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Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1β and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-β expression, as well as the magnitude of IFN-β cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-β via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Additionally, we observed that ATF3 itself is a type I IFN–inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFNβ, as well as the expression of genes downstream of IFN signaling.

Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.

NLRP3 is an innate immune receptor that needs to be tightly regulated to prevent overshooting immune responses. Stutz et al. demonstrate that NLRP3 is phosphorylated as a safeguard against accidental activation, and that dephosphorylation involving PP2A activity is required for NLRP3 activation., NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge–charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.

We synthesized a series of amino substituted triarylboranes (TABs) 1-3 by copper(I)-catalyzed cross-coupling reactions. The title compounds were investigated by means of cyclic voltammetry (CV) and UV-visible absorption and fluorescence spectroscopy. Electrochemical oxidation of tris(4-carbazolyl-2,6-dimethylphenyl)-borane (3) leads to the formation of an electroactive polymer film on the electrode surface. The charge-transfer (CT) absorption band of all three TABs shows a pronounced negative solvatochromism, while the emission is positively solvatochromic. By combining Jortner's theory, AMI computations, and electrooptical absorption measurements (EOAM), this unexpected behavior was shown to be due to a dipole inversion upon S 0 →S 1 excitation. Furthermore, polarized steady-state fluorescence spectroscopy and EOAM prove that the ground-state geometry of 3 is of lower symmetry than D 3 and that the excitation energy can be transferred from one subchromophore to another within the lifetime of the excited state. Exciton-coupling theory was used to quantitatively analyze this excitation transfer.

Three 3,4-bis(alkylthio)pyrroles (5a−c) were synthesized by a nonclassical pyrrole ring formation reaction followed by alkylation of the dithiol-2-one functional group. UV−vis absorption studies together with time dependent density-functional theory (TD-DFT) computations suggest that the monomers (5a−c) display low-energy π−σ* transitions (5a, 254 nm; 5b, 232 nm; 5c, 240 nm) compared to pyrrole (208 nm). Cyclic voltammetry investigations show that the monomers 5a−c possess significantly lower oxidation potentials (0.60 V for 5a, 0.58 V for 5b, and 0.71 V for 5c) than unsubstituted pyrrole (1.0 V). The corresponding polymers were successfully prepared by anodic electropolymerization and/or chemical oxidation (FeCl3 as oxidant) and investigated by cyclic voltammetry, spectroelectrochemistry and in situ conductivity studies. Our investigations of monomeric 3,4-disubstituted pyrroles and corresponding polymers suggest that the electron donating alkylthio substituents at the 3- and 4-positions of the pyrrole r...

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid–sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid–sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.

Grundlage dieses Artikels ist eine umfangreiche empirische Untersuchung zum Losen einfacher linearer Gleichungen. Mit Hilfe eines selbstentwickelten Diagnosesoftware gelang es zusatzlich zum Losungsprodukt den Losungsprozess zu analysieren. Dieser Beitrag widmet sich den Losungsstrategien beim Losen einfacher linearer Gleichungen und zeigt auf, dass das Verwenden geeigneter Losungstrategien den Losungserfolg steigert. Im ersten Teil werden die Anforderungen, die an die Schulerinnen und Schuler bei der unterrichtlichen Behandlung gestellt werden, dargestellt. Im zweiten Teil werden Ergebnisse zu den Losungsstrategien bei linearen Gleichungen vorgestellt. Zum Schluss wird ein neuer unterrichtspraktischer Vorschlag entwickelt.

In sugar beet genotypes with the ‘Holly’ type of resistance to rhizomania, a disease due to infection of the beet necrotic yellow vein virus (BNYVV), the major gene rrl is responsible for resistance. Twelve RAPD markers linked to rrl were selected by BSA and mapped on linkage group IV using a segregating population previously analysed by the same group. Markers F61050 and N9600 were tightly linked, respectively in coupling and repulsion, to the Rrl allele (recombination values of 1.4 cM for both markers). After sequencing the products amplified by F61050 and N9600, new PCR primers were used to generate the two SCAR markers F6 and N9. The simultaneous use of these markers in a PCR reaction allows the correct fingerprinting of rrl rrl, Rrl rrl and Rrl Rrl sugar beet plants in populations segregating for the ‘Holly’ resistance. In a group of sugar beet elite lines containing the ‘Holly’ type of rhizomania resistance, SCAR F6 is always present whereas the SCAR N9 fragment is absent. Thus, in marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele.

