A method for traceable protein quantification using isotope dilution ICP-MS and its application on the tau protein

In dieser Arbeit wurde ein Verfahren zur rückführbaren Proteinquantifizierung unter Verwendung von Schwefelisotopenverdünnung mit induktiv gekoppelter Plasmamassenspektrometrie (ID-ICP-MS) entwickelt. Die Methode dient zur zuverlässigen Quantifizierung laborinterner Proteinstandards. Sie eignet sich nur zur Analyse reiner Proteine, deren Stöchiometrie bekannt ist, da die Proteinkonzentration aus dem Schwefelgehalt bestimmt wird. Nicht proteingebundener Schwefel wird durch Membranfiltration von der Proteinfraktion abgetrennt und mit ID-ICP-MS quantifiziert. Der Gesamtschwefelgehalt in der Probe wird ebenfalls mittels ID-ICP-MS quantifiziert und um die Menge an ungebundenem Schwefel korrigiert. Die Optimierung der Probenvorbereitung zeigte, dass ein Aufschluss die Unsicherheit des Ergebnisses verbessert. Für die Methodenentwicklung wurden ein zertifiziertes Rinderserumalbumin-Referenzmaterial (BSA), sowie ein kommerzielles Avidin verwendet. Die Bestimmung der Proteinmassenfraktionen und Unsicherheitsbudgets erfolgte nach Korrektur für den ungebundenen Schwefel (0,4 % für BSA, 30 % für Avidin). Die Methode wurde auf das Tau-Protein angewendet, das ein Biomarker für die sogenannten „Tauopathien“ ist – eine Gruppe neurodegenerativer Krankheiten, die die Alzheimer-Krankheit und die frontotemporale Demenz einschließen. Durch Verwendung eines isotopenangereicherten Spikes, der an das SI-System angebunden ist, wurde die metrologische Rückführbarkeit für den Massenanteil des Tau-Proteins erreicht. Dieser Tau-Standard wurde zur absoluten Quantifizierung toxischer transgener Tau-Spezies in der löslichen Hirnfraktion transgener Mäuse verwendet. Die entwickelte Methode ist für die rückführbare Quantifizierung von Proteinen zur Verwendung als Standards in der biologischen und medizinischen Forschung anwendbar. Sie zeigte eine ähnliche Präzision wie etablierte Verfahren, erforderte jedoch weniger Probenvorbereitung und keine spezies-spezifischen Standards.In this work, a method for traceable protein quantification using sulphur isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) was developed. The method is intended for the reliable quantification of in-house protein standards. It is only suited for pure protein formulations for proteins of known stoichiometry because the protein concentration is determined from the sulphur content. Non-protein bound sulphur is separated from the protein fraction by membrane filtration and is quantified by ID-ICP-MS. The total sulphur content in the sample is as well quantified by ID-ICP-MS and corrected for the amount of non-protein bound sulphur. Optimisation of the sample preparation showed that digestion improves the uncertainty of the result. For method development, a certified reference material bovine serum albumin (BSA) and a commercial avidin were used. The protein mass fractions with full uncertainty budgets were determined after correction for unbound sulphur. The developed procedure was applied to the tau protein, which is a biomarker fort he so-called „tauopathies“ – a group of neurodegenerative diseases including Alzheimer’s disease and Frontotemporal dementia. By employing an isotopically enriched spike, which is linked to the SI system, full metrological traceability was achieved for the tau mass fraction. The mass fraction of (0.328 ± 0.036) g kg-1 for tau was determined by ID-ICP-MS and confirmed by amino acid analysis. This tau standard was used for the absolute quantification of toxic transgenic tau species in the soluble brain fraction of transgenic mice. The developed method is applicable for the traceable quantification of proteins for use as standards in biological and medical research. The precision of the method was similar to established absolute protein quantification procedures while requiring less sample preparation and no species-specific standards

Differential compartmental processing and phosphorylation of pathogenic human tau and native mouse tau in the Line 66 model of frontotemporal dementia

Funding Information: Funding and additional information—This work was supported by EMPIR programme in Research Project 15HLT02 ReMiND cofinanced by the Participating States and the European Union's Horizon 2020 research and innovation programme (to N. L.). Work was also supported by WisTa Laboratories Ltd. (to V. M., D. L., M. M., C. R. H., G. R., C. M. W., F. T., and K. S.). Conflict of interest—This work was sponsored by WisTa Laboratories Ltd., an affiliate of TauRx Therapeutics Ltd. C. R. H. and C. M. W. are employees and officers of TauRx Therapeutics Ltd.Peer reviewedPublisher PD

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014–2021)

HBM4EU is co-financed under Horizon 2020 (grant agreement No 733032).As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants from three age groups: (i) 3,576 children aged 6-12 years, (ii) 3,117 teenagers aged 12-18 years, and (iii) 4,102 young adults aged 20-39 years. The participants were recruited between 2014 and 2021 in 11-12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, and benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs, and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with the highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European-wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability, and will give leverage to national policymakers for the implementation of targeted measures.info:eu-repo/semantics/publishedVersio

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

Harmonized human biomonitoring in European children, teenagers and adults : EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014–2021)

