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Developing HaloTag and SNAP-Tag Chemical Inducers of Dimerization to Probe Receptor Oligomerization and Downstream Signaling
Get PDFControlling protein–protein interactions is critical for dissecting signaling pathways, especially those initiated by ligand-receptor interactions, which alter receptor oligomerization and drive downstream signaling cascades. Traditional methods for driving protein–protein complexes use antibodies that face limitations in terms of stoichiometry, geometric rigidity, and antibody specificity. Chemical inducers of dimerization (CIDs) for fusion proteins such as HaloTag (Halo) and SNAP-Tags (SNAP) offer precise and covalent control of protein proximities, overcoming limitations of antibody-dependent methods. In this study, we expand the toolkit of Halo and SNAP CIDs with (1) benzylguanine (BG) and HaloTag ligand (HTL) crosslinkers featuring varying polyethylene glycol linker lengths and update this kit with (2) a FRET-based dimerizing sensor to induce and verify protein proximity. Here we establish our CIDs on extracellularly Halo- and SNAP-tagged TGFβ, BMP, neurotrophic factor, and metabotropic glutamate receptors, thereby elucidating the signaling potential of ligand-independent dimerization in a heteromeric fashion
Differential Diagnoses in Critically Ill Adults with Hyperferritinemia
Get PDFHintergrund: Hyperferritinämie ist bei kritisch kranken Patienten häufig zu beobachten. Da bisher wenig über die zugrunde liegenden Ursachen bekannt war, zielten die vorliegenden Analysen darauf ab, Patienten mit Hyperferritinämie zu charakterisieren und Faktoren zu identifizieren, die neben der Hämophagozytischen Lymphohistiozytose (HLH) zur Hyperferritinämie führen. Zur sicheren Unterscheidung der HLH zielt die Analyse dar-über hinaus auf die Validierung der Diagnose-Scores bei kritisch kranken Erwachsenen ab. Das übergeordnete Ziel ist somit die Verbesserung der Differentialdiagnostik in dieser Patientengruppe.
Methodik: Die vorliegenden Studien sind Primär- und Sekundäranalysen einer retrospektiven Observationsstudie, die erwachsene Patienten von Januar 2006 bis August 2018 einschloss, die auf einer der Intensivstationen der Charité ─ Universitätsmedizin Berlin mindestens eine Ferritinmessung ≥ 500 μg/l hatten. Nicht-HLH-Patienten wurden nach dem Auftreten einer Sepsis bzw. eines septischen Schocks, Immunsuppression und 17 weiteren Erkrankungsgruppen klassifiziert. Es wurden Gruppenvergleiche durchgeführt und ROC-Analysen angefertigt zur Identifikation des Ferritinwertes, der am besten zwischen HLH und anderen Diagnosen unterscheiden kann. Es wurden uni-/multivariable lineare Regressionsanalysen zu Untersuchung der Differentialdiagnosen sowie ROC-Analysen zur Validierung von HLH-2004-Kriterien und HScore bei kritisch kranken Patienten durchgeführt.
Ergebnisse: 2623 Patienten mit Hyperferritinämie, davon 40 Patienten mit HLH, wurden analysiert. Ein Ferritingrenzwert von 9083 μg/l eignet sich mit einer Sensitivität von 92,5% und einer Spezifität von 91,9% als Biomarker für HLH. Die multivariable lineare Regression zeigte jeweils positive Assoziationen für den maximalen Sequential Organ Failure Assessment (SOFA)-Score, Sepsis bzw. septischen Schock, Lebererkrankung und hämatologisches Malignom mit dem maximalen Ferritinwert. Zur sicheren Unterscheidung von HLH und deren Differentialdiagnosen konnten HLH-2004-Kriterien und HScore auch für kritisch kranke Patienten validiert werden. Vier von acht erfüllten Kriterien und ein HScore von 168 eignen sich zur Diagnosestellung einer HLH.
Schlussfolgerung: Hyperferritinämie ist ein geeigneter Screeningparameter für HLH. Neben der HLH müssen Sepsis bzw. septischer Schock, Lebererkrankung und hämatologische Malignome als wichtigste Differentialdiagnosen bei kritisch kranken Patienten mit Hyperferritinämie bedacht werden. Da diese Erkrankungen jeweils auch in Kombination
mit HLH auftreten können, ist es bei diesen Patienten besonders wichtig, die HLH-2004-Kriterien anzuwenden. Im Anschluss an einen HLH-Ausschluss sollte eine weiterführende Diagnostik in Hinblick auf Sepsis bzw. septischen Schock, Lebererkrankung sowie hämatologische Malignome erfolgen. Diese Erkrankungen bilden das Quartett der Hyperferritinämie und erfordern aufgrund der hohen Letalität dieser Patienten eine schnelle diagnostische Abklärung.Background: Hyperferritinemia is frequently seen in critically ill patients. Nonetheless, underlying conditions of higher ferritin levels remain unclear. Hence, the study aims for characterisation of patients with hyperferritinemia, for identification of underlying conditions of hyperferritinemia besides HLH and for validation of HLH-2004 criteria and HScore in critically ill patients. The overall aim is the improvement of differential diagnostics.