Our body is continuously exposed to a diverse array of infectious agents, foreign antigens, and host-derived danger signals. The innate immune system plays a vital role as the first line of defence against these insults by protecting the host from infection and maintaining tissue homeostasis. Families of signalling receptors (e.g. TLRs) recognize invading microbes and co-ordinate an appropriate inflammatory response via the production of pro-inflammatory cytokines and type I interferons (IFNs) to clear infection. However, inappropriate activation of innate immune receptors and unregulated production of inflammatory mediators is thought to be the basis for many acute and chronic inflammatory conditions. Indeed, excessive IFN production is implicated in the pathogenesis of lupus and atherosclerosis. Maintaining a balance between the beneficial and detrimental effects of IFNs is therefore crucial and their production must be tightly regulated. Yet these precise regulatory mechanisms remain poorly understood. We have discovered that macrophages deficient in the transcriptional modulator, ATF3, display significantly greater IFN β production following activation of TLRs and other innate immune receptors, as well as reduced LCMV viral infection. In contrast, overexpression of ATF3 in macrophages reduced the induction of IFN β and increased viral replication. Utilizing a range of molecular biology, biochemical and immunological techniques, integrated with powerful bioinformatics approaches we have investigated the molecular mechanisms responsible for ATF3-mediated regulation of IFN β . Our ATF3 ChIP-seq data has revealed that ATF3 regulates IFN β by binding directly to its promoter. Furthermore, we have found that ATF3 itself is regulated by IFNs and thus forms part of a negative feedback loop during the induction of IFN signalling. Together our findings indicate ATF3 is a critical regulator of the IFN innate immune response. A better understanding of how the innate immune response is regulated is imperative for the development of future therapeutic strategies against autoimmune and inflammatory diseases.

NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. Because of its involvement in many diseases such as atherosclerosis and Alzheimer’s Disease, a better understanding of NLRP3 regulation may help to shape therapeutic approaches. The aim of this study was to identify post-translational modifications on NLRP3 and their impact on inflammasome activation. We applied mass spectrometry of NLRP3 to identify post-translational modifications. We identified ubiquitinated and phosphorylated residues. To analyze the function of the modifications, we mutated the residues in NLRP3 expression plasmids and used them for reconstitution of NLRP3-deficient macrophages or overexpression studies in HEK293T cells. Ubiquitinylated residues were found in the NACHT and LRR domains of NLRP3 and mutation of these residues resulted in hyperactive NLRP3. In addition, mutation of one of the phosphorylated residues to a phosphomimetic residue showed reduced IL-1beta release and caspase-1 cleavage upon NLRP3 inflammasome activation, whereas mutation to alanine had no effect. Further studies in HEK293T cells found that the recruitment of the downstream adaptor ASC as well as the self-interaction of NLRP3 were reduced with the phosphomimetic residue. Thus, NLRP3 is likely regulated by post-translational modification and needs to be deubiquitinylated and possibly dephosphorylated before activation. Knowledge about the modifications and the pathways involved may help shape novel strategies for treating diseases that involve NLRP3.

Patterns and sites of T-DNA integrations into the barley genome from single and double cassette vectors are of interest for the identification of cultivars with value added properties as well as for the production of selection marker-free transgenic lines that can be retransformed. T-DNA/Plant DNA junctions were obtained by capturing a single-stranded DNA with a biotinylated primer annealing to the vector adjacent to the border and an adaptor ligated to a restriction site overhang in the flanking barley DNA. The captured junction was converted into a double strand and sequenced. Fifty left and right border junctions from plants transgenic for one of five human genes were analyzed. Primers of 15–30 nucleotides designed from the genomic DNA at the insertion site can PCR amplify fragments that identify unequivocally any transformant. Adjacent transgene insertions with single cassette vectors were always in tandem direct repeat configuration. With regard to T-DNA integration the patterns were comparable to the variations found in dicotyledonous plants. Twelve of the 46 integrations characterized by blast searches were within different regions of the BARE - 1 retrotransposon element occurring with a frequency of 2 × 10 5 copies in the barley genome. The use of border junctions to identify number of copies and loci of integrates in transformants is discussed.

