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Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins
(2018)
With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the "labeling barrier" and to bypass photobleaching in multi-plane, whole-cell 3D experiments.
Inflammatory activation of astroglia adds to the pathology of various neurological diseases. Astrocytes respond to microglia-derived cytokines such as interleukin-1α (IL-1α) with enhanced inflammatory signaling. This provokes pro-inflammatory gene expression of, among others, the eicosanoid-generating enzyme prostaglandin endoperoxide synthase 2 (Ptgs2). Whereas metabolic regulation of innate immune cell inflammatory responses is intensely studied, pathways related to how metabolism modulates inflammatory signaling in astrocytes are underexplored. Here, we examined how mitochondrial oxidative phosphorylation affects inflammatory responses towards IL-1α and tumor necrosis factor α in neonatal rat astrocytes. Blocking respiratory complex I and III or adenosine triphosphate (ATP) synthase did not affect activation of inflammatory signaling by IL-1α, but did elicit differential effects on inflammatory gene mRNA expression. Remarkably, mRNA and protein expression of Ptgs2 by IL-1α was consistently up-regulated when oxidative phosphorylation was inhibited. The increase of Ptgs2 resulted from mRNA stabilization. Mitochondrial inhibitors also increased IL-1α-triggered secretion of eicosanoids, such as prostaglandin E2, prostaglandin F2α, and 6-keto-prostaglandin F1α, as assessed by liquid chromatography/mass spectrometry. Mechanistically, attenuating oxidative phosphorylation elevated adenosine monophosphate (AMP) and activated AMP-activated protein kinase (AMPK). AMPK silencing prevented Ptgs2 up-regulation by mitochondrial inhibitors, while AMPK activators recapitulated Ptgs2 mRNA stability regulation. Our data indicate modulation of astrocyte inflammatory responses by oxidative metabolism, with relevance towards eicosanoid production.
Lipoxygenases (LOXs) catalyze the stereo-specific peroxidation of polyunsaturated fatty acids (PUFAs) to their corresponding hydroperoxy derivatives. Human macrophages express two arachidonic acid (AA) 15-lipoxygenating enzymes classified as ALOX15 and ALOX15B. ALOX15, which was first described in 1975, has been extensively characterized and its biological functions have been investigated in a number of cellular systems and animal models. In macrophages, ALOX15 functions to generate specific phospholipid (PL) oxidation products crucial for orchestrating the nonimmunogenic removal of apoptotic cells (ACs) as well as synthesizing precursor lipids required for production of specialized pro-resolving mediators (SPMs) that facilitate inflammation resolution. The discovery of ALOX15B in 1997 was followed by comprehensive analyses of its structural properties and reaction specificities with PUFA substrates. Although its enzymatic properties are well described, the biological functions of ALOX15B are not fully understood. In contrast to ALOX15 whose expression in human monocyte-derived macrophages is strictly dependent on Th2 cytokines IL-4 and IL-13, ALOX15B is constitutively expressed. This review aims to summarize the current knowledge on the regulation and functions of ALOX15 and ALOX15B in human macrophages.
Myeloid-specific deletion of the AMPK2 subunit alters monocyte protein expression and atherogenesis
(2019)
The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.
