PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis

A missense mutation in the highly conserved TNF-like domain of Ectodysplasin A is the candidate causative variant for X-linked hypohidrotic ectodermal dysplasia in Limousin cattle: Clinical, histological, and molecular analyses

Ectodysplasin A related hypohidrotic ectodermal dysplasia (XLHED) is a well-studied fetal developmental disorder in mammals that mainly affects ectodermal structures. It has been identified in a variety of species, including mice, rats, dogs, cattle, and humans. Here, we report the clinical, histological, and molecular biological analyses of a case of XLHED in Limousin cattle. An affected Limousin calf showed pathognomonic signs of ectodermal dysplasia, i.e. sparse hair and characteristic dental aplasia. Histopathologic comparison of hairy and glabrous skin and computed tomography of the mandible confirmed the phenotypic diagnosis. In addition, a keratoconjunctivitis sicca was noted in one eye, which was also confirmed histopathologically. To identify the causative variant, we resequenced the bovine X-chromosomal ectodysplasin A gene ( EDA ) of the affected calf and compared the sequences to the bovine reference genome. A single missense variant (rs439722471) at position X:g.80411716T>C (ARS-UCD1.3) was identified. The variant resulted in an amino acid substitution from glutamic acid to glycine within the highly conserved TNF-like domain. To rule out the possibility that the variant was relatively common in the cattle population we genotyped 2,016 individuals including 40% Limousin cattle by fluorescence resonance energy transfer analysis. We also tested 5,116 multibreed samples from Run9 of the 1000 Bull Genomes Project for the said variant. The variant was not detected in any of the cattle tested, confirming the assumption that it was the causative variant. This is the first report of Ectodysplasin A related hypohidrotic ectodermal dysplasia in Limousin cattle and the description of a novel causal variant in cattle.Open-Access-Publikationsfonds 202

Particle based Cultivation of Lentzea aerocolonigenes in Membrane aerated stirred Bioreactors for increased Rebeccamycin Production

Filamentous microorganisms represent the majority of natural producers of antibiotics and other active pharmaceutical ingredients (APIs). Lentzea aerocolonigenes is a filamentous actinomycete producing the antitumor antibiotic rebeccamycin. The addition of glass beads (100 g/L, ⌀ = 969 μm) in shake flask cultivations led to a significant increase in rebeccamycin production compared to an unsupplemented cultivation by inducing mechanical stress. To achieve larger amounts of rebeccamycin a scale-up was conducted. A bubble free membrane aeration was chosen to reduce the power input to stirrer and particle induced stress. Glass bead addition (50 g/L, ⌀ = 969 μm) in this membrane aerated stirred bioreactor increased rebeccamycin concentration compared to an unsupplemented cultivation as well. Moreover it reduced biomass growth on the aeration membrane which is a widespread challenge in membrane aerated bioreactors

Production and Characterization of Bacterial Cellulose Separators for Nickel-Zinc Batteries

The need for energy-storing technologies with lower environmental impact than Li-ion batteries but similar power metrics has revived research in Zn-based battery chemistries. The application of bio-based materials as a replacement for current components can additionally contribute to an improved sustainability of Zn battery systems. For that reason, bacterial cellulose (BC) was investigated as separator material in Ni-Zn batteries. Following the biotechnological production of BC, the biopolymer was purified, and differently shaped separators were generated while surveying the alterations of its crystalline structure via X-ray diffraction measurements during the whole manufacturing process. A decrease in crystallinity and a partial change of the BC crystal allomorph type Iα to II was determined upon soaking in electrolyte. Electrolyte uptake was found to be accompanied by dimensional shrinkage and swelling, which was associated with partial decrystallization and hydration of the amorphous content. The separator selectivity for hydroxide and zincate ions was higher for BC-based separators compared to commercial glass-fiber (GF) or polyolefin separators as estimated from the obtained diffusion coefficients. Electrochemical cycling showed good C-rate capability of cells based on BC and GF separators, whereas cell aging was pronounced in both cases due to Zn migration and anode passivation. Lower electrolyte retention was concluded as major reason for faster capacity fading due to zincate supersaturation within the BC separator. However, combining a dense BC separator with low zincate permeability with a porous one as electrolyte reservoir reduced ZnO accumulation within the separator and improved cycling stability, hence showing potentials for separator adjustment

Particle-based production of antibiotic rebeccamycin with Lechevalieria aerocolonigenes

Rebeccamycin is a promising antibiotic synthesized by the filamentous bacterium Lechevalieria aerocolonigenes. To date, the main investigations were focused on the biological effect or the structure elucidation of rebeccamycin. The aim of this study was to develop an efficient cultivation process based on shake flask experiments. The relationship between morphology and productivity and the positive effects of micro- and macroparticle enhanced cultivation on rebeccamycin production with this strain in the pellet-like morphology was investigated. The addition of talc microparticles increased the rebeccamycin concentration to 120\ua0mg\ua0L and 3-fold compared to the control without microparticles. An up to 9-fold increase in rebeccamycin concentration was achieved using surface modified talc particles. Through the addition of glass beads as macroparticles, the highest rebeccamycin concentration of approximately 120\ua0mg\ua0L was achieved by the induction of mechanical stress. Cultivation with glass beads enhanced the rebeccamycin concentration 22-fold compared to the control without macroparticles. By further increasing the diameter of the glass beads and increasing mechanical stress, the morphology was driven toward mycelial growth and the productivity was decreased. An adjusted mechanical stress, through the addition of macro-shaped glass bead particles, led to the desired increase in productivity

Cystobactamid 507: Concise Synthesis, Mode of Action and Optimization toward More Potent Antibiotics.

Lack of new antibiotics and increasing antimicrobial resistance are the main concerns of healthcare community nowadays, which necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids - a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe a concise total synthesis of cystobactamid 507, the identification of the bioactive conformation using non-covalently bonded rigid analogs, the first structure–activity relationship (SAR) study for cystobactamid 507 leading to new analogs with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor groove binding as part of the drug–target interaction without showing significant intercalation. By designing a new analog of cystobactamid 919-2 we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high: In vivo efficacy

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL−1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL−1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.Deutsches Zentrum für Infektionsforschun

4Pi Microscopy of the Nuclear Pore Complex

To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110–130 nm and a two-color localization accuracy of 5–10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 ± 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 ± 12 (Potorous tridactylus cells) or 91 ± 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 ± 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general