Methods: The primary and two secondary analyses of a retrospective observational study which was conducted at Charité ─ Universitätsmedizin Berlin contained adult critically ill patients, who had at least one ferritin measurement ≥ 500 μg/l in one of the medical or surgical intensive care units between January 2006 and August 2018. Non HLH patients were classified into groups with sepsis or septic shock and 17 other disease groups and pre-existing immunosuppression. Groups were compared, ROC analyses were performed to identify the ferritin value best predictive for HLH, uni- and multivariable linear regression analyses were performed to investigate differential diagnoses and ROC analyses were conducted for validation of HLH-2004 criteria and HScore in the critically ill.
Results: 2623 patients with hyperferritinemia were included in the analyses, 40 of them had HLH. Ferritin values of 9083 μg/l were best predictive for HLH with sensitivity of 92,5% and specificity of 91,9%. Univariable linear regression analysis preselected factors for the following multivariable linear regression analysis in order to identify differential diagnoses besides HLH. The multivariable analysis revealed positive associations of maximum SOFA score, sepsis, septic shock, liver disease (except hepatitis), and hematological malignancy with maximum ferritin levels. HLH-2004 criteria and HScore could be validated for a safe differentiation of HLH in critically ill. Four out of eight criteria and HScore of 168 are suitable for HLH diagnosis.
Conclusion: Hyperferritinemia can be used as a biomarker for HLH. Sepsis, septic shock, liver disease and hematological malignancy are the main drivers of hyperferritinemia in critically ill patients besides HLH. As these conditions also occur in conjunction with HLH, these patients need special consideration and the application of HLH-2004 criteria. The exclusion of HLH should be followed by a further diagnostic workup in terms of sepsis or septic shock, liver disease and hematological malignancy. These four diagnoses combine into the quartet of hyperferritinemia and require prompt evaluation because of increased mortality in patients with hyperferritinemia
Global patterns of nutrient limitation in soil microorganisms
Get PDFThe availability of nitrogen (N) and phosphorus (P) is essential for soil microbial activity and growth, yet global patterns of N and P limitation in soil microbial metabolism remain largely unknown. We modeled ecoenzyme stoichiometry data from 5,259 field observations of natural ecosystems to assess microbial N and P limitation in global surface soils. We found that microbial P limitation, which was especially strong at low latitudes, was more prevalent globally than microbial N limitation, which prevailed in cold environments. We also found widespread N and P colimitation in soil microorganisms in the tropics, contradicting the long-held paradigm that P, and not N, is the primary limiting nutrient at low latitudes. This colimitation could be attributable to elevated microbial N demand for the synthesis of P-acquiring enzymes under P limitation. Upscaling (0.1 × 0.1° spatial resolution) suggested that soil microorganisms were limited by N and P in 39% and 57%, respectively, of natural terrestrial surface areas, with 21% of areas with N and P colimitation. As a global assessment of spatial variation in microbial N and P limitation, our results highlight the importance of N availability in supporting microbial P acquisition at low latitudes and improve our understanding of microbial nutrient limitation on a global scale
Critical spin models from holographic disorder
Get PDFDiscrete models of holographic dualities, typically modeled by tensor networks on hyperbolic tilings, produce quantum states with a characteristic quasiperiodic disorder not present in continuum holography. In this work, we study the behavior of XXZ spin chains with such symmetries, showing that lessons learned from previous non-interacting (matchgate) tensor networks generalize to more generic Hamiltonians under holographic disorder: While the disorder breaks translation invariance, site-averaged correlations and entanglement of the disorder-free critical phase are preserved at a plateau of nonzero disorder even at large system sizes. In particular, we show numerically that the entanglement entropy curves in this disordered phase follow the expected scaling of a conformal field theory (CFT) in the continuum limit. This property is shown to be non-generic for other types of quasiperiodic disorder, only appearing when our boundary disorder ansatz is described by a "dual" bulk hyperbolic tiling. Our results therefore suggest the existence of a whole class of critical phases whose symmetries are derived from models of discrete holography
Applying an Anti-Kasha Model Resolves Differences Between Photosynthetic and Artificial Pigments
No full textDataset required for reproducing the title paper; containing the scripts and data for exciton analysis, as well as the structures used to computed the FRET coupling element
Death and destruction in Dayvd-Haradok. German crimes, local complicity in the Holocaust, and survivors’ search for justice in a former Polesian shtetl
Get PDFThis article provides a microhistory of Davyd-Haradok during and after the Second World War. Within post-1945 Soviet Belarus, the case of Davyd-Haradok in August 1941 constituted one of the most extreme examples of local participation in the Holocaust, displaying a brutality and intimacy of violence previously known mostly from western Ukraine and the Białystok region. This article sheds light on this little-known history. By situating it in a larger context, in particular the 1941 wave of pogroms in the East European borderlands, it contributes to studies that examine variations in local complicity in German atrocities across time and space
The expression of information structure in Bantu
Get PDFSynopsis:
The Bantu language family is spread over a large area of Africa, stretching from Cameroon to Kenya to South Africa, and comprises an estimated 555 languages. The languages show a large amount of small-scale variation while at the same time forming part of one relatively uniform family within Niger-Congo. Interestingly, the morphosyntax of these languages has been observed to be heavily influenced by information structure. Studying the expression of information structure in Bantu is therefore of great importance not only for developing cognitive models of the role of information structure in language, but also for understanding the basic grammatical structure of the Bantu languages themselves. Before modelling the interaction between syntax and information structure in Bantu, however, a thorough empirical description of the expression of information structure in Bantu should be available. That description is what this book aims to provide.