Debatten über den relativen Beitrag von Konfessionalisierung und Säkularisierung zum Werden der modernen Welt bleiben von der Frage bestimmt, in welchem Verhältnis die theologischen Debatten der Reformation und die Gestalt der entstehenden Konfessionskirchen zu unseren Vorstellungen der Moderne stehen. Dabei muss berücksichtigt werden, dass Reformation und Konfessionalisierung den Glaubensgegnern, und dann auch protestantischen Juristen in Konflikten mit ihren eigenen Konfessionskirchen, Argumente zur Verteidigung ihrer Positionen aufzwangen, die sich nach wie vor auf Grundpositionen und zentrale Autoritäten der eigenen Glaubensüberzeugung beriefen, aber der Sache nach tiefgreifende Transformationen im Verhältnis von Glauben, Gesellschaft und Bürger begründeten. Solche Argumente wurzelten in Reformation und Konfessionalisierung nicht allein deswegen, weil sie ohne diese nie entwickelt worden wären, sondern legten durch ihre Berufung beispielsweise auf Luther auch die Entwicklungspotentiale der Reformation frei, so wenig Ziele und Wünsche der Reformatoren umstandslos mit denen der sich auf sie berufenden Späteren gleichgesetzt werden können. Die frühe Neuzeit darf denn auch nicht in eine mehr dem Mittelalter angehörende frühere Epoche – etwa bis zum Westfälischen Frieden – und eine schließlich in die Aufklärung und den modernen Staat mündende Epoche des späteren 17. und des 18. Jahrhunderts zweigeteilt werden. ...
In den großen Linien ist die Geschichte, die Christopher Brooks erzählt, bekannt. Es sind die Details, das Hinabtauchen in unzählige bislang unberücksichtigte Manuskripte und damit vor allem die Berücksichtigung zahlreicher Akteure in den Grafschaften, Städten und selbst Dörfern, die Reiz, Wert und besonderes Schwergewicht seiner Darstellung ausmachen – und ihm eine erstaunliche Pointe der Argumentation ermöglichen. ...
Martin Heckels monumentale Kompilation kennt – soweit ich sehe – kein vergleichbares Werk, weder zum Thema noch dem Genre nach. Auf nicht ganz tausend Seiten – rund 170 davon eine inhaltlich strukturierte Literaturübersicht – verhandelt der Autor in großer Quellennähe, beeindruckender analytischer Durchdringung und präziser Diktion die drei großen Sachbereiche, welche der Untertitel nennt: die "Entwicklung der Theologie Luthers", die "Auswirkung auf das Recht", die Rahmenbedingungen Reichsreform und Territorialstaatsbildung, und die Auseinandersetzung mit "Rom und den Schwärmern". ...
It was seventeen years ago when the first same-sex marriage was celebrated in a civil ceremony in Amsterdam, right after the Dutch Parliament passed legislation that legalized same-sex marriages. Since then, same-sex marriage has become legal in over two dozen countries worldwide. Last year, the German Bundestag added Germany to the growing list of countries where same-sex couples can obtain a legal marriage license. The past decades have indeed witnessed social mobilizations around the globe for LGBTI+ rights. Whether through legislation, court rulings, or popular referenda, 25 countries grant full juridical marital recognitions only recently enjoyed by opposite-sex partners to all citizens, regardless of their gender and sexual preferences. However, this legal evolution has been uneven. Currently, in many countries, LGBTI+ relations not only contravene moral codes but are still punishable crimes with varying amounts of prison time, fines, and in a few cases, with the death penalty. ...
Los últimos años han sido testigos de un creciente interés por el derecho indiano desde el mundo académico anglosajón. Varios autores han tocado distintos aspectos de la construcción de un imperio legal en la América española (véase por ejemplo los trabajos de Woodrow Borah, Charles Cutter, Tamar Herzog, Susan Kellog, Brian P. Owensby y Yanna Yanakakis), buscando comprender las complejidades detrás de la formación de un régimen normativo colonial cimentado sobre la traslación de sistemas jurídicos europeos, un aparato de prácticas locales y la efervescencia de normatividades contingentes al proceso colonial. En esta dirección se han estudiado las instituciones jurídicas de la América española, los agentes jurídicos coloniales, y más recientemente, los actores indígenas, estos últimos no solo como usuarios del sistema legal, sino también como contribuyentes netos a la vertebración del nuevo orden normativo en la América española. Estos estudios aducen que en estos territorios el derecho castellano se adaptó a los nuevos escenarios coloniales en parte por la coexistencia de diferentes niveles normativos basados en los usos y costumbres, las compilaciones legales y el ius commune, además de códigos normativos que emanaban de las autoridades religiosas. Este último aspecto, sobre todo lo relacionado con el derecho canónico y la teología moral, sólo recientemente ha sido objeto de estudio como parte intrínseca de las multinormatividades que enmarcaban las vidas diarias de las personas en el periodo moderno. Por todo esto el nuevo libro del profesor Osvaldo F. Pardo, Honor and Personhood in Early Modern Mexico, un gran conocedor de los procesos culturales ocurridos en el México del siglo XVI, resulta muy bienvenido. ...
