Distinguishing enzymes using metabolome data for the hybrid dynamic/static method

BACKGROUND: In the process of constructing a dynamic model of a metabolic pathway, a large number of parameters such as kinetic constants and initial metabolite concentrations are required. However, in many cases, experimental determination of these parameters is time-consuming. Therefore, for large-scale modelling, it is essential to develop a method that requires few experimental parameters. The hybrid dynamic/static (HDS) method is a combination of the conventional kinetic representation and metabolic flux analysis (MFA). Since no kinetic information is required in the static module, which consists of MFA, the HDS method may dramatically reduce the number of required parameters. However, no adequate method for developing a hybrid model from experimental data has been proposed. RESULTS: In this study, we develop a method for constructing hybrid models based on metabolome data. The method discriminates enzymes into static modules and dynamic modules using metabolite concentration time series data. Enzyme reaction rate time series were estimated from the metabolite concentration time series data and used to distinguish enzymes optimally for the dynamic and static modules. The method was applied to build hybrid models of two microbial central-carbon metabolism systems using simulation results from their dynamic models. CONCLUSION: A protocol to build a hybrid model using metabolome data and a minimal number of kinetic parameters has been developed. The proposed method was successfully applied to the strictly regulated central-carbon metabolism system, demonstrating the practical use of the HDS method, which is designed for computer modelling of metabolic systems

Conformational Epitopes of Pemphigus Antigens (Dsg1 and Dsg3) Are Calcium Dependent and Glycosylation Independent

The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the antigenicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculoproteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens

Isotopic evidence of plutonium release into the environment from the Fukushima DNPP accident

The Fukushima Daiichi nuclear power plant (DNPP) accident caused massive releases of radioactivity into the environment. The released highly volatile fission products, such as 129mTe, 131I, 134Cs, 136Cs and 137Cs were found to be widely distributed in Fukushima and its adjacent prefectures in eastern Japan. However, the release of non-volatile actinides, in particular, Pu isotopes remains uncertain almost one year after the accident. Here we report the isotopic evidence for the release of Pu into the atmosphere and deposition on the ground in northwest and south of the Fukushima DNPP in the 20–30 km zones. The high activity ratio of 241Pu/239+240Pu (> 100) from the Fukushima DNPP accident highlights the need for long-term 241Pu dose assessment, and the ingrowth of 241Am. The results are important for the estimation of reactor damage and have significant implication in the strategy of decontamination

Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks

Osteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo

Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.Uenaka M., Yamashita E., Kikuta J., et al. Osteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo. Nature Communications 13, 1066 (2022); https://doi.org/10.1038/s41467-022-28673-2

Impacts of Fukushima Nuclear Accident on Freshwater Environments

A large amount of radiocaesium (137Cs + 134Cs) was released into the atmosphere and diffused mainly in the Tohoku and Kanto regions after the TEPCO’s Fukushima Daiichi Nuclear Power Plant accident in March 2011. Fallout radiocaesium flows into rivers and lakes over time and have been taken up by living organisms. Although as of September 2019, more than 8 years have passed since the accident, freshwater fish contamination with radiocaesium is still serious issue in some area because shipping restrictions and restraint have continued. Internal radiation exposure through the consumption of contaminated fish is of the public concerns. Therefore, it is important to understand the current contamination state in freshwater fish and to predict the trend of the contamination in future. Freshwater fishes have been captured from September 2015 in Lake Inba, Chiba Prefecture. Activity concentrations of 137Cs in the muscle of funa remained constant after November 2016. The similar trends were found for catfish, snakehead, largemouth bass, bluegill and so on. In addition, effective half-life of 137Cs in muscle tissues of funa from Lake Inba was calculated about 9,900 days. These results suggest that the same concentration level of 137Cs in 2018 will be seen for those fishes in the future. It is still important to monitor 137Cs in freshwater fish to provide information for predicting future concentration changes