Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A

AbstractAdipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein–protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites

Skin Barrier Development Depends on CGI-58 Protein Expression during Late-Stage Keratinocyte Differentiation

Adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) are limiting in cellular triglyceride catabolism. Although ATGL deficiency is compatible with normal skin development, mice globally lacking CGI-58 die postnatally and exhibit a severe epidermal permeability barrier defect, which may originate from epidermal and/or peripheral changes in lipid and energy metabolism. Here, we show that epidermis-specific disruption of CGI-58 is sufficient to provoke a defect in the formation of a functional corneocyte lipid envelope linked to impaired ω-O-acylceramide synthesis. As a result, epidermis-specific CGI-58-deficient mice show severe skin dysfunction, arguing for a tissue autonomous cause of disease development. Defective skin permeability barrier formation in global CGI-58-deficient mice could be reversed via transgenic restoration of CGI-58 expression in differentiated but not basal keratinocytes suggesting that CGI-58 is essential for lipid metabolism in suprabasal epidermal layers. The compatibility of ATGL deficiency with normal epidermal function indicated that CGI-58 may stimulate an epidermal triglyceride lipase beyond ATGL required for the adequate provision of fatty acids as a substrate for ω-O-acylceramide synthesis. Pharmacological inhibition of ATGL enzyme activity similarly reduced triglyceride-hydrolytic activities in wild-type and CGI-58 overexpressing epidermis implicating that CGI-58 participates in ω-O-acylceramide biogenesis independent of its role as a coactivator of epidermal triglyceride catabolism

Novel metallo-porphyrin based colourimetric amine sensors and their processing via plasma enhanced chemical vapour deposition at atmospheric pressure : synthesis, characterisation and mechanistic studies

Volatile amines are prominent indicators of food freshness, as they are produced during many microbiological food degradation processes. Monitoring and indicating the volatile amine concentration within the food package by intelligent packaging solutions might therefore be a simple yet powerful way to control food safety throughout the distribution chain.rnrnIn this context, this work aims to the formation of colourimetric amine sensing surfaces on different substrates, especially transparent PET packaging foil. The colour change of the deposited layers should ideally be discernible by the human eye to facilitate the determination by the end-user. rnrnDifferent tailored zinc(II) and chromium(III) metalloporphyrins have been used as chromophores for the colourimetric detection of volatile amines. A new concept to increase the porphyrins absorbance change upon exposure to amines is introduced. Moreover, the novel porphyrins’ processability during the deposition process is increased by their enhanced solubility in non-polar solvents.rnrnThe porphyrin chromophores have successfully been incorporated into polysiloxane matrices on different substrates via a dielectric barrier discharge enhanced chemical vapour deposition. This process allows the use of nitrogen as a cheap and abundant plasma gas, produces minor amounts of waste and by-products and can be easily introduced into (existing) roll-to-roll production lines. The formed hybrid sensing layers tightly incorporate the porphyrins and moreover form a porous structure to facilitate the amines diffusion to and interaction with the chromophores.rnrnThe work is completed with the thorough analysis of the porphyrins’ amine sensing performance in solution as well as in the hybrid coatings . To reveal the underlying interaction mechanisms, the experimental results are supported by DFT calculations. The deposited layers could be used for the detection of NEt3 concentrations below 10 ppm in the gas phase. Moreover, the coated foils have been tested in preliminary food storage experiments. rnrnThe mechanistic investigations on the interaction of amines with chromium(III) porphyrins revealed a novel pathway to the formation of chromium(IV) oxido porphyrins. This has been used for electrochemical epoxidation reactions with dioxygen as the formal terminal oxidant.rnDa flüchtige Amine als Abbauprodukte einer Vielzahl von mikrobiellen Zersetzungsprozessen von Lebensmitteln auftreten, können diese als Indikatoren für die Frische von Lebensmitteln genutzt werden. Intelligente Verpackungssysteme, die die Aminkonzentration in Lebensmittelverpackungen überwachen und sichtbar machen, könnten daher ein einfacher aber wirkungsvoller Weg sein die Lebensmittelsicherheit zu erhöhen.rnrnIn diesem Zusammenhang ist es das Ziel dieser Arbeit die Oberflächen unterschiedlicher Substrate, z.B. transparenter PET Folien, mit Sensorbeschichtungen zur farbmetrischen Detektion von flüchtigen Aminen zu funktionalisieren. Der Farbumschlag des Sensors sollte idealer Weise mit bloßem Auge zu erkennen sein, um eine einfache Nutzung durch den Verbraucher zu gewährleisten. rnrnUnterschiedliche maßgeschneiderte Zink(II) und Chrom(III)-Metalloporphyrine wurden für die farbmetrische Detektion von flüchtigen Aminen eingesetzt. Dabei wurde ein neues Konzept vorgestellt, um deren Absorptionsänderung durch die Wechselwirkung mit Aminen zu verstärken. Außerdem wurde die Verarbeitung der Porphyrine in dem Beschichtungsprozess verbessert indem Ihre Löslichkeit in unpolaren Lösungsmitteln erhöht wurde.rnrnDie Porphyrinchromophore wurden mit Hilfe einer plasma-gestützten chemischen Gasphasenabscheidung in Polysiloxan-Matrizen auf unterschiedlichen Substraten eingebaut. Für diesen Prozess kann Stickstoff als gut verfügbares und günstiges Plasmagas verwendet werden. Zudem wird nur eine geringe Menge an Abfall oder Nebenprodukten produziert und das Verfahren kann leicht in (bestehende) roll-to-roll Produktionslinien eingebaut werden. Die Porphyrine sind stabil in den hergestellten hybriden Sensorbeschichtungen eingeschlossen. Außerdem zeigen die Matrizen eine poröse Struktur, die eine Diffusion der Amine zu und eine Wechselwirkung mit den Chromophoren erlaubt.rnrnDie Arbeit wird durch intensive Untersuchungen der Möglichkeit zur Amindetektion mit Hilfe der Porphyrine in Lösung und mit den hybriden Beschichtungen abgerundet. Um die zu Grunde liegenden Mechanismen aufzuklären wurden die experimentellen Ergebnisse von DFT Berechnungen unterstützt. Die beschichteten Sensorfolien konnten erfolgreich für die Detektion von NEt3 bis zu einer Konzentration von 10ppm in der Gasphase eingesetzt werden. Schließlich wurden die Folien zudem für die Überwachung von ersten Lebensmittelproben getestet.rnrnIm Rahmen der mechanistischen Untersuchungen der Wechselwirkung zwischen Chrom(III)-Porphyrinen mit Aminen wurden zusätzlich ein neuer Reaktionspfad für die Herstellung von Chrom(IV)-oxidoporphyrinen aufgedeckt. Dieser konnte für elektrochemische Epoxidierungen mit Luftsauerstoff als terminales Oxidationsmittel eingesetzt werden.r

