation of cell extension by ethylene and auxin in aquatic plants

Elongation of the shoots of three aquatic plants (Hydrocharis morsus-ranae, Regnellidium diphyllum and Ranunculus sceleratus) is stimulated by treatment with ethylene or IAA. The effects of the two hormones are additive, and experiments with an ethylene biosynthesis inhibitor and silver ions indicate that the mechanisms by which ethylene and IAA stimulate growth may be different. Hydrocharis and Ranunculus leaf discs synthesize [¹⁴C]ethylene from [¹⁴C]methionine, but no [¹⁴C]ethylene is formed by Regnellidium, suggesting the existence of an alternative pathway of ethylene biosynthesis in the fern.Abbreviations IAA Indole-3-acetic acid - RBA 1,2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid     

The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.     

Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication?

Leaf area expansion is affected by environmental conditions because of differences in cell number and/or cell size. Increases in the DNA content (ploidy) of a cell by endoreduplication are related to its size. The aim of this work was to determine how cell ploidy interacts with the regulation of cell size and with leaf area expansion. The approach used was to grow Arabidopsis thaliana plants performing increased or decreased rounds of endoreduplication under shading and water deficit. The shading and water deficit treatments reduced final leaf area and cell number; however, cell area was increased and decreased, respectively. These differences in cell size were unrelated to alterations of the endocycle, which was reduced by these treatments. The genetic modification of the extent of endoreduplication altered leaf growth responses to shading and water deficit. An increase in the extent of endoreduplication in a leaf rendered it more sensitive to the shade treatment but less sensitive to water deficit conditions. The link between the control of whole organ and individual cell expansion under different environmental conditions was demonstrated by the correlation between the plasticity of cell size and the changes in the duration of leaf expansion.     

Hexokinases link DJ-1 to the PINK1/parkin pathway

Background

Hyperexcitability of neuronal networks can lead to excessive release of the excitatory neurotransmitter glutamate, which in turn can cause neuronal damage by overactivating NMDA-type glutamate receptors and related signaling pathways. This process (excitotoxicity) has been implicated in the pathogenesis of many neurological conditions, ranging from childhood epilepsies to stroke and neurodegenerative disorders such as Alzheimer’s disease (AD). Reducing neuronal levels of the microtubule-associated protein tau counteracts network hyperexcitability of diverse causes, but whether this strategy can also diminish downstream excitotoxicity is less clear.

Methods

We established a cell-based assay to quantify excitotoxicity in primary cultures of mouse hippocampal neurons and investigated the role of tau in exicitotoxicity by modulating neuronal tau expression through genetic ablation or transduction with lentiviral vectors expressing anti-tau shRNA or constructs encoding wildtype versus mutant mouse tau.

Results

We demonstrate that shRNA-mediated knockdown of tau reduces glutamate-induced, NMDA receptor-dependent Ca²⁺ influx and neurotoxicity in neurons from wildtype mice. Conversely, expression of wildtype mouse tau enhances Ca²⁺ influx and excitotoxicity in tau-deficient (Mapt ^−/−) neurons. Reconstituting tau expression in Mapt ^−/− neurons with mutant forms of tau reveals that the tau-related enhancement of Ca²⁺ influx and excitotoxicity depend on the phosphorylation of tau at tyrosine 18 (pY18), which is mediated by the tyrosine kinase Fyn. These effects are most evident at pathologically elevated concentrations of glutamate, do not involve GluN2B–containing NMDA receptors, and do not require binding of Fyn to tau’s major interacting PxxP motif or of tau to microtubules.

Conclusions

Although tau has been implicated in diverse neurological diseases, its most pathogenic forms remain to be defined. Our study suggests that reducing the formation or level of pY18-tau can counteract excitotoxicity by diminishing NMDA receptor-dependent Ca²⁺ influx.

     

A qualitative and quantitative study of the cellular fatty acids of 'Streptococcus milleri' with capillary gas chromatography

Fatty acid analysis was done with GC and GC-MS on 21 strains of 'Streptococcus milleri', representative of the various proposed species. Although no qualitative differences were found in the fatty acid profiles, discriminant analysis of the quantitative data revealed three groups. Streptococcus anginosus and Streptococcus constellatus were indistinguishable but separated from the other two groups which comprised Streptococcus intermedius, with a wide fermentation pattern and Streptococcus intermedius with a narrow fermentation pattern. Three of the strains could be distinguished from the others by a 'fingerprint' of a particularly prominent fatty acid peak. The results support the suggestion that there is more than one species in this group of organisms and that the technique might be of value in epidemiological investigations of 'S. milleri'.     

Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis

The etiological agent of whooping cough, Bordetella pertussis, destroys the ciliated epithelial cells lining the large airways of infected individuals. This cytopathology can be reproduced in respiratory epithelium by tracheal cytotoxin (TCT), a small peptidoglycan-related molecule purified from the culture supernatant of growing B. pertussis organisms. Using fast atom bombardment mass spectrometry, we analyzed the positive- and negative-ion spectra of the purified, biologically active material and assigned a mass of 921 daltons to TCT. Analysis of fragment ions in these spectra as well as the spectra of the methyl ester and acetylated derivatives of TCT unambiguously defined the primary structure of TCT as N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-gamma- glutamyldiaminopimelylalanine. TCT is therefore identical with the ciliostatic anhydropeptidoglycan monomer released by Neisseria gonorrhoeae and with the neurologically active slow-wave sleep-promoting factor FSu. These and other structurally related glycopeptides containing muramic acid thus form a family of molecules with remarkably diverse biological activities.