Substrate-derived Sortase A inhibitors: targeting an essential virulence factor of Gram-positive pathogenic bacteria

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed in vitro. Among our panel, we identified several promising inhibitors with IC50 values below 200 mu M, with our strongest inhibitor - LPRDSar - having an IC50 of 18.9 mu M. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 mu g mL(-1), manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA

The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

National Institutes of Health (U.S.) (GM088204

Protein cohabitation: long-term immunoglobulin G storage at room temperature

Long-term functional storage of therapeutic proteins at room temperature has been an eternal challenge. Inspired by the cellular cooperativity of proteins, we have taken a step forward to address this challenge by cohabitating Immunoglobulin G (IgG1) with a food protein gelatin in the solid-state at room temperature. Interestingly, IgG1 remained functionally active for a record 14 months revealed from the western-blot assay. Further quantification by HP-LC analysis showed 100% structural integrity of IgG1 with no degradation in the gelatin matrix during this period. The developed formulation has a direct application in oral medical nutrition therapy to cure gastrointestinal microbial infections. Also the strategy provides a robust energy economic alternative to the protein engineering methods for long-term functional storage of therapeutic proteins at room temperature

Non-destructive assay of nuclear waste containers using muon scattering tomography in the Horizon2020 CHANCE project

Methods for the non-destructive assay of nuclear waste drums are of great importance to the nuclear waste management community, especially where loss in continuity of knowledge about the content of drums happened or chemical processes altering the contents of the drums may occur. Muon scattering tomography has been shown to be a promising technique for the non-destructive assay of nuclear waste drums in a safe way. By measuring tracks of muons entering and leaving the probed sample and extracting scattering angles from the tracks, it is possible to draw conclusions about the contents of the sample and its spatial arrangement. Within the CHANCE project, a newly built large-scale mobile detector system for scanning and imaging the contents of nuclear waste drums using atmospheric muons is currently undergoing commissioning

Comparison of diagnostic value of 68 Ga-DOTATOC PET/MRI and standalone MRI for the detection of intracranial meningiomas

To evaluate the diagnostic performance of magnetic resonance imaging (MRI) alone in comparison to positron emission tomography/ magnetic resonance imaging (PET/MRI) in patients with meningiomas. 57 patients with a total of 112 meningiomas of the brain were included. PET/MRI, including a fully diagnostic contrast enhanced MRI and PET, were acquired. PET/MRI was used as reference standard. The size and location of meningiomas was recorded. Likelihood-ratio chi-square tests were used to calculate p-values within logistic regression in order to compare different models. A multi-level logistic regression was applied to comply the hierarchical data structure. Multi-level regression adjusts for clustering in data was performed. The majority (n=103) of meningiomas could be identified based on standard MRI sequences compared to PET/MRI. MRI alone achieved a sensitivity of 95% (95% CI 0.78, 0.99) and specificity of 88% (95% CI 0.58, 0.98). Based on intensity of contrast medium uptake, 97 meningiomas could be diagnosed with intense uptake (93.75%). Sensitivity was lowest with 74% for meningiomas2cm(3) and highest with 100% for meningiomas 0.5-1.0 cm(3). Petroclival meningiomas showed lowest sensitivity with 88% compared to sphenoidal meningiomas with 94% and orbital meningiomas with 100%. Specificity of meningioma diagnostic with MRI was high with 100% for sphenoidal and hemispherical-dural meningiomas and meningiomas with 0.5-1.0 and 1.0-2.0 cm(3). Overall MRI enables reliable detection of meningiomas compared to PET/MRI. PET/MRI imaging offers highest sensitivity and specificity for small or difficult located meningiomas

Von Fachlicher Risikoanalyse zu Politischer Risikobewertung: Die Vermittlung von Risikoanalysen im Bevölkerungsschutz in die Bundespolitik