DMC - The Digital Sensor Technology of Z/I-Imaging

Aerial cameras manufactured by Carl Zeiss have been successfully used around the world for many decades. Z/IImaging is continuing this tradition with the digital camera system DMC. The DMC uses a modular design to achieve high geometrical resolution together with multispectral capabilities. It comprises a variable number of synchronously operating CCD-matrix based array cameras that can be built together in different configurations. Four parallel cameras can generate multi-spectral R,G,B and Near Infrared imagery for the acquisition of color composites. Four panchromatic images from converging cameras, are mosaiced digitally to form a single high resolution image. The color composite image and the composed panchromatic image have the same ground coverage. The resulting image is based on central perspective view and can be handled by existing photogrammetric workstations. Based on its outstanding electronic Forward Motion Compensation the DMC reaches ground resolutions better than 2 inches. Thus it can be used for the same wide range of applications as film-based aerial camera systems like the RMK-TOP which are mostly used for mapping applications with photo scale between 1:5.000 and 1:15.000. The paper describes the properties of the camera system and modules. Test results with system components will be discussed

The Patatin–Like Phospholipase Domain Containing Protein 7 Regulates Macrophage Classical Activation through SIRT1/NF-κB and p38 MAPK Pathways

Lysophosphatidylcholine (LPC) is a bioactive lipid that modulates macrophage polarization during immune responses, inflammation, and tissue remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) is a lysophospholipase with a preference for LPC. However, the role of PNPLA7 in macrophage polarization as an LPC hydrolase has not been explored. In the current study, we found that PNPLA7 is highly expressed in naïve macrophages and downregulated upon lipopolysaccharide (LPS)-induced polarization towards the classically activated (M1) phenotype. Consistently, overexpression of PNPLA7 suppressed the expression of proinflammatory M1 marker genes, including interleukin 1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor α (TNF-α), whereas knockdown of PNPLA7 augmented the inflammatory gene expression in LPS-challenged macrophages. PNPLA7 overexpression and knockdown increased and decreased Sirtuin1 (SIRT1) mRNA and protein levels, respectively, and affected the acetylation of the nuclear factor-kappa B (NF-κB) p65 subunit, a key transcription factor in M1 polarization. In addition, the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) were suppressed and enhanced by PNPLA7 overexpression and knockdown, respectively. Taken together, these findings suggest that PNPLA7 suppresses M1 polarization of LPS-challenged macrophages by modulating SIRT1/NF-κB- and p38 MAPK-dependent pathways

Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets

Neuropathy target esterase (NTE) is an endoplasmic reticulum (ER)-localized phospholipase that deacylates phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Loss-of-function mutations in the human NTE gene have been associated with a spectrum of neurodegenerative disorders such as hereditary spastic paraplegia, ataxia and chorioretinal dystrophy. Despite this, little is known about structure–function relationships between NTE protein domains, enzymatic activity and the interaction with cellular organelles. In the current study we show that the C-terminal region of NTE forms a catalytically active domain that exhibits high affinity for lipid droplets (LDs), cellular storage organelles for triacylglycerol (TAG), which have been recently implicated in the progression of neurodegenerative diseases. Ectopic expression of the C domain in cultured cells decreases cellular PC, elevates TAG and induces LD clustering. LD interactions of NTE are inhibited by default by a non-enzymatic regulatory (R) region with three putative nucleotide monophosphate binding sites. Together with a N-terminal TMD the R region promotes proper distribution of the catalytic C-terminal region to the ER network. Taken together, our data indicate that NTE may exhibit dynamic interactions with the ER and LDs depending on the interplay of its functional regions. Mutations that disrupt this interplay may contribute to NTE-associated disorders by affecting NTE positioning

Molecular identification of transmembrane protein 68 as an endoplasmic reticulum-anchored and brain-specific protein.

