Age-Related Differences in Plasma Proteins: How Plasma Proteins Change from Neonates to Adults

The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.GE Healthcare Life Sciences Australia funded Sherif Tawfilis' time in the initial laboratory aspects of this project, some aspects of data analysis and preparation of the manuscript. This study was funded by internal Haematology Research Team funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Ultramarine, a chromoprotein acceptor for Forster resonance energy transfer

We have engineered a monomeric blue non-fluorescent chromoprotein called Ultramarine (fluorescence quantum yield, 0.001; epsilon (585) (nm), 64,000 M(-1). cm(-1)) for use as a Forster resonance energy transfer acceptor for a number of different donor fluorescent proteins. We show its use for monitoring activation of caspase 3 in live cells using fluorescence lifetime imaging. Ultramarine has the potential to increase the number of cellular parameters that can be imaged simultaneously

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

MEDiate: A diagnosis and assessment of the primary types of interacting hazards related to European areas : Deliverable D2.2

The MEDiate project aims to develop a decision-support system (DSS) for disaster-risk management that provides local authorities, businesses, and citizens with a comprehensive web-based platform to assess the risks posed by natural hazards. Within the project, Work Package 2 (WP2) is designed for the Assessment of current and future multi-hazard interactions and cascading impacts. It diagnoses the primary forms of interacting natural hazards across Europe, drawing examples from four specific testbed locations. Within WP2, the focus of this report, designated as deliverable 2.2 (D2.2), lies in analysing interactions between hydrological, meteorological, and climatological hazards, including mass movements. Specifically, D2.2 seeks to understand the mechanistic dependencies between interacting natural hazards, such as a storm inducing flooding leading to subsequent landslides. The report elucidates the interconnections between primary and associated hazards across various spatial and temporal scales. Furthermore, it provides projections regarding changes in hazard frequency and severity, grounded in the principal natural hazards identified within each testbed. The findings of this report serve as input data for Work Package 3 (WP3), which is tasked with developing a people-centred risk assessment for each testbed. This assessment is then integrated into the DSS portal to enhance local and regional disaster risk management as part of Work Package 4 (WP4)

Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells

The misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous publishedmethodswere adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate ofα-synuclein fibrillization and produces species that harbourenhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live nontransgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfoldedα-synucleincauses hyperactive mitochondrial respiration without causing any functional deficit.These datagive strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies

Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity

The consolidated European synthesis of CH4and N2O emissions for the European Union and United Kingdom : 1990-2017

Acknowledgements FAOSTAT statistics are produced and disseminated with the support of its member countries to the FAO regular budget. The views expressed in this publication are those of the author(s) and do not necessarily reflect the views or policies of FAO. We acknowledge the work of the entire EDGAR group (Marilena Muntean, Diego Guizzardi, Edwin Schaaf and Jos Olivier). Financial support Philippe Ciais received support of the European Research Council Synergy project SyG-2013-610028 IMBALANCE-P and from the ANR CLAND Convergence Institute. Prabir Patra received support of the Environment Research and Technology Development Fund (JPMEERF20172001, JPMEERF20182002) of the Environmental Restoration and Conservation Agency of Japan. David Basviken received support of the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 725546). David Bastviken was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 725546). Tuula Aalto received support from the Caroline Herschel Framework Partnership Agreement under the EU Horizon 2020 program (FPCUP, grant no. 809596) and Academy of Finland (SOMPA, grant no. 312932). Christine D. Groot Zwaaftink received support by the Norwegian Research Council (ICOS-Norway, project 245927). Joe McNorton received financial support from the Horizon2020 CHE project (776186).Peer reviewe