Stefanie Schlager

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [(3)H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [(3)H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.

Abstract We present results for direct bio‐electrocatalytic reduction of CO 2 to C 1 products using electrodes with immobilized enzymes. Enzymatic reduction reactions are well known from biological systems where CO 2 is selectively reduced to formate, formaldehyde, or methanol at room temperature and ambient pressure. In the past, the use of such enzymatic reductions for CO 2 was limited due to the necessity of a sacrificial co‐enzyme, such as nicotinamide adenine dinucleotide (NADH), to supply electrons and the hydrogen equivalent. The method reported here in this paper operates without the co‐enzyme NADH by directly injecting electrons from electrodes into immobilized enzymes. We demonstrate the immobilization of formate, formaldehyde, and alcohol dehydrogenases on one‐and‐the‐same electrode for direct CO 2 reduction. Carbon felt is used as working electrode material. An alginate–silicate hybrid gel matrix is used for the immobilization of the enzymes on the electrode. Generation of methanol is observed for the six‐electron reduction with Faradaic efficiencies of around 10 %. This method of immobilization of enzymes on electrodes offers the opportunity for electrochemical application of enzymatic electrodes to many reactions in which a substitution of the expensive sacrificial co‐enzyme NADH is desired.

Microglia, the immunocompetent cells of the CNS, rapidly respond to brain injury and disease by altering their morphology and phenotype to adopt an activated state. Microglia can exist broadly between two different states, namely the classical (M1) and the alternative (M2) phenotype. The first is characterized by the production of pro-inflammatory cytokines/chemokines and reactive oxygen and/or nitrogen species. In contrast, alternatively activated microglia are typified by an anti-inflammatory phenotype supporting wound healing and debris clearance. The objective of the present study was to determine the outcome of lysophosphatidic acid (LPA)-mediated signaling events on microglia polarization.LPA receptor expression and cyto-/chemokine mRNA levels in BV-2 and primary murine microglia (PMM) were determined by qPCR. M1/M2 marker expression was analyzed by Western blotting, immunofluorescence microscopy, or flow cytometry. Cyto-/chemokine secretion was quantitated by ELISA.BV-2 cells express LPA receptor 2 (LPA2), 3, 5, and 6, whereas PMM express LPA1, 2, 4, 5, and 6. We show that LPA treatment of BV-2 and PMM leads to a shift towards a pro-inflammatory M1-like phenotype. LPA treatment increased CD40 and CD86 (M1 markers) and reduced CD206 (M2 marker) expression. LPA increased inducible nitric oxide synthase (iNOS) and COX-2 levels (both M1), while the M2 marker Arginase-1 was suppressed in BV-2 cells. Immunofluorescence studies (iNOS, COX-2, Arginase-1, and RELMα) extended these findings to PMM. Upregulation of M1 markers in BV-2 and PMM was accompanied by increased cyto-/chemokine transcription and secretion (IL-1β, TNFα, IL-6, CCL5, and CXCL2). The pharmacological LPA5 antagonist TCLPA5 blunted most of these pro-inflammatory responses.LPA drives BV-2 and PMM towards a pro-inflammatory M1-like phenotype. Suppression by TCLPA5 indicates that the LPA/LPA5 signaling axis could represent a potential pharmacological target to interfere with microglia polarization in disease.

In humans, mutations in ATGL lead to TG accumulation in LDs of most tissues and cells, including peripheral blood leukocytes. This pathologic condition is called Jordans' anomaly, in which functional consequences have not been investigated. In the present study, we tested the hypothesis that ATGL plays a role in leukocyte LD metabolism and immune cell function. Similar to humans with loss-of-function mutations in ATGL, we found that global and myeloid-specific Atgl(-/-) mice exhibit Jordans' anomaly with increased abundance of intracellular TG-rich LDs in neutrophil granulocytes. In a model of inflammatory peritonitis, lipid accumulation was also observed in monocytes and macrophages but not in eosinophils or lymphocytes. Neutrophils from Atgl(-/-) mice showed enhanced immune responses in vitro, which were more prominent in cells from global compared with myeloid-specific Atgl(-/-) mice. Mechanistically, ATGL(-/-) as well as pharmacological inhibition of ATGL led to an impaired release of lipid mediators from neutrophils. These findings demonstrate that the release of lipid mediators is dependent on the liberation of precursor molecules from the TG-rich pool of LDs by ATGL. Our data provide mechanistic insights into Jordans' anomaly in neutrophils and suggest that ATGL is a potent regulator of immune cell function and inflammatory diseases.

Abstract In the recent decade, CO 2 has increasingly been regarded not only as a greenhouse gas but even more as a chemical feedstock for carbon‐based materials. Different strategies have evolved to realize CO 2 utilization and conversion into fuels and chemicals. In particular, biological approaches have drawn attention, as natural CO 2 conversion serves as a model for many processes. Microorganisms and enzymes have been studied extensively for redox reactions involving CO 2 . In this review, we focus on monitoring nonliving biocatalyzed reactions for the reduction of CO 2 by using enzymes. We depict the opportunities but also challenges associated with utilizing such biocatalysts. Besides the application of enzymes with co‐factors, resembling natural processes, and co‐factor recovery, we also discuss implementation into photochemical and electrochemical techniques.

Abstract The alkynyl‐substituted Re I complex [Re(5,5′‐bisphenylethynyl‐2,2′‐bipyridyl)(CO) 3 Cl] was immobilized by electropolymerization onto a Pt‐plate electrode. The polymerized film exhibited electrocatalytic activity for the reduction of CO 2 to CO. Cyclic voltammetry studies and bulk controlled‐potential electrolysis experiments were performed by using a CO 2 ‐saturated acetonitrile solution. The CO 2 reduction, determined by cyclic voltammetry, occurs at approximately −1150 mV versus the normal hydrogen electrode (NHE). Quantitative analysis by GC and IR spectroscopy was used to determine a Faradaic efficiency of approximately 33 % for the formation of CO. Both values of the modified electrode were compared to the performance of the homogenous monomer [Re(5,5′‐bisphenylethynyl‐2,2′‐bipyridyl)(CO) 3 Cl] in acetonitrile. The polymer formation and its properties were studied by using SEM, AFM, and attenuated total reflectance (ATR) FTIR and UV/Vis spectroscopy.

Carbon dioxide has evolved from being considered as a greenhouse gas to valuable carbon feedstock for the generation of artificial fuels and valuable chemicals.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH.We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils.Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus.Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes.The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis.To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/-mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2.Acute inhibition of LAL resulted in reduced release of 18:2-and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages.We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.

The discovery of significant amounts of metabolically active brown adipose tissue (BAT) in adult humans renders it a promising target for anti-obesity therapies by inducing weight loss through increased energy expenditure. The components of the N-acetylaspartate (NAA) pathway are highly abundant in BAT. Aspartate N-acetyltransferase (Asp-NAT, encoded by Nat8l) synthesizes NAA from acetyl-CoA and aspartate and increases energy expenditure in brown adipocytes. However, the exact mechanism how the NAA pathway contributes to accelerated mobilization and oxidation of lipids and the physiological regulation of the NAA pathway remained elusive. Here, we demonstrate that the expression of NAA pathway genes corresponds to nutrient availability and specifically responds to changes in exogenous glucose. NAA is preferentially produced from glucose-derived acetyl-CoA and aspartate and its concentration increases during adipogenesis. Overexpression of Nat8l drains glucose-derived acetyl-CoA into the NAA pool at the expense of cellular lipids and certain amino acids. Mechanistically, we elucidated that a combined activation of neutral and lysosomal (acid) lipolysis is responsible for the increased lipid degradation. Specifically, translocation of the transcription factor EB to the nucleus activates the biosynthesis of autophagosomes and lysosomes. Lipid degradation within lysosomes accompanied by adipose triglyceride lipase-mediated lipolysis delivers fatty acids for the support of elevated mitochondrial respiration. Together, our data suggest a crucial role of the NAA pathway in energy metabolism and metabolic adaptation in BAT.