Funding Information: The authors would like to thank everybody who contributed to the HBM4EU Aligned Studies: the participating children, teenagers, adults and their families, the fieldworkers that collected the samples and database managers that made the information available to HBM4EU, the HBM4EU project partners, especially those from WP7 for developing all materials supporting the fieldwork, WP9 for organizing the QA/QC scheme under HBM4EU and all laboratories who performed the analytical measurements. We would like to acknowledge Sun Kyoung Jung from the National Institute of Environmental Research of South-Korea for providing the KoNEHS Cycle III results (crt adjusted). HBM4EU is co-financed under Horizon 2020 (grant agreement No 733032). The authors thank all principal investigators of the contributing studies for their participation and contribution to the HBM4EU Aligned Studies and the national program owners for their financial support. Further details on funding for all the participating studies can be found in the Supplemental Material, Table S12. Funding Information: The authors would like to thank everybody who contributed to the HBM4EU Aligned Studies: the participating children, teenagers, adults and their families, the fieldworkers that collected the samples and database managers that made the information available to HBM4EU, the HBM4EU project partners, especially those from WP7 for developing all materials supporting the fieldwork, WP9 for organizing the QA/QC scheme under HBM4EU and all laboratories who performed the analytical measurements. We would like to acknowledge Sun Kyoung Jung from the National Institute of Environmental Research of South-Korea for providing the KoNEHS Cycle III results (crt adjusted). HBM4EU is co-financed under Horizon 2020 (grant agreement No 733032). The authors thank all principal investigators of the contributing studies for their participation and contribution to the HBM4EU Aligned Studies and the national program owners for their financial support. Further details on funding for all the participating studies can be found in the Supplemental Material, Table S12 . Publisher Copyright: © 2023 The AuthorsAs one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6–12 years, (ii) 3,117 teenagers aged 12–18 years and (iii) 4,102 young adults aged 20–39 years. The participants were recruited between 2014 and 2021 in 11–12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures.Peer reviewe

Generalized epilepsy and myoclonic seizures in 22q11.2 deletion syndrome

Prompted by the observations of juvenile myoclonic epilepsy (JME) in 22q11.2 deletion syndrome (22q11DS) and recurrent copy number variants in genetic generalized epilepsy (GGE), we searched for further evidence supporting a possible correlation of 22q11DS with GGE and with myoclonic seizures. Through routine diagnostics, we identified 3 novel individuals with the seemingly uncommon combination of 22q11DS and JME. We subsequently screened the literature for reports focussing on the epilepsy phenotype in 22q11DS. We additionally screened a database of 173 22q11DS patients and identified a fourth individual with JME as well as 2 additional cases with GGE. We describe 6 novel and 22 published cases with co-occurrence of 22q11DS and GGE. In many patients, GGE was associated with myoclonic seizures allowing for a diagnosis of JME in at least 6 individuals. Seventeen of the 173 22q11DS cases (10%) had a diagnosis of either focal or generalized epilepsy. In these cases, focal epilepsy could often be attributed to syndrome-associated hypocalcaemia, cerebral bleeds, or structural brain anomalies. However, the cause of GGE remained unclear. In this study, we describe and review 28 individuals with 22q11DS and GGE (especially JME), showing that both disorders frequently co-occur. Compared to the reported prevalence of 15-21%, in our case series only 10% of 22q11DS individuals were found to have epilepsy, often GGE. Since 22q11.2 does not contain convincing GGE candidate genes, we discuss the possibility of an aetiological correlation through a possibly disturbed interaction with the GABAB receptor

Multiply Intercalator-Substituted Cu(II) Cyclen Complexes as DNA Condensers and DNA/RNA Synthesis Inhibitors

Many drugs that are applied in anticancer therapy such as the anthracycline doxorubicin contain DNA-intercalating 9,10-anthraquinone (AQ) moieties. When Cu­(II) cyclen complexes were functionalized with up to three (2-anthraquinonyl)­methyl substituents, they efficiently inhibited DNA and RNA synthesis resulting in high cytotoxicity (selective for cancer cells) accompanied by DNA condensation/aggregation phenomena. Molecular modeling suggests an unusual bisintercalation mode with only one base pair between the two AQ moieties and the metal complex as a linker. A regioisomer, in which the AQ moieties point in directions unfavorable for such an interaction, had a much weaker biological activity. The ligands alone and corresponding Zn­(II) complexes (used as redox inert control compounds) also exhibited lower activity

Tubby regulates microglial phagocytosis through MerTK

Immunologically-silent microglial phagocytosis of apoptotic cells and cellular debris is critical for CNS homeostasis and innate immune balance. The beneficial and detrimental effects of microglial phagocytosis on neurons remain controversial. Phagocytosis ligands are the key to selecting extracellular cargos, initiating the engulfment process, defining phagocyte functional roles and regulating phagocyte activities with therapeutic potentials. Here we characterized tubby as a new ligand to regulate microglial phagocytosis through MerTK receptor, which is well known for its immunosuppressive signaling. Tubby at 0.1 nM significantly induced microglial phagocytosis of apoptotic cells with a maximal activity at 10 nM. Tubby activated MerTK with receptor autophosphorylation in a similar dose range. Excessive soluble MerTK extracellular domain blocked tubby-mediated microglial phagocytosis of plasma membrane vesicles as cellular debris. Immunocytochemistry revealed that the ingested cargos were co-localized with MerTK-dependent non-muscle myosin II, whose rearrangement is necessary for cargo engulfment. Phagosome biomarker Rab7 was colocalized with cargos, suggesting that internalized cargos were targeted to phagocytic pathway. Tubby stimulated phagocytosis by neonatal and aged microglia with similar activities, but not by MerTK(−/−) microglia. These results suggest that tubby is a ligand to facilitate microglial phagocytosis through MerTK for the maintenance of CNS homeostasis