This book follows from a systematic investigation of information structure in the languages of the BaSIS research project (Bantu Syntax and Information Structure). The data come from original field research conducted using the BaSIS methodology, which was specifically developed to investigate the expression of information structure in Bantu. The book contains a comprehensive introduction chapter which explains the main terms and issues in the field of information structure, the methodology employed in the project, and common structures which characterise topic and focus expression in Bantu. The introduction is then followed by eight chapters which each give detailed descriptive overviews of the expression of information structure in a different Bantu language, namely Tunen (Guthrie classification A44, Cameroon), Teke-Kukuya (B77, Congo), Kîîtharaka (E54, Kenya), Kirundi (JD62, Burundi), Rukiga (JE14, Uganda), Kinyakyusa (M31, Tanzania), Makhuwa-Enahara (P31, Mozambique), and Cicopi (S61, Mozambique).
Taken together, the book provides detailed information on the expression of information structure in the Bantu family. It is intended both to inform future theoretical work and to provide a methodology and model for the investigation of information structure that can be used in studies of other languages
Toxicity study of TEoS-DAZA, a chemical precursor for functional liver imaging with PET/CT
Get PDFBackground
N,1,4-Tri(4-ethoxy-2-hydroxybenzyl)-1,4-diazepan-6-amine (TEoS-DAZA), a novel radiopharmaceutical precursor for a liver-specific 68Ga-based diagnostic radiopharmaceutical, was tested for toxicity in rats to ensure its safe applicability and to fulfil the preclinical requirements in preparation of a clinical study. The study was performed according to EMA draft Guideline on the non-clinical requirements for radiopharmaceuticals, as well as to the so-called microdosing approach of the ICH guideline M3 (R2).
Results
This randomized study was conducted using Wistar rats. The test item was administered intravenously at three different dose levels, the vehicle solution was administered to a separate group as control. Toxicity assessment included a 24 h observation period in three dose groups, and a 14-day recovery period in the high dose group. Animals were monitored regarding clinical behaviour, bodyweight, food and water consumption, additionally undergoing modified IRWIN, grip-strength and beam-walking tests. Following euthanisation, extensive haematological and clinical biochemical parameters were analysed. Necropsy and histopathology were performed. There was no evidence to any test-item related adversities at any dose level. No delayed effects were identified in any animal at the end of the recovery phase. Some small, albeit significant changes in haematology and clinical biochemistry could not be related to the test item administration. The NOAEL of TEoS-DAZA was determined at 1.4 mg/kg bodyweight.
Conclusions
Administration of a thousandfold clinical dose of TEoS-DAZA in rats did not cause any observable adverse events. An injectable solution of [68Ga]Ga-TEoS-DAZA containing 100 µg of the precursor is safe for clinical application to humans from the pharmacological point of view. Subsequent dosimetry studies need to be undertaken to reveal any radiation related toxicity
Cyclo-P5− Revisited: The Surprisingly Stable Uncoordinated Pentaphospholide Anion
Get PDFThe addition of [2.2.2]cryptand to alkali metal heptaphosphides M3P7 (M = Na, K) leads to the formation of the salts [M([2.2.2]cryptand)][cyclo-P5]. Although the cyclo-P5− anion is spectroscopically known since the 1980s it was so far prepared and handled only in solution. By complexing the alkali metal cation with [2.2.2]cryptand, the product was found to be surprisingly stable in the solid state. Cyclo-P5−, which is isolobal to the well-known cyclopentadienide anion, was now characterized crystallographically for the first time. The structural elucidation proves its planar D5h symmetry, not only as ligand in different sandwich complexes but also in its uncoordinated form. Cyclo-P5− was further characterized by UV-vis spectroscopy in solution and in the solid-state by Raman and 31P MAS NMR spectroscopy. Accompanying DFT studies were performed to support the experimental results