Sensing of nucleic acids (NAs) by Toll-like receptors (TLRs) is crucial in the host defence against viruses and bacteria. Yet, these TLRs can contribute to autoimmunity. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the ER into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 show defective signalling of endosomal TLRs and are at increased risk to develop herpes simplex virus encephalitis. To date, molecular insights into UNC93B1-mediated regulation of human NA-sensing TLRs are lacking. In this study, we aimed to investigate the role of trafficking motifs and post-translational modifications of UNC93B1 for human endosomal TLR activity. To this end, we utilised mass spectrometry in order to identify post-translational modification sites and prepared mutant forms of UNC93B1 by site-directed mutagenesis. EBV immortalized B cells from UNC93B1-deficient patients as well as UNC93B1 KO THP1s generated by CRISPR-Cas-based genome editing were reconstituted with wild type or mutant forms of UNC93B1 and assessed for endosomal TLR activity. The localization of UNC93B1 was analysed by confocal microscopy. The destruction of either trafficking motifs or post-translational modification sites resulted in receptor-, ligand-, and cell-type specific defects of human endosomal TLRs. Furthermore, mass spectrometry revealed that wild type versus mutant forms of UNC93B1 differed in terms of interaction partners and cellular compartment signatures. Our study reveals another level of complexity of UNC93B1-mediated TLR regulation and thereby contributes to the understanding of nucleic-acid sensing TLR biology.

Endosomal toll-like receptors, namely human TLR3, TLR7, TLR8, and TLR9 sense nucleic acids (NAs) derived from viruses and bacteria. Yet, these TLRs can also contribute to autoimmunity by recognizing host NAs. NA-sensing TLR activity is highly regulated by the correct subcellular localization. Experimental mislocalization of endosomal TLRs to the cell surface leads to insufficient microbial recognition and inflammatory disease due to the recognition of endogenous NAs. Under physiological conditions, efficient activation of murine TLR7 and TLR9 is restricted to acidic compartments due to the need for proteolytic processing. TLR trafficking within the endolysosomal system further determines the nature of signaling events, as type I IFN and proinflammatory responses are initiated from different compartments. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the endoplasmic reticulum (ER) into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 display defective signaling of endosomal TLRs, and are at increased risk of developing herpes simplex virus encephalitis. The precise mechanism of how UNC93B1 regulates TLR trafficking remains unknown. Recently, a highly conserved tyrosine-based motif of UNC93B1 was identified. This motif is essential for the correct localization and function of murine TLR9, but dispensable for murine TLR7, TLR11, TLR12, and TLR13. TLR11, TLR12, and TLR13 are not expressed in humans. In contrast, human TLR8 plays a crucial role in the initiation of immune responses to certain pathogens. To date, molecular insights into UNC93B1-mediated trafficking of human endosomal TLRs are missing. Our study reveals that the tyrosine-based motif of UNC93B1 differentially regulates human TLR7, TLR8, and TLR9 responses and thereby contributes to the understanding of human TLR trafficking.

Das vorliegende Ubungsbuch erarbeitet einige vielfaltig nutzbare Problemlosungen mit dem HP 28C sowie HP 28S. Der Umfang dieses Buchs gestattet nicht, das Leistungsspektrum des HP 28 wirklich zu entfalten. Dafur setzt der Rechner zuviele neue Masstabe und sprengt bisherige Leistungsgrenzen. Der groste Teil der Beispiele beruht auf Problemen, die Sie mit Taschenrechnern bisherigen Zuschnitts nicht losen konnten. Die Beispiele sind nicht neu, aber der Rechner bietet neue und oft uberraschende Perspektiven fur den Losungsweg. Einen fast beliebig komplexen Funktionsterm in symbolischer Form speichern, ihn auf Nullstellen, Ableitungen, Extremwerte und Integrale untersuchen, die symbolisch ermittelte Ableitungsfunktion in die Formel eines Linienintegrals direkt einsetzen oder die Jacobideterminante nutzen, die numerische Losung einer Differentialgleichung graphisch darstellen, fur naherungsweise bestimmte Funktionen das Integral mit einer einfachen Anweisung berechnen — das sind Moglichkeiten, die man mit Taschenrechnern nicht assoziiert. Die dargestellten Problemlosungen konnen in viele Anwendungen einbezogen werden, da die Formulierung der Aufgabenstellungen jeweils nur den mathematischen Kern hervorhebt. Die Beispiele zeigen daruber hinaus, das eine systematische Problemlosung mit dem HP 28 nicht kunstlich in ein problemfremdes Sprachkorsett gezwangt werden mus, sondern unter Nutzung der dargebotenen Funktionen intuitiv formuliert und schlieslich auch durchgefuhrt werden kann.