uORF-tools—workflow for the determination of translation-regulatory upstream open reading frames
(2019)
Ribosome profiling (ribo-seq) provides a means to analyze active translation by determining ribosome occupancy in a transcriptome-wide manner. The vast majority of ribosome protected fragments (RPFs) resides within the protein-coding sequence of mRNAs. However, commonly reads are also found within the transcript leader sequence (TLS) (aka 5’ untranslated region) preceding the main open reading frame (ORF), indicating the translation of regulatory upstream ORFs (uORFs). Here, we present a workflow for the identification of translation-regulatory uORFs. Specifically, uORF-Tools uses Ribo-TISH to identify uORFs within a given dataset and generates a uORF annotation file. In addition, a comprehensive human uORF annotation file, based on 35 ribo-seq files, is provided, which can serve as an alternative input file for the workflow. To assess the translation-regulatory activity of the uORFs, stimulus-induced changes in the ratio of the RPFs residing in the main ORFs relative to those found in the associated uORFs are determined. The resulting output file allows for the easy identification of candidate uORFs, which have translation-inhibitory effects on their associated main ORFs. uORF-Tools is available as a free and open Snakemake workflow at https://github.com/Biochemistry1-FFM/uORF-Tools. It is easily installed and all necessary tools are provided in a version-controlled manner, which also ensures lasting usability. uORF-Tools is designed for intuitive use and requires only limited computing times and resources.
Macrophage S1PR1 signaling alters angiogenesis and lymphangiogenesis during skin inflammation
(2019)
The bioactive lipid sphingosine-1-phosphate (S1P), along with its receptors, modulates lymphocyte trafficking and immune responses to regulate skin inflammation. Macrophages are important in the pathogenesis of psoriasiform skin inflammation and express various S1P receptors. How they respond to S1P in skin inflammation remains unknown. We show that myeloid specific S1P receptor 1 (S1PR1) deletion enhances early inflammation in a mouse model of imiquimod-induced psoriasis, without altering the immune cell infiltrate. Mechanistically, myeloid S1PR1 deletion altered the formation of IL-1β, VEGF-A, and VEGF-C, and their receptors’ expression in psoriatic skin, which subsequently lead to reciprocal regulation of neoangiogenesis and neolymphangiogenesis. Experimental findings were corroborated in human clinical datasets and in knockout macrophages in vitro. Increased blood vessel but reduced lymph vessel density may explain the exacerbated inflammatory phenotype in conditional knockout mice. These findings assign a novel role to macrophage S1PR1 and provide a rationale for therapeutically targeting local S1P during skin inflammation.
Für die Rechtsgeschichte in Lateinamerika scheint heutzutage eine neue Phase anzubrechen; jüngere Forscher erweitern ihr Betätigungsfeld hinsichtlich der Themen, Chronologie und Methodologie, und ihre Forschungsarbeiten führen zu einer neuen Auseinandersetzung mit der aktuellen Aufgabe des Rechtshistorikers. Es handelt sich um sehr unterschiedliche Arbeiten, was Charakteristika, Ansätze, Ausrichtung, Voraussetzungen und Kontext anbelangt; sie illustrieren Rechtserfahrungen, die – obwohl relativ homogen – nur zum Teil auf ein kontinentales unicum zurückgeführt werden können. ...