In Deutschland werden seit 2010 im Auftrag des Bundesministeriums des Innern und für Heimat (bis 8. Dezember 2021 Bundesministerium des Innern, für Bau und Heimat), unter der Koordination des Bundesamtes für Bevölkerungsschutz und Katastrophenhilfe (BBK) regelmäßig Risikoanalysen zu unterschiedlichen Risiken erarbeitet und zur politischen Bewertung vor allem an den Innenausschuss des Deutschen Bundestages gesandt. Diese Studie untersucht explorativ die Rahmenbedingungen der politischen Arbeit von Abgeordneten des Deutschen Bundestages und geht der Frage nach, welche Gründe und Bedingungen die Wahrnehmung der Risikoanalysen beeinflussen und die Verwendung dieser für die Entscheidungsfindung und Politikgestaltung erschweren oder erleichtern. Zur Beantwortung dieser Frage wurden 15 leitfadengestützte Interviews mit aktiven und ehemaligen Bundestagsabgeordneten sowie zwei Gruppendiskussionen mit wissenschaftlichen Mitarbeitenden von Bundestagsabgeordneten durchgeführt. Auf der Grundlage eines heuristischen Modells erfolgte eine strukturierte qualitative Inhaltsanalyse des Materials entlang festgelegter Auswertungskategorien. Durch die Analyse konnten grundlegende Rahmenbedingungen für die Arbeits- und Verhaltensweisen der Abgeordneten herausgearbeitet und Gründe aufgezeigt werden, die eine politische Diskussion der Risikoanalysen in der Vergangenheit erschwert haben. Gleichzeitig erzielte die Analyse eine große Anzahl von Anhaltspunkten für direkte oder indirekte Verbesserungen in der kommunikativen Begleitung der Risikoanalysen sowie deren inhaltlicher und visueller Aufbereitung. Zusätzliche Aspekte hinsichtlich der politischen Wahrnehmung und Bewertung der Risikoanalysen konnten im Rahmen einer Analyse politischer Dokumente erarbeitet werden, die über das Dokumentations- und Informationssystem für Parlamentarische Vorgänge (DIP) abrufbar sind. Die Studie stellt abschließend Handlungsempfehlungen für einen erfolgreichen Transfer der Inhalte der Risikoanalysen in die Bundespolitik vor

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid– dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other G subunits, we here set out to rationally design G16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gq/G11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold– based inhibitors, provided that these inhibitors mimic the interaction of FR with G proteins harboring engineered FR-binding sites

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble

Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells

© 2018, American Society for Clinical Investigation. This article has been published in final form at https://doi.org/10.1172/JCI120612First-generation immune checkpoint inhibitors, including anti-CTLA-4 and anti-programmed death 1 (anti-PD-1) antibodies, have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) are pattern-recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not until recently considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs, including Siglec-9, on tumor-infiltrating T cells from non-small cell lung cancer (NSCLC), colorectal, and ovarian cancer patients. Siglec-9-expressing T cells coexpressed several inhibitory receptors, including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anticancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with reduced survival, and Siglec-9 polymorphisms showed association with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as a potential target for improving T cell activation for immunotherapy.Peer reviewe

Apparent Alkyl Transfer and Phenazine Formation via an Aryne Intermediate

Treatment of chlorotriaryl derivatives 3a and 3d or fluorotriaryl derivatives 3b and 3e with potassium diisopropylamide afforded alkyl-shifted phenazine derivatives 5a/5b, rather than the expected 9-membered triazaorthocyclophane 2a. The phenazine derivatives were isolated in 78–98% yield depending on the halogen and alkyl group present. In the absence of the halogen (chloro or fluoro), the apparent alkyl shift proceeds more slowly and cannot proceed via the intermediacy of the aryne intermediate. Mechanistic possibilities include intramolecular nucleophilic attack on an aryne intermediate leading to a zwitterionic intermediate and alkyl transfer via a 5-endo-tet process, or via a Smiles rearrangement