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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.

Carbon dioxide has evolved from being considered as a greenhouse gas to a valuable carbon feedstock for the generation of artificial fuels and valuable chemicals. In this work, we review the biocatalytic approaches towards CO2 conversion into chemicals and fuels. We display the opportunities and challenges of using biocatalysts. Our work especially focuses on bio-electrocatalytic systems. These electrochemical applications of biocatalysts gain increasing interest, as electrochemical redox processes can avoid expensive mediators and co-factors. This is also a pathway for renewable energy storage because wind or solar energy can possibly be applied as electrical sources for the electrochemical CO2 conversion systems. Biocatalytic CO2 conversion together with renewable energy storage represents a viable and sustainable route for the generation of chemicals and fuels.

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 [3H]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.

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.

Summary 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.

A broad review of homogeneous and heterogeneous catalytic approaches toward CO2 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.

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.

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.

Background 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. Methods 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. Results 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. Conclusions 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.

We present a study on a microbial electrolysis cell with methanogenic microorganisms adapted to reduce CO2 to CH4 with the direct injection of electrons and without the artificial addition of H2 or an additional carbon source except gaseous CO2. This is a new approach in comparison to previous work in which both bicarbonate and gaseous CO2 served as the carbon source. The methanogens used are known to perform well in anaerobic reactors and metabolize H2 and CO2 to CH4 and water. This study shows the biofilm formation of those microorganisms on a carbon felt electrode and the long‐term performance for CO2 reduction to CH4 using direct electrochemical reduction. CO2 reduction is performed simply by electron uptake with gaseous CO2 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 CH4 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.

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.

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.

We present results for direct bio‐electrocatalytic reduction of CO2 to C