Abstract The electrocatalytic reduction of CO 2 to methanol is explored by the direct comparison of protonated pyridazine and pyridine for their capabilities towards CO 2 reduction. The two materials inherit a significant difference in their pKa values adding valuable information to the ongoing discussion on the nature of CO 2 reduction catalyzed by pyridinium and similar nitrogen containing heteroaromatic systems. Cyclic voltammetry studies as well as bulk controlled‐potential electrolysis experiments were performed combined with product analysis using gas chromatography. Methanol was detected as main CO 2 reduction product in all cases.

Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s).We studied metabolic adaptations in Lal (-/-) mice.Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels.Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.

Abstract We present a study on a microbial electrolysis cell with methanogenic microorganisms adapted to reduce CO 2 to CH 4 with the direct injection of electrons and without the artificial addition of H 2 or an additional carbon source except gaseous CO 2 . This is a new approach in comparison to previous work in which both bicarbonate and gaseous CO 2 served as the carbon source. The methanogens used are known to perform well in anaerobic reactors and metabolize H 2 and CO 2 to CH 4 and water. This study shows the biofilm formation of those microorganisms on a carbon felt electrode and the long‐term performance for CO 2 reduction to CH 4 using direct electrochemical reduction. CO 2 reduction is performed simply by electron uptake with gaseous CO 2 as the sole carbon source in a defined medium. This “electrometabolism” in such microbial electrolysis cells depends strongly on the potential applied as well as on the environmental conditions. We investigated the performance using different adaption mechanisms and a constant potential of −700 mV vs. Ag/AgCl for CH 4 generation at 30–35 °C. The experiments were performed by using two‐compartment electrochemical cells. Production rates with Faradaic efficiencies of around 22 % were observed.

<h2>Abstract</h2><h3>Background and aims</h3> Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading monoglyceride (MG) to glycerol and fatty acid. MGL also hydrolyzes and thereby deactivates 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid in the mammalian system. 2-AG acts as full agonist on cannabinoid receptor type 1 (CB1R) and CB2R, which are mainly expressed in brain and immune cells, respectively. Thus, we speculated that in the absence of MGL, increased 2-AG concentrations mediate CB2R signaling in immune cells to modulate inflammatory responses, thereby affecting the development of atherosclerosis. <h3>Methods and results</h3> We generated apolipoprotein E (ApoE)/MGL double-knockout (DKO) mice and challenged them with Western-type diet for 9 weeks. Despite systemically increased 2-AG concentrations in DKO mice, CB2R-mediated signaling remains fully functional, arguing against CB2R desensitization. We found increased plaque formation in both <i>en face</i> aortae (1.3-fold, p = 0.028) and aortic valve sections (1.5-fold, p = 0.0010) in DKO mice. Interestingly, DKO mice also presented reduced lipid (12%, p = 0.031) and macrophage content (18%, p = 0.061), elevated intraplaque smooth muscle staining (1.4-fold, p = 0.016) and thicker fibrous caps (1.8-fold, p = 0.0032), together with a higher ratio of collagen to necrotic core area (2.5-fold, p = 0.0003) and expanded collagen content (1.6-fold, p = 0.0007), which suggest formation of less vulnerable atherosclerotic plaques. Treatment with a CB2R inverse agonist prevents these effects in DKO mice, demonstrating that the observed plaque phenotype in DKO mice originates from CB2R activation. <h3>Conclusion</h3> Loss of MGL modulates endocannabinoid signaling in CB2R-expressing cells, which concomitantly affects the pathogenesis of atherosclerosis. We conclude that despite larger lesion size loss of MGL improves atherosclerotic plaque stability. Thus, pharmacological MGL inhibition may be a novel intervention to reduce plaque rupture.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations.Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients.An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL.However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors.Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo.We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest.Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics.Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/ Cas9 systems for probing oncogene addiction.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

Abstract A broad review of homogeneous and heterogeneous catalytic approaches toward CO 2 reduction using organic, organometallic, and bioorganic systems is provided. Electrochemical, bioelectrochemical and photoelectrochemical approaches are discussed in terms of their faradaic efficiencies, overpotentials and reaction mechanisms. Organometallic complexes as well as semiconductors and their homogeneous and heterogeneous catalytic activities are compared to enzymes. In both cases, their immobilization on electrodes is discussed and compared to homogeneous catalysts in solution.

Mast cells are potent effectors of immune reactions and key players in various inflammatory diseases such as atherosclerosis, asthma, and rheumatoid arthritis. The cellular defense response of mast cells represents a unique and powerful system, where external signals can trigger cell activation resulting in a stimulus-specific and highly coordinated release of a plethora of bioactive mediators. The arsenal of mediators encompasses preformed molecules stored in cytoplasmic secretory granules, as well as newly synthesized proteinaceous and lipid mediators. The release of mediators occurs in strict chronological order and requires proper coordination between the endomembrane system and various enzymatic machineries. For the generation of lipid mediators, cytoplasmic lipid droplets have been shown to function as a major intracellular pool of arachidonic acid, the precursor for eicosanoid biosynthesis. Recent studies have revealed that not only phospholipids in mast cell membranes, but also triglycerides in mast cell lipid droplets are a substrate source for eicosanoid formation. The present review summarizes current knowledge about mast cell lipid droplet biology, and discusses expansions and challenges of traditional mechanistic models for eicosanoid production.