Acyltransferases catalyze essential reactions in the buildup and remodeling of glycerophospholipids and contribute to the maintenance and diversity of cellular membranes. Transmembrane protein 68 (TMEM68) is an evolutionarily conserved protein of unknown function, that forms a distinct subgroup within the glycerophospholipid acyltransferase family. In the current study we expressed murine TMEM68 for the first time in mammalian cells to characterize its subcellular localization, topology, and possible biological function(s). We show that TMEM68 is an integral membrane protein and orients both, the N- and C-terminus towards the cytosol. Live cell imaging demonstrated that TMEM68 is localized mainly at the endoplasmic reticulum (ER), but not at cellular lipid droplets (LDs). The positioning of TMEM68 at the ER was dependent on its first transmembrane domain (TMD), which by itself was sufficient to target cytosolic green fluorescence protein (GFP) to the ER. In contrast, a second TMD was dispensable for ER localization of TMEM68. Finally, we found that among multiple murine tissues the expression level of TMEM68 transcripts was highest in brain. We conclude that TMEM68 is an integral ER membrane protein and a putative acyltransferase involved in brain glycerolipid metabolism

Performance of antigen testing for diagnosis of COVID-19: a direct comparison of a lateral flow device to nucleic acid amplification based tests

Abstract Objectives The gold standard for diagnosing an infection with SARS-CoV-2 is detection of viral RNA by nucleic acid amplification techniques. Test capacities, however, are limited. Therefore, numerous easy-to-use rapid antigen tests based on lateral flow technology have been developed. Manufacturer-reported performance data seem convincing, but real-world data are missing. Methods We retrospectively analysed all prospectively collected antigen tests results performed between 23.06.2020 and 26.11.2020, generated by non-laboratory personnel at the point-of-care from oro- or nasopharyngeal swab samples at the University Hospital Augsburg and compared them to concomitantly (within 24 h.) generated results from molecular tests. Results For a total of 3630 antigen tests, 3110 NAAT results were available. Overall, sensitivity, specificity, NPV and PPV of antigen testing were 59.4%, 99.0%, 98.7% and 64.8%, respectively. Sensitivity and PPV were lower in asymptomatic patients (47.6% and 44.4%, respectively) and only slightly higher in patients with clinical symptoms (66.7% and 85.0%, respectively). Some samples with very low Ct-values (minimum Ct 13) were not detected by antigen testing. 31 false positive results occurred. ROC curve analysis showed that reducing the COI cut-off from 1, as suggested by the manufacturer, to 0.9 is optimal, albeit with an AUC of only 0.66. Conclusion In real life, performance of lateral-flow-based antigen tests are well below the manufacturer's specifications, irrespective of patient’s symptoms. Their use for detection of individual patients infected with SARS-CoV2 should be discouraged. This does not preclude their usefulness in large-scale screening programs to reduce transmission events on a population-wide scale

Subcellular localization of TMEM68-GFP.

<p>(A) Detection of TMEM68-GFP and GFP expression by immunoblotting. 48-h post transfection, cells were harvested, homogenized, and subjected to immunoblotting using an anti-GFP antibody. (B) Subcellular localization of TMEM68-GFP in mammalian cells. COS-7 cells were transfected with plasmids encoding for GFP, TMEM68-GFP, and DsRed-ER as indicated and imaged by confocal fluorescence microscopy. For the detection of LDs, cells were incubated with OA and stained with LipidTOX Deep Red. Scale bar = 10 μm. Figures are representative of three separate experiments.</p

Targeting of GFP to the ER by the first TMD of TMEM68.

<p>(A) Scheme of the constructs expressing the first (TMD1), the second (TMD2), and both TMDs (TMD1+2) tagged with GFP. Black boxes represent the first and second TMDs of TMEM68. (B) Post-nuclear supernatants of COS-7 cells expressing TMD1-GFP, TMD2-GFP or TMD1+2-GFP were fractionated into cytosolic and membrane fractions and analyzed by immunoblotting using antibodies against GFP or the ER protein calnexin. (C) COS-7 cells were co-transfected with TMD1-GFP, TMD2-GFP or TMD1+2-GFP and the ER marker DsRed–ER as indicated and visualized by a confocal fluorescence microscopy. Scale bar = 10 μm. Figures are representative of three separate experiments.</p