Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.     

Viability of a marine microbial biofilm

The formation of a microbial biofilm on glass surfaces arranged in lamellar piles parallel with circulating sea water (3 cm·sec^–1) was studied. The increase in dry weight, protein content, nucleotide content (ATP, ADP), and diatoms was followed over a period of 62 days. Dry weight and protein were estimates of the total biofilm development, whereas the nucleotide measurements revealed the viability of the biofilm and reflected the dynamics in the community structure.     

GROWTH AND ORGANIC OSMOLYTES OF GEOGRAPHICALLY DIFFERENT ISOLATES OF MICROCOLEUS CHTHONOPLASTES (CYANOBACTERIA) FROM BENTHIC MICROBIAL MATS:RESPONSE TO SALINITY CHANGE1

The comparative growth and osmotic acclimation often culture strains of the marine benthic cyanobacterium Microcoleus chthonoplastes Thuret isolated from microbial mats in Germany, Spain, Egypt, the United States, Mexico, Chile, and Australia were investigated in salinities ranging from freshwater to twice seawater. All isolates showed a broad growth versus salinity response consistent with the dominance of this species in intertidal and hypersaline microbial communities. Growth optima, salinity preferences, and maximum growth rates differed for each isolate and could be related to the habitat from which they were isolated. This is most obvious when comparing strains from brackish habitats with those from a hypersaline lake. While the former isolates exhibited sharply pronounced growth optima under hyposaline conditions, cultures from the hypersaline environment grew best in salinity more than double seawater. The major low-molecular weight organic compounds present in all M. chthonoplastes strains were the carbohydrates glycosylglycerol and trehalose. This was proven by using ¹³C-nuclear magnetic resonance spectroscopy. Glycosylglycerol was synthesized and accumulated with increasing salinities, indicating its role as an osmolyte. In contrast, trehalose was present in relatively high concentrations under hyposaline conditions only. Differences in the patterns of growth versus salinity, as well as in those of osmotic acclimation among the M. chthonoplastes isolates, point to the development of different physiological ecotypes within the species.     

Desert breath—How fog promotes a novel type of soil biocenosis,forming the coastal Atacama Desert’s living skin

The Atacama Desert is the driest non‐polar desert on Earth, presenting precarious conditions for biological activity. In the arid coastal belt, life is restricted to areas with fog events that cause almost daily wet–dry cycles. In such an area, we discovered a hitherto unknown and unique ground covering biocenosis dominated by lichens, fungi, and algae attached to grit‐sized (~6 mm) quartz and granitoid stones. Comparable biocenosis forming a kind of a layer on top of soil and rock surfaces in general is summarized as cryptogamic ground covers (CGC) in literature. In contrast to known CGC from arid environments to which frequent cyclic wetting events are lethal, in the Atacama Desert every fog event is answered by photosynthetic activity of the soil community and thus considered as the desert's breath. Photosynthesis of the new CGC type is activated by the lowest amount of water known for such a community worldwide thus enabling the unique biocenosis to fulfill a variety of ecosystem services. In a considerable portion of the coastal Atacama Desert, it protects the soil from sporadically occurring splash erosion and contributes to the accumulation of soil carbon and nitrogen as well as soil formation through bio‐weathering. The structure and function of the new CGC type are discussed, and we suggest the name grit–crust. We conclude that this type of CGC can be expected in all non‐polar fog deserts of the world and may resemble the cryptogam communities that shaped ancient Earth. It may thus represent a relevant player in current and ancient biogeochemical cycling.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

MicroRNA-138 Regulates Hypoxia-Induced Endothelial Cell Dysfunction By Targeting S100A1

The Ca²⁺ sensor S100A1 is essential for proper endothelial cell (EC) nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown. Here we show that ECs transfected with a S100A1–3′ untranslated region (UTR) luciferase reporter construct display significantly reduced gene expression when subjected to low oxygen levels or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (MiR-138) could target the 3′UTR of S100A1. Patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased MiR-138 levels and decreased S100A1 protein expression. Consistent with this finding, hypoxia greatly increased MiR-138 levels in ECs, but not in skeletal muscle C2C12 myoblasts or differentiated myotubes or primary human vascular smooth muscle cells. Transfection of a MiR-138 mimic into ECs reduced S100A1–3 ‘UTR reporter gene expression, while transfection of an anti MiR-138 prevented the hypoxia-induced downregulation of the reporter gene. Deletion of the 22 nucleotide putative MiR-138 target site abolished the hypoxia-induced loss of reporter gene expression. Knockdown of Hif1-α mediated by siRNA prevented loss of hypoxia-induced reporter gene expression. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression even in the absence of hypoxia. Finally, primary ECs transfected with a MiR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. We conclude that hypoxia-induced MiR-138 is an essential mediator of EC dysfunction via its ability to target the 3′UTR of S100A1.     

Generative FDG-PET and MRI Model of Aging and Disease Progression in Alzheimer's Disease

The failure of current strategies to provide an explanation for controversial findings on the pattern of pathophysiological changes in Alzheimer''s Disease (AD) motivates the necessity to develop new integrative approaches based on multi-modal neuroimaging data that captures various aspects of disease pathology. Previous studies using [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) and structural magnetic resonance imaging (sMRI) report controversial results about time-line, spatial extent and magnitude of glucose hypometabolism and atrophy in AD that depend on clinical and demographic characteristics of the studied populations. Here, we provide and validate at a group level a generative anatomical model of glucose hypo-metabolism and atrophy progression in AD based on FDG-PET and sMRI data of 80 patients and 79 healthy controls to describe expected age and symptom severity related changes in AD relative to a baseline provided by healthy aging. We demonstrate a high level of anatomical accuracy for both modalities yielding strongly age- and symptom-severity- dependant glucose hypometabolism in temporal, parietal and precuneal regions and a more extensive network of atrophy in hippocampal, temporal, parietal, occipital and posterior caudate regions. The model suggests greater and more consistent changes in FDG-PET compared to sMRI at earlier and the inversion of this pattern at more advanced AD stages. Our model describes, integrates and predicts characteristic patterns of AD related pathology, uncontaminated by normal age effects, derived from multi-modal data. It further provides an integrative explanation for findings suggesting a dissociation between early- and late-onset AD. The generative model offers a basis for further development of individualized biomarkers allowing accurate early diagnosis and treatment evaluation.