Summary The finP gene of plasmid R1 is located between the genes traM and traJ , partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5′ non‐translated sequences, the ribosome‐binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1‐19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R l‐19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1‐19 intrans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice). CGI-58−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL−/− macrophages. In contrast to ATGL−/− macrophages, however, CGI-58−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency. Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice). CGI-58−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL−/− macrophages. In contrast to ATGL−/− macrophages, however, CGI-58−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency. Monocyte-derived macrophages are present in virtually all tissues, where they remove apoptotic cells and cellular debris generated by tissue remodelling and/or cellular necrosis. Under pathophysiological conditions, macrophages play a key role during atherogenesis by internalizing modified lipoproteins or lipoprotein remnants that have invaded the vessel wall to form cholesterol-rich foam cells. This (along with other disease-related functions for this immune cell) has prompted profound research into the role of the macrophage, and how its functions are regulated during disease progression (1McLaren J.E. Michael D.R. Ashlin T.G. Ramji D.P. Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy.Prog. Lipid Res. 2011; 50: 331-347Crossref PubMed Scopus (278) Google Scholar). Comparative gene identification-58 (CGI-58) is the coactivator of adipose TG lipase (ATGL), the major TG hydrolase for the initial and rate-limiting step in lipolysis (2Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). Lipolysis has been extensively studied in adipocytes, where under basal conditions CGI-58 binds to the surface of lipid droplets through interaction with perilipin1. Hormonal stimulation of lipolysis leads to phosphorylation of perilipin1 and hormone-sensitive lipase, resulting in the release of CGI-58 from perilipin1 to interact with and activate ATGL, which then converts TG to diacylglycerol and FA (3Zechner R. Zimmermann R. Eichmann T.O. Kohlwein S.D. Haemmerle G. Lass A. Madeo F. FAT SIGNALS–lipases and lipolysis in lipid metabolism and signaling.Cell Metab. 2012; 15: 279-291Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). Both mice and humans affected with ATGL or CGI-58 deficiency suffer from systemic TG accumulation, a condition called neutral lipid storage disease (NLSD) in humans (4Schweiger M. Lass A. Zimmermann R. Eichmann T.O. Zechner R. Neutral lipid storage disease: genetic disorders caused by mutations in adipose triglyceride lipase/PNPLA2 or CGI-58/ABHD5.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E289-E296Crossref PubMed Scopus (215) Google Scholar). Of note, specific phenotypical alterations are observed depending on whether ATGL or CGI-58 is defective. The most apparent difference is the severe epidermal skin defect observed in mice and humans with CGI-58 deficiency (2Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 5Radner F.P. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), which is absent in both species lacking ATGL (6Fischer J. Lefevre C. Morava E. Mussini J.M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Crossref PubMed Scopus (365) Google Scholar, 7Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar). This finding resulted in different classifications of the respective human diseases, namely NLSD with myopathy in ATGL deficiency (6Fischer J. Lefevre C. Morava E. Mussini J.M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Crossref PubMed Scopus (365) Google Scholar), whereas CGI-58 deficiency leads to NLSD with ichthyosis (8Lefèvre C. Jobard F. Caux F. Bouadjar B. Karaduman A. Heilig R. Lakhdar H. Wollenberg A. Verret J.L. Weissenbach J. et al.Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.Am. J. Hum. Genet. 2001; 69: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Several ongoing studies using tissue-specific CGI-58 and ATGL-deficient (−/−) mice aim at studying the proteins' shared and individual contribution to lipid metabolism. In addition, functional differences observed in hepatocytes between mice with CGI-58 and ATGL deficiency argue for an ATGL-independent function of CGI-58 in this cell population (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Lord C.C. Brown J.M. Distinct roles for alpha-beta hydrolase domain 5 (ABHD5/CGI-58) and adipose triglyceride lipase (ATGL/PNPLA2) in lipid metabolism and signaling.Adipocyte. 2012; 1: 123-131Crossref PubMed Google Scholar) and maybe other tissues as well (11Zierler K.A. Zechner R. Haemmerle G. Comparative gene identification-58/alpha/beta hydrolase domain 5: more than just an adipose triglyceride lipase activator?.Curr. Opin. Lipidol. 2014; 25: 102-109Crossref PubMed Scopus (9) Google Scholar). Because CGI-58−/− mice die shortly after birth due to a severe skin barrier defect (5Radner F.P. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), we generated myeloid-specific CGI-58 (macCGI-58)−/− mice to investigate the consequences of CGI-58 deficiency in macrophages. In the present study, we examined: i) whether CGI-58−/− macrophages mimic the TG accumulation phenotype observed in ATGL−/− macrophages; ii) whether CGI-58 deficiency affects macrophage function; and iii) whether the altered phenotype culminates in increased atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. We have previously shown that loss of ATGL in macrophages affects macrophage phenotype and function, such as TG-rich lipid droplet accumulation, increased apoptosis (12Aflaki E. Radovic B. Chandak P.G. Kolb D. Eisenberg T. Ring J. Fertschai I. Uellen A. Wolinski H. Kohlwein S.D. et al.Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages.J. Biol. Chem. 2011; 286: 7418-7428Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and endoplasmic reticulum (ER) stress (13Aflaki E. Doddapattar P. Radovic B. Povoden S. Kolb D. Vujic N. Wegscheider M. Koefeler H. Hornemann T. Graier W.F. et al.C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.Cell Death Dis. 2012; 3: e280Crossref PubMed Scopus (42) Google Scholar), reduced migration (14Aflaki E. Balenga N.A. Luschnig-Schratl P. Wolinski H. Povoden S. Chandak P.G. Bogner-Strauss J.G. Eder S. Konya V. Kohlwein S.D. et al.Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis.Cell. Mol. Life Sci. 2011; 68: 3933-3947Crossref PubMed Scopus (52) Google Scholar), and decreased phagocytosis ability (15Chandak P.G. Radovic B. Aflaki E. Kolb D. Buchebner M. Frohlich E. Magnes C. Sinner F. Haemmerle G. Zechner R. et al.Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.J. Biol. Chem. 2010; 285: 20192-20201Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In addition, transplantation of ATGL−/− bone marrow into LDL receptor (LDLR)−/− mice revealed that the lack of ATGL in immune cells attenuates atherosclerosis susceptibility (16Lammers B. Chandak P.G. Aflaki E. Van Puijvelde G.H. Radovic B. Hildebrand R.B. Meurs I. Out R. Kuiper J. Van Berkel T.J. et al.Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 67-73Crossref PubMed Scopus (40) Google Scholar). Being the coactivator of ATGL, we predicted that the absence of CGI-58 in macrophages leads to TG-rich lipid droplet accumulation. We hypothesized that loss of the ATGL coactivator CGI-58 in myeloid cells affects macrophage function in vitro and in vivo and impacts atherosclerosis susceptibility. Mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice) were generated by crossing CGI-58flox/flox mice (17Zierler K.A. Jaeger D. Pollak N.M. Eder S. Rechberger G.N. Radner F.P. Woelkart G. Kolb D. Schmidt A. Kumari M. et al.Functional cardiac lipolysis in mice critically depends on comparative gene identification-58.J. Biol. Chem. 2013; 288: 9892-9904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (provided by Dr. Guenther Haemmerle, University of Graz, Austria) with transgenic mice that express Cre recombinase under the control of the murine M lysozyme promoter (18Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1563) Google Scholar) (LysMCre, C57BL/6 background; provided by Dr. Thomas Ruelicke, University of Veterinary Medicine, Vienna, Austria). CGI-58flox/flox [wild-type (Wt)] mice were used as controls. Experiments were performed with female animals. To investigate atherosclerosis susceptibility, we generated CGI-58flox/flox/ApoE−/− (designated as ApoE−/−) and macCGI-58/ApoE-DKO mice by crossing CGI-58flox/flox and macCGI-58-KO mice with ApoE−/− mice (Jackson Laboratory, Bar Harbor, ME). For genotyping, the following primers were used: CGI-58flox/flox-forward, 5′-GTCATGGTTGT­GG­GGAAATC-3′; CGI-58flox/flox-reverse, 5′-GACTGGAAG­GA­TTT­GA­GGGG-3′; Cre-mut, 5′-CCCAGAAATGCCAGATTACG-3′; Cre-comm, 5′-CTTGGGCTGCCAGAATTTCTC-3′; Cre-Wt, 5′-TTACAGTCGGCCAGGCTGAC-3′; ApoE-forward, 5′-GCCTAGCCGAG­GGA­GA­GCCG-3′; ApoE-reverse, 5′-TGTGACTTGGGAGCTCTGCAGC-3′; and ApoE-neo, 5′-GCCGCCCCGACTGCATCT-3′. Female mice were either fed a standard chow diet [containing 4% fat and 21% protein (R/M H; Ssniff, Soest, Germany)], challenged with a Western type diet (WTD) [TD88137mod; 21% fat, 0.2% cholesterol (Ssniff)] or a high-fat/high-cholesterol diet (HF/HCD) (E15126-34 EF R/M; 30% fat, 1% cholesterol) for 10–30 weeks starting at the age of 4–6 weeks. Mice were kept with water ad libitum on a regular light-dark cycle (12 h light, 12 h dark) in a clean environment. Body weights were measured weekly and plasma lipid parameters once a month. For atherosclerosis studies using antisense oligonucleotide (ASO)-mediated knockdown of CGI-58, 6-week-old male LDLR−/− mice were fed a diet enriched in 0.2% (w/w) cholesterol and 20% of energy as lard for 16 weeks in conjunction with weekly injections (50 mg/kg) of either a nontargeting control ASO or CGI-58 ASO, as previously described (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Plasma samples were collected by submandibular vein puncture at baseline (chow-fed animals, 6 weeks of age), and after 4, 8, and 16 weeks of diet and ASO treatment for subsequent lipid and lipoprotein analyses. The majority of animal experiments were performed according to the standards set by the Austrian Federal Ministry of Science and Research, Division of Genetic Engineering and Animal Experiments, Vienna, Austria (BMWF-66.010/0039-II/10b/2009, BMWF-66.010/0057-II/3b/2011). The ASO-mediated knockdown studies of CGI-58 were conducted in an American Association for Accreditation of Laboratory Animal Care-approved animal facility, and all experimental protocols were approved by the Institutional Animal Care and Use Committee at either the Wake Forest University School of Medicine or the Cleveland Clinic Lerner Research Institute. Peritoneal macrophages were collected after an ip injection of 2.5 ml 3% thioglycolate. After 3 days, the peritoneum was flushed with 10 ml PBS containing 1 mM EDTA. The cells were cultivated in DMEM (Gibco, Invitrogen, Carlsbad, CA) containing 10% lipoprotein-deficient serum (LPDS) and 1% penicillin/streptomycin for 2–3 h. Thereafter, the cells were washed three times with prewarmed PBS and the adherent cells (macrophages) were cultured in DMEM containing 25 mM glucose, 4 mM glutamine, 1 mM pyruvate, 10% LPDS, and 1% penicillin/streptomycin for 24 h. Bone marrow-derived macrophages were isolated from femur and tibia flushed with sterile PBS. Cells were cultured in DMEM containing 10% LPDS, 1% penicillin/streptomycin, and 10 ng/ml macrophage colony-stimulating factor for 7 days. To assess in vitro lipopolysaccharide (LPS)-induced acute-phase response, macrophages were treated with saline (control) or LPS (100 ng/ml) for 16 h. IL-6 concentrations in supernatants were determined by ELISA (Enzo Life Sciences, Lausen, Switzerland). For the studies using ASO-mediated knockdown, elicited peritoneal macrophages were collected 4 days after injection of 1 ml of 10% thioglycolate into the peritoneal cavities of C57BL/6 mice that had been treated with ASOs and fed a chow diet for 6 weeks, as previously described (19Brown J.M. Chung S. Sawyer J.K. Degirolamo C. Alger H.M. Nguyen T. Zhu X. Duong M.N. Wibley A.L. Shah R. et al.Inhibition of stearoyl-coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis.Circulation. 2008; 118: 1467-1475Crossref PubMed Scopus (126) Google Scholar). Following 2 h of culture, nonadherent cells were removed by washing three times with PBS, and remaining adherent macrophages were harvested for Western blotting using methods previously described (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Blood was collected from 12 h-fasted mice or from 12 h-fasted/2 h-refed mice, and plasma was prepared by centrifugation at 5,200 g for 7 min at 4°C. Plasma TG, total cholesterol (TC), free cholesterol (FC), and nonesterified FA concentrations were measured enzymatically by commercially available kits (DiaSys, Holzheim, Germany; Wako Chemicals GmbH, Neuss, Germany). For atherosclerosis studies using ASO-mediated knockdown of CGI-58, total plasma concentrations of TC and TG were measured enzymatically by commercially available kits (Wako Chemicals, Richmond, VA). In addition, plasma lipoproteins were separated by fast protein liquid chromatography, and cholesterol concentrations in lipoprotein fractions were measured using an enzymatic assay as previously described (19Brown J.M. Chung S. Sawyer J.K. Degirolamo C. Alger H.M. Nguyen T. Zhu X. Duong M.N. Wibley A.L. Shah R. et al.Inhibition of stearoyl-coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis.Circulation. 2008; 118: 1467-1475Crossref PubMed Scopus (126) Google Scholar). Macrophages were plated for 2 h in serum-free DMEM. After washing the cells three times with PBS, lipids were extracted with 2 ml hexane:isopropanol (3:2, v:v) for 1 h at 4°C. One hundred microliters of 1% Triton X-100 in chloroform were added and the lipid extract was dried under a stream of nitrogen. The samples were dissolved in 100 μl ddH2O for 15 min at 37°C in a water bath. TG, TC, and FC concentrations were measured enzymatically by using 30 μl of the sample with the above mentioned kits. The readings were normalized to protein concentrations. Protein was quantitated using a Lowry assay (Bio-Rad Laboratories, Hercules, CA) after dissolving the proteins of cells in 2 ml NaOH (0.3 M) for 2 h at room temperature. FA composition in the TG fraction was quantitated by GC-flame ionization detection. Briefly, lipid extracts were separated by thin layer chromatography (hexane:diethylether:acetic acid, 70:30:1, v:v:v) and the band comigrating with tri-C16:0 TG was scraped, extracted with CHCl3/methanol (2:1, v:v), dried, and transesterified in BF3/toluene. Pentadecanoic acid was used as internal standard. Separation and quantitation were performed as previously described (20Sattler W. Puhl H. Hayn M. Kostner G.M. Esterbauer H. Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography: BF3/methanol transesterification of lyophilized samples instead of Folch extraction gives higher yields.Anal. Biochem. 1991; 198: 184-190Crossref PubMed Scopus (84) Google Scholar). Macrophages were plated on chamber slides in DMEM containing 10% LPDS and 1% penicillin/streptomycin for 24 h. Cells were washed three times with PBS and fixed with 10% formalin (30 min). Lipid droplets were visualized after Nile Red staining (2.5 μg/ml) by confocal laser scanning microscopy using an LSM 510 META microscope system (Carl Zeiss GmbH, Vienna, Austria). Pictures (×63 magnification) were taken at excitation 543 nm and signals were recorded using a 560 nm long pass filter. Macrophages were lysed with 100 μl of lysis buffer [100 mM potassium phosphate, 250 mM sucrose, 1 mM EDTA, 0.1 mM DTT (pH 7)], sonicated on ice twice for 10 s with 10 s interval, and protein concentrations were measured using a Lowry assay (BioRad Laboratories). The TG substrate contained 17 nmol triolein/assay and 2,000 cpm/nmol of [9,10-3H(N)]triolein (Perkin Elmer, Waltham, MA). The cholesteryl ester (CE) substrate contained 20 nmol cholesteryl oleate per assay and 1,000 cpm/nmol of cholesteryl [1-14C]oleate (Amersham Biosciences, Piscataway, NJ). Fifty micrograms of protein from cell lysates was mixed with 100 μl of substrate and incubated in a water bath for 1 h at 37°C. The reaction was stopped by the addition of 3.25 ml stop solution (methanol:chloroform:n-heptane, 10:9:7, v:v:v) and 1 ml of 0.1 M potassium carbonate and 0.1 M boric acid (pH 10.5) (21Schweiger M. Eichmann T.O. Taschler U. Zimmermann R. Zechner R. Lass A. Measurement of lipolysis.Methods Enzymol. 2014; 538: 171-193Crossref PubMed Scopus (114) Google Scholar). The tubes were vortexed for 10–15 s and centrifuged at 800 g for 20 min at 4°C. The radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting, and the release of FAs was calculated. Macrophages were incubated in 6-well plates with 300 μl medium, 2% FA-free BSA (Sigma-Aldrich, St. Louis, MO), and 2 units/ml of heparin for 1 h at 37°C under continuous shaking. For the substrate preparation per sample, 0.6 μCi [3H]triolein, 920 ng glycerol trioleate, and 0.1% Triton X-100 in chloroform were evaporated under a stream of nitrogen. Forty microliters of 1 M Tris-HCl (pH 8.6) and 80 μl ddH2O were added, and the mixture was sonicated six times (1 min on and 1 min off) on ice. Then 40 μl of heat-inactivated human serum containing ApoC-II as activator (obtained from a pool of donors, heated at 50°C for 1 h, and stored at 20°C) and 40 μl of 10% FA-free BSA were added to the substrate. Analysis was performed as previously described (15Chandak P.G. Radovic B. Aflaki E. Kolb D. Buchebner M. Frohlich E. Magnes C. Sinner F. Haemmerle G. Zechner R. et al.Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.J. Biol. Chem. 2010; 285: 20192-20201Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Total RNA from macrophages was isolated using a PerfectPure RNA cultured cell kit (5Prime, Hamburg, Germany). RNA concentrations were measured at 260 nm on a NanoDrop instrument (Thermo Scientific, Wilmington, DE). Two micrograms of total RNA were reverse transcribed by using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR was performed on a LightCycler 480 (Roche Diagnostics, Rotkreuz, Switzerland) using the QuantifastTM SYBR® Green PCR kit (Qiagen, Hilden, Germany). Amplification of murine hypoxanthine-guanine phosphoribosyltransferase (HPRT) as housekeeping gene was performed on all samples as internal controls for variations in mRNA amounts. Expression profiles and associated statistical parameters were determined using the public domain program Relative Expression Software Tool-REST 2008 (22Pfaffl M.W. Horgan G.W. Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google Scholar). Primer sequences are listed in the supplementary material. Protein samples of lysed macrophages from the different genotypes (40 or 50 μg protein/lane) were separated by SDS-PAGE (15%). Proteins were transferred to polyvinylidene difluoride or nitrocellulose membranes. Blots were incubated with monoclonal anti-mouse antibodies against β-actin (1:20,000) (Santa Cruz, Heidelberg, Germany), ABHD5/CGI-58 (1:1,000) (Abnova GmbH, Heidelberg, Germany), and CCAAT/enhancer-binding protein homologous protein (CHOP) (1:1,000) (Cell Signaling Technology, Danvers, MA), or anti-rabbit polyclonal antibodies against Bax (1:1,000), cytochrome C (1:1,000), inositol-requiring enzyme 1α (IRE1α) (1:1,000), CHOP (1:1,000), and ATGL (1:200) (Cell Signaling Technology). HRP-conjugated goat anti-rabbit (1:5,000) or rabbit anti-mouse antibodies (1:1,000) (Dako, Glostrup, Denmark) were visualized by enhanced chemiluminescence detection (ClarityTM Western ECL substrate; Bio-Rad) using a ChemiDocTM MP imaging system (Bio-Rad). Macrophages were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience®, North Billerica, MA) at a density of 60,000 cells per well. After 24 h, cells were washed and preincubated for 30 min in XF assay medium supplemented with sodium pyruvate (1 mM) with or without glutamine (2 mM) and glucose (25 mM) at 37°C in a nonCO2 environment. The oxygen consumption rate (OCR) was subsequently measured every 7 min using an XF96 extracellular flux analyzer (Seahorse Bioscience®). A standard protocol with 15 min basal measurement followed by 10 μM oligomycin, addition of 0.3 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 2.5 μM antimycin A was performed. Oxygen consumption was either normalized to protein content (pmol O2/min × μg protein) or expressed as a percentage of the maximal mitochondrial respiration in the presence of 0.3 μM FCCP. Macrophages were plated in black 96-well μClear plates (Greiner Bio-One GmbH, Solingen, Germany). After 24 h of preincubation in DMEM, 10% LPDS, and 25 mM glucose, cells were incubated in DMEM, 10% LPDS, and 0, 6, or 25 mM glucose for 1 h, respectively. Cells were washed and incubated with 100 μl of fluorescein-labeled E. coli BioParticles (VybrantTM phagocytosis assay, Molecular Probes, Invitrogen; suspended in Hanks' balanced salt solution; 2 h). The suspension was removed and subsequently 100 μl of trypan blue was added (1 min) to quench the extracellular probe. After aspiration of trypan blue, the fluorescence was measured at 484 nm (excitation) and 535 nm (emission) on a Victor 1420 multilabel counter (PerkinElmer Life Sciences, Turku, Finland). Fluorescence was normalized to the protein content of each well. To analyze phagocytosis in vivo, mice were injected intraperitoneally with 200 μl of fluorescein-labeled E. coli BioParticles suspended in Hanks' balanced salt solution. After 2 h, macrophages were collected by flushing the peritoneal cavity with 10 ml PBS containing 1 mM EDTA and incubated in DMEM containing 25 mM glucose and 10% LPDS for 90 min. The cells were washed three times with PBS, and fluorescence was measured before and after adding trypan blue to obtain total and intracellular fluorescence, respectively. Experimental readings were normalized to protein content. Apoptosis was assayed by annexin V and propidium iodide (PI) costaining (Annexin-V-Fluor staining kit; Roche, Vienna, Austria). Two hundred thousand cells were washed twice with 200 μl PBS; 50 μl staining buffer was added and cells were incubated for 10 min. Macrophages were immediately analyzed on a FACScalibur flow cytometer (BD Biosciences, San José, CA). Animals were fasted for 6 h (6 AM to 12 PM) with free access to drinking water. Blood was taken from the tail vein before and 15, 30, 60, 120, and 180 min after an ip injection of glucose (2.0 g/kg body weight). Glucose concentrations from blood were determined using a portable glucometer (AccuCheck). We analyzed atherosclerotic lesions in the aortic root and aorta of ApoE−/− and macCGI-58/ApoE-DKO animals after 10 weeks of HF/HCD feeding. Mice were euthanized and the arterial tree was perfused in situ with PBS (100 mm Hg) for 10 min via a cannula in the left ventricular apex. Mice were perfused with 10% formalin (Carl Roth GmbH, Vienna, Austria) for 15 min. After fixing the hearts in 10% formalin, serial sections (8 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany). Images of the atherosclerotic lesion areas in Oil Red O-stained (Sigma-Aldrich) sections were taken with ScanScope T3 whole slide scanner (Aperio Technologies, Bristol, UK). Plaque areas were quantitated by ImageJ software. Mean lesion area was calculated from 10 consecutive Oil Red O-stained sections, starting at the appearance of the tricuspid valves. Sections were stained immunohistochemically for the presence of macrophages using a monoclonal rat anti-mouse Moma-2 antibody (1:600) (Acris, Hiddenhausen, Germany), as well as for collagen content using Masson's trichrome staining kit (Sigma-Aldrich). For en face analysis in macCGI-58/ApoE-DKO mice, aortas were dissected and plaques were stained with Oil Red O as described recently (23Kratzer A. Buche

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis “master-regulator” PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.

The synthesis, structure, photophysics, and spectroscopic characterization of an organometallic rhenium multichromophore compound carrying a central 2,2′-bipyridyl acceptor moiety with additional phenylethynyl substituents conjugated at the 4,4′-positions of the acceptor ligands and its effect on the electron density of the central rhenium atom as metal center for CO2 reduction is reported. The results were compared to fac-(2,2′-bipyridyl)Re(CO)3Cl and fac-(5,5′-bisphenylethynyl-2,2′-bipyridyl)Re(CO)3Cl. Cyclovoltammetric studies and rotating disk electrochemistry were performed for electrochemical characterization. Ultraviolet and visible (UV-vis) absorption, Fourier transform infrared (FTIR), and luminescence measurements were carried out for a spectroscopic characterization and compared to theoretical calculations at the density functional theory (DFT) level. In addition, the rhenium complex fac-(4,4′-bisphenyl-ethynyl-2,2′-bipyridyl)-Re(CO)3Cl was used as a novel catalyst for the electrochemical reduction of CO2 in homogeneous solution. Results showed an 11-fold increase in the current density under CO2 saturation and a catalytic second-order rate constant for CO formation of about 560 M−1 s−1 on a Pt working electrode. For further characterization of the CO2 reduction capabilities, bulk controlled potential electrolysis experiments were performed using a CO2-saturated acetonitrile electrolyte solution. The headspace product gas analysis yields CO as main reduction product with faradaic efficiencies of about 12 % over 5-h electrolysis time.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with that of cells infected with wild-type virus. Switching to Cp, as well as EBNA1 gene transcription, was observed upon infection of EBV-negative Burkitt's lymphoma (BL) cell lines with EBNA2- EBNA4mut virus, thus establishing a correlation between early EBNA1 gene transcription and upregulation of transcription initiation from Cp. However, in EBV-negative BL cell lines infected with EBNA2- EBNA4mut virus, transcription of the EBNA1 gene at early time points postinfection initiated from Qp, the EBNA1 gene promoter active in group I BL cells (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995), rather than from Wp. The data support a model in which EBNA1 plays an important role in the cascade of events leading to successful switching from Wp to Cp and subsequent immortalization of the infected B cell.

Abstract Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO 2 ‐reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate–silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO 2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO 2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid‐injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %.

Third-generation polythiophenes, bearing Rhenium carbonyl complexes and pyridinium as pendant active sites were used to drive the photoelectrochemical reduction of CO2. Cyclic voltammetry and controlled potential electrolysis experiments were performed in CO2-saturated acetonitrile, acetonitrile-water and aqueous solutions under illumination as well as in dark. The formation of CO was confirmed with IR spectroscopy and quantified with gas chromatography in the case of poly-[Re-(4-methyl-4′-(7-(thiophen-3-yl)heptyl)-2,2′-bipyridyl)tricarbonylchloride] giving a max. Faradaic efficiency of 2.5% and a Turnover number of ∼20. In the case of poly- [4-(7-(thiophen-3-yl)heptyl)pyridine] which was supposed to catalyse reduction reaction of CO2 to MeOH, no products were observed. Although the initial efficiencies are not high due to the surface limited processes, this immobilization of such catalysts on the photoelectrode might be industrially attractive.

Abstract Interferon-based therapies, such as ropeginterferon alfa-2b have emerged as promising disease-modifying agents for myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET). Current ET treatments aim to normalize hematological parameters and reduce the thrombotic risk, but they do not modify the natural history of the disease and hence, have no impact on disease progression. Ropeginterferon alfa-2b (trade name BESREMi®), a novel, monopegylated interferon alfa-2b with an extended administration interval, has demonstrated a robust and sustained efficacy in polycythemia vera (PV) patients. Given the similarities in disease pathophysiology and treatment goals, ropeginterferon alfa-2b holds promise as a treatment option for ET. The ROP-ET trial is a prospective, multicenter, single-arm phase III study that includes patients with ET who are intolerant or resistant to, and/or are ineligible for current therapies, such as hydroxyurea (HU), anagrelide (ANA), busulfan (BUS) and pipobroman, leaving these patients with limited treatment options. The primary endpoint is a composite response of hematologic parameters and disease-related symptoms, according to modified European LeukemiaNet (ELN) criteria. Secondary endpoints include improvements in symptoms and quality of life, molecular response and the safety profile of ropeginterferon alfa-2b. Over a 3-year period the trial assesses longer term outcomes, particularly the effects on allele burden and clinical outcomes, such as disease-related symptoms, vascular events and disease progression. No prospective clinical trial data exist for ropeginterferon alfa-2b in the planned ET study population and this study will provide new findings that may contribute to advancing the treatment landscape for ET patients with limited alternatives. Trial registration EU Clinical Trials Register; EudraCT, 2023-505160-12-00; Registered on October 30, 2023.

A new total synthesis of (–)-hennoxazole A is reported. The synthetic approach is based on the preparation of three similarly sized fragments resulting in a fast and convergent assembly of the natural product. The three key reactions of the synthesis include a highly stereoselective 1,5-<i>anti</i> aldol coupling, a gold-catalyzed alkoxycyclization reaction, and a stereocontrolled diene cross-meta­thesis. The synthesis involves integrated batch and flow chemistry methods leading to the natural product in 16 steps longest linear ­sequence and 2.8% overall yield.

Electron-phonon interactions of free charge-carriers in doped pi-conjugated polymers are conceptually described by 1-dimensional (1D) delocalization. Thereby, polaronic transitions fit the 1D-Froehlich model in quasi-confined chains. However, recent developments in conjugated polymers have diversified the backbones to become elaborate heterocylcic macromolecules. Their complexity makes it difficult to investigate the electron-phonon coupling. In this work we resolve the electron-phonon interactions in the ground and doped state in a complex push-pull polymer. We focus on the polaronic transitions using in-situ spectroscopy to work out the differences between single-unit and push-pull systems to obtain the desired structural- electronic correlations in the doped state. We apply the classic 1D-Froehlich model to generate optical model fits. Interestingly, we find the 1D-approach in push-pull polarons in agreement to the model, pointing at the strong 1D-character and plain electronic structure of the push-pull structure. In contrast, polarons in the single-unit polymer emerge to a multi- dimensional problem difficult to resolve due to their anisotropy. Thus, we report an enhancement of the 1D-character by the push-pull concept in the doped state - an important view in light of the main purpose of push-pull polymers for photovoltaic devices.

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

ABSTRACT An anthracene‐containing poly(arylene‐ethynylene)‐ alt ‐poly(arylene‐vinylene) (PAE‐PAV) of general constitutional unit (PhCCAnthrCCPhCHCHAnthrCHCH) n bearing two 2‐ethylhexyloxy solubilizing side chains on each phenylene (Ph) unit has been synthesized and characterized. The basic electrochemical characterization was done, showing the existence of two non‐reversible oxidation and one reversible reduction peaks. The optical properties, the real and imaginary part of the dielectric function, were probed using spectroscopic ellipsometry (SE). The vibrational structure of the undoped/doped polymer was investigated using Fourier transformed infrared spectroscopy. A strong change in the polaronic absorption was observed during the doping, which after modeling revealed the existence of two separated transitions. The optical changes upon doping were additionally recorded using the SE technique. Similar to the results from FT‐IR spectroscopy, two new in‐the‐gap absorptions were found. Moreover, the electrical conductivity as well as the mobility of positive carriers were measured. In the undoped state, the conductivity of the polymer was found to be below the detection limit (&lt;μS cm −1 ), after doping the conductivity increased to 0.69 S cm −1 . © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52 , 338–346

According to genome-wide RNA sequencing data from human and mouse platelets, adipose triglyceride lipase (ATGL), the main lipase catalyzing triglyceride (TG) hydrolysis in cytosolic lipid droplets (LD) at neutral pH, is expressed in platelets. Currently, it is elusive to whether common lipolytic enzymes are involved in the degradation of TG in platelets. Since the consequences of ATGL deficiency in platelets are unknown, we used whole-body and platelet-specific (plat)Atgl-deficient (-/-) mice to investigate the loss of ATGL on platelet function. Our results showed that platelets accumulate only a few LD due to lack of ATGL. Stimulation with platelet-activating agonists resulted in comparable platelet activation in Atgl-/-, platAtgl-/-, and wild-type mice. Measurement of mitochondrial respiration revealed a decreased oxygen consumption rate in platelets from Atgl-/- but not from platAtgl-/- mice. Of note, global loss of ATGL was associated with an anti-thrombogenic phenotype, which was evident by reduced thrombus formation in collagen-coated channels in vitro despite unchanged bleeding and occlusion times in vivo. We conclude that genetic deletion of ATGL affects collagen-induced thrombosis without pathological bleeding and platelet activation.

The final step of triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs). We have previously shown that ApoE-/-Dgat1-/- mice are protected from developing atherosclerosis in association with reduced foam cell formation. However, the role of DGAT1, specifically in myeloid and other hematopoietic cell types, in determining this protective phenotype is unknown. To address this question, we reconstituted the bone marrow of irradiated Ldlr–/–mice with that from wild-type (WT→ Ldlr–/–) and Dgat1–/–(Dgat1–/–→ Ldlr–/–) donor mice. We noted that DGAT1 in the hematopoietic compartment exerts a sex-specific effect on systemic cholesterol homeostasis. However, both male and female Dgat1–/–→ Ldlr–/–mice had higher circulating neutrophil and lower lymphocyte counts than control mice, suggestive of a classical inflammatory phenotype. Moreover, specifically examining the aortae of these mice revealed that Dgat1–/–→ Ldlr–/–mice have atherosclerotic plaques with increased macrophage content. This increase was coupled to a reduced plaque collagen content, leading to a reduced collagen-to-macrophage ratio. Together, these findings point to a difference in the inflammatory contribution to plaque composition between Dgat1–/–→ Ldlr–/–and control mice. By contrast, DGAT1 deficiency did not affect the transcriptional responses of cultured macrophages to lipoprotein treatment in vitro, suggesting that the alterations seen in the plaques of Dgat1–/–→ Ldlr–/–mice in vivo do not reflect a cell intrinsic effect of DGAT1 in macrophages. We conclude that although DGAT1 in the hematopoietic compartment does not impact the overall lipid content of atherosclerotic plaques, it exerts reciprocal effects on inflammation and fibrosis, two processes that control plaque vulnerability.

Pyridine (1) and pyridazine (2) are investigated as electrocatalysts for the reduction of CO2 to methanol in aqueous electrolyte solution by E. Portenkirchner et al. on p. 1543. The picture shows the cyclic voltammogram of the one-electron reduction of pyridinium at different scan rates.

Objectives: Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading monoacylglycerol (MG) to glycerol and free fatty acid. MGL also deactivates the endocannabinoid 2-arachidonoyl glycerol (2-AG), which acts as a full agonist on both cannabinoid receptor (CBR) 1 and 2, predominantly expressed in brain and immune cells, respectively. Taking into account the role of MGL in lipolysis and the multiple effects of the cannabinoid system on lipid homeostasis and immunity, the lack of MGL may promote or prevent lipotoxicity and atherosclerosis.

Objectives: Monoglyceride lipase (MGL) mediates the last step of lipolysis by hydrolyzing monoglyceride to glycerol and fatty acid. Among substrates for MGL activity is 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid in mammals. 2-AG acts as full agonist on cannabinoid receptors (CBR) 1 and 2, which are biphasically distributed in brain and immune cells, respectively. Based on the direct involvement of MGL in lipolysis and its indirect effects on endocannabinoid homeostasis and thus immune response, we hypothesized that MGL deficiency modulates CB2R-mediated signaling and atherogenesis.

Objectives: Lysosomal acid lipase (LAL) is highly expressed in mouse peritoneal macrophages (MPM) and hydrolyzes exogenous (via lipoproteins) or endogenous (via autophagy) originated triacylglycerols (TG) and cholesteryl esters (CE). Human LAL deficiency causes severe atherosclerotic lesions and macrophages play a central role in the pathology evolution, becoming hyperplasic and hypertrophic with increasing lysosomal lipid accumulation. We used macrophages from LAL deficient (Lal-/-) mice to investigate the role of the enzyme in lipid metabolism.

Diese Diplomarbeit behandelt das Thema e-Tandem via Skype. Die Autorin versucht zu zeigen, auf welche Art e-Tandem-Sprachaustausche im schulischen Fremdsprachenunterricht integriert werden konnen und inwiefern dies zum Erwerb sprachlicher und interkultureller Kompetenzen sowie jener zum autonomen Fremdsprachenlernen beitragen kann. So werden im theoretischen Teil der Arbeit zunachst die rechtlichen Rahmenbedingungen fur den Schulunterricht diskutiert. Danach werden die neuesten Erkenntnisse zum nachhaltigen Kompetenzerwerb im Fremdsprachenunterricht prasentiert, wobei der Fokus vor allem auf die konstruktivistische Auffassung dessen gerichtet ist. Anschliesend werden die Tandem-Methode naher vorgestellt, Aspekte des e-Learnings erortert sowie Studien, welche den positiven Nutzen von e-Tandem bestatigen, prasentiert. Im praktischen Teil der Arbeit wird anschliesend auf Grundlage dessen ein didaktisches Konzept fur das Jahresprojekt e-Tandem Sprachaustausch vorgelegt. Dieses beinhaltet die Projektplanung – also die zeitliche Grobplanung, die Aufgaben der Akteure sowie die Evaluierungsmethode – Vorschlage zur Suche nach Tandempartnerinnen und Tandempartnern sowie die dafur notigen Materialien und deren didaktische Begrundung.