The assumed space : pre-reflective spatiality and doctrinal configurations in juridical experience
(2015)
The purpose of this contribution is to analyse, by means of the legal-historical perspective, the relationship between the pre-reflections of space and the configurations of legal concepts and categories. Three examples of the interplay between doctrinal configurations and the spatial dimension within the context of three different historical periods will be illustrated: given space in the Middle Ages, possible space in the Modern Age and decided space in the Contemporary Age. From this basis, the essay considers the heuristic importance of such an analytical approach – mindful of the profiles of presupposition, such as the space assumption, underlying the conceptualisation of ideas – for a history attentive to the constraints of the theoretical sustainability of legal concepts.
Invincible ignorance is defined as the state in which one cannot overcome his ignorance, despite one’s utmost diligence, and hence cannot be blamed for the acts resulting from that circumstance. It is particularly relevant with regard to law and principles that one is bound to know. The main problem with the admission that such a state may occur results from the difficulty of assessing the subjective element present in such a state: How can we know that one applied his diligence to the utmost extent?
This notion emerged in the 12th century. But while medieval theologians elaborated such a notion, they nonetheless stressed that in reality no one could be guiltlessly ignorant of natural and divine law. The arrival of the Spaniards to the Americas triggered the awareness that entire nations could, in fact, be invincibly ignorant of Christianity. The Spanish theologians then started to use this notion, admitting the existence of invincible ignorance of some principles of divine and natural law. Their argumentative strategy rested on emphasising the subjective element of invincible ignorance.
In this article, I examine Vitoria’s Relectio de Indis against the medieval doctrinal background. I also analyse Vitoria’s, Domingo de Soto’s and Juan Gil de Nava’s unedited lectures on Aquinas’s Summa theologiae as well as the works by Matías de Paz, Juan López de Palacios Rubios, Juan de Celaya and Bartolomé de Las Casas.
The sphingolipid sphingosine-1-phosphate (S1P) is produced by sphingosine kinases to either signal through intracellular targets or to activate a family of specific G-protein-coupled receptors (S1PR). S1P levels are usually low in peripheral tissues compared to the vasculature, forming a gradient that mediates lymphocyte trafficking. However, S1P levels rise during inflammation in peripheral tissues, thereby affecting resident or recruited immune cells, including macrophages. As macrophages orchestrate initiation and resolution of inflammation, the sphingosine kinase/S1P/S1P-receptor axis emerges as an important determinant of macrophage function in the pathogenesis of inflammatory diseases such as cancer, atherosclerosis, and infection. In this review, we therefore summarize the current knowledge how S1P affects macrophage biology.
Over the last years, many microRNAs (miRNAs) have been identified that regulate the formation of bioactive lipid mediators such as prostanoids and leukotrienes. Many of these miRNAs are involved in complex regulatory circuits necessary for the fine-tuning of biological functions including inflammatory processes or cell growth. A better understanding of these networks will contribute to the development of novel therapeutic strategies for the treatment of inflammatory diseases and cancer. In this review, we provide an overview of the current knowledge of miRNA regulation in eicosanoid pathways with special focus on novel miRNA functions and regulatory circuits of leukotriene and prostaglandin biosynthesis.
The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. Therefore, we analyzed the coupling of peptide binding, transport and ATP hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical Km values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. TAPL shows a basal ATPase activity, which is inhibited in the presence of longer peptides. Uncoupling between ATP hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate.
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.
A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.
Das griechische Recht ist, so stellt der Verfasser dieser Passauer juristischen Dissertation zu Recht fest, nicht hinreichend erforscht worden. Althistoriker behandeln es nebenher mit, typischerweise ohne juristische Expertise; Spezialisten für antike Rechtsgeschichte wenden sich zumeist anderen Rechtskulturen zu. Zeitler will in diese Lücke vorstoßen, mit einem Schwerpunkt auf dem Prozess des Sokrates – nun gerade einer der meistdiskutierten Fälle griechischen Rechts. Doch sind diesem gerade knapp 30 Seiten von etwas mehr als 200 gewidmet. ...