Abstract The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). It is a DNA-binding protein that represses gene transcription through the recruitment of co-repressor proteins via its BTB domain. Disruption of the interaction of BCL6 with co-repressor proteins interferes with the physiological and oncogenic functions of BCL6. We have used structure-based drug desgin to develop potent compounds that block this interaction with half maximal inhibitory concentration values (IC50s) below 3 nM. In addition to inhibiting co-repressor binding, a subset of the identified inhibitors also caused rapid disappearance of BCL6 protein in cells. This effect was mediated by compound-induced multi-ubiquitylation of BCL6 and degradation by the proteasome and was dependent on the presence of a functional DNA binding domain on BCL6. Compounds that induced BCL6 degradation displayed significantly stronger induction of expression of BCL6-repressed genes than compounds that merely inhibited co-repressor interaction. BCL6-degrading inhibitors had anti-proliferative effects in several DLBCL cell lines, while non-degrading BCL6 inhibitors only had minor effects on proliferation. The fact that the magnitude of effects elicited by this novel class of BCL6 degrading compounds greatly exceeds that of our equipotent classical co-repressor interaction inhibitors offers exciting new opportunities for the development of BCL6-based lymphoma therapeutics. Citation Format: Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Helmut Berger, Sophia Blake, Maureen Caligiuri, John Engen, Peter Ettmayer, Thomas Gerstberger, Daniel Gerlach, Roxana Jacob, Simon Lucas, Moriz Mayer, Mischerikow Nikolai, Rumpel Klaus, Scharn Dirk, Schnitzer Renate, Tilman Voss, Zoephel Andreas, Norbert Kraut, Darryl McConnell, Pearson Mark, Manfred Koegl. Chemically induced degradation of the oncogenic transcription factor BCL6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2017-1525

The Cover Feature illustrates the consequences of CO2 release into the atmosphere from man-made sources. With humanity re-emitting, within a couple of decades, large amounts of CO2 that was fixated in millions of years though natural photosynthesis, there is a clear need for cyclic usage of CO2 to create a sustainable future employing photo- and bio-electrocatalysis. More information can be found in the Review by D. H. Apaydin et al. on page 3094 in Issue 22, 2017 (DOI: 10.1002/cphc.201700148).

Lipoproteinrezeptoren sind Plasmamembranproteine, welche fur die spezifische Bindung und Aufnahme der im Blutkreislauf zirkulierenden Lipoproteine in hepatische und extrahepatische Gewebe verantwortlich sind. Dieser physiologische Vorgang wird als rezeptorvermittelte Endozytose bezeichnet und basiert auf der Interaktion zwischen Mitgliedern der sogenannten Low Density Lipoprotein (LDL) Rezeptor Familie und Lipoproteinen im Plasma. Der als erster entdeckte und am besten charakterisierte Lipoproteinrezeptor ist der LDL Rezeptor (LDLR). Dieser Rezeptor spielt eine wichtige Rolle in der Regulation der Cholesterinspiegel durch die Bindung und Internalisierung von Lipoproteinen, welche Apolipoprotein- B (apoB) und /oder -E (apoE) als Proteinkomponente besitzen. In der vorliegenden Arbeit wurde das legende Huhn, ein gut etablierter Modellorganismus zur Erforschung des Lipidstoffwechsels, herangezogen um die Interaktion des LDL Rezeptors mit Apolipoprotein B- und -E haltigen Lipoproteinen naher zu charakterisieren. Fruheren Studien zufolge gibt es zahlreiche Oberflachenrezeptoren im Huhn, die eine deutliche Homologie zur Familie der LDL Rezeptorproteine von Saugern aufweisen. Der zum menschlichen LDL Rezeptor homologe Rezeptor im Huhn wurde erstmals im Jahr 2003 beschrieben und weist die klassischen Strukturmerkmale der Sauger-LDL Rezeptoren auf, was auf eine hohe evolutionare Konservierung deutet. Alle Mitglieder der LDL Rezeptorfamilie besitzen einen charakteristischen modularen Aufbau aus vier bis funf unterschiedlichen Domanen. Die amino-terminale Region enthalt die Ligandenbindungsdomane des Rezeptors und besteht aus mehreren cysteinreichen Abschnitten, den sogenannten LA-repeats, welche die Interaktion mit verschiedenen Proteinen, wie Apolipoprotein, Proteasen und Signalproteinen vermitteln. Mutationsanalysen zeigen, dass die einzelnen Repeats unterschiedliche funktionelle Bedeutungen bei der Ligandenbindung besitzen. Bindungsstudien im Huhnersystem ergaben, dass einige Rezeptoren der LDLR Familie Apolipoprotein E binden, ein in Saugern, jedoch nicht im Huhn produziertes Protein. Andere Rezeptoren wiederum besitzen diese Bindungseigenschaft nicht. Dieses unterschiedliche Verhalten weist darauf hin, dass bestimmte Variationen in der ligandenbindenden Domane des Rezeptors einen entscheidenden Einfluss auf die Ligandenbindung besitzen. In der vorliegenden Arbeit habe ich mich damit beschaftigt, die strukturellen Unterschiede zwischen ApoE-bindenden Rezeptoren und Rezeptoren, welche die Fahigkeit ApoE zu binden nicht besitzen, zu ermitteln. Gemas den Ergebnissen aus fruheren Mutationsanalysen wurde die Ligandenbindungsdomane des Huhner- LDLR an vermeintlich wichtigen Bindungsregionen mutiert. Die bakteriell exprimierten mutanten Rezeptorproteine wurden durch Affninitatschromatographie gereinigt und, um die physiologische Funktionsweise zu erlangen, einem Faltungsprozess unterzogen, welcher durch das molekulare Chaperon RAP (Receptor-associated Protein) unterstutzt wurde. Schlieslich wurden die Rezeptormutanten hinsichtlich der Ligandenbindung von Apolipoprotein B- und -E -haltigen Lipoproteinen untersucht. Diese Bindungsanalysen ergaben, dass das erste cysteinreiche LA Repeat (LA1) nicht an der Binding von Apolipoprotein B beteiligt scheint. Indessen fuhrten Mutationen in den Repeats 4 und 5 (LA4, LA5), sowie in der Linker-Region zwischen diesen Repeats zu erheblichen Einschrankung bezuglich der Bindung von ApoB-haltigen Lipoproteinen. Bindungsanalysen zur Erforschung der Interaktion zwischen ApoE und den Rezeptormutanten lieferten bisher keine reproduzierbaren Ergebnisse, und erfordern daher weitere Untersuchungen. Die Daten, die im Zuge dieser Arbeit durch Experimente im Huhn ermittelt wurden, tragen zum Verstandnis des molekularen Mechanismus der LDL Rezeptor-Ligand Interaktion bei und stehen im Einklang mit Untersuchungsergebnissen am menschlichen LDL Rezeptor, welche eine grose Bedeutung des funften LA Repeats (LA5) fur die Ligandenbindung beschreiben.

Abstract The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We have used structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also cause rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and antiproliferative effects than compounds that merely inhibited co-repressor interaction. The fact that the magnitude of effects elicited by this class of BCL6 degrading compounds greatly exceeds that of our equipotent non-degrading inhibitors offers exciting opportunities for the development of BCL6-based lymphoma therapeutics. To support further research, the most potent BCL6 degrading inhibitor is made freely available to the research community as an in vitro tool compound. Please see http://www.opnMe.com for further infos. Citation Format: Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Maureen Caligiuri, Christian Dank, John R. Engen, Peter Ettmayer, Daniel Gerlach, Thomas Gerstberger, Bingsong Han, Roxana E. Iacob, Dirk Kessler, David R. Lancia, Mayer Moriz, Nikolai Mischerikow, Klaus Rumpel, Renate Schnitzer, Tilman Voss, Xiaozhang Zheng, Andreas Zoephel, Norbert Kraut, Darryl McConnell, Mark Pearson, Manfred Koegl. Chemically induced degradation of the transcription factor BCL6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3354.

Background and Aims: In platelets, the presence of cytosolic lipid droplets and the role of their lipolytic machinery to degrade triglycerides are still elusive. According to genome-wide RNA-sequencing data from human platelets, the main neutral lipase adipose triglyceride lipase (ATGL) is expressed in platelets. However, its role in platelet function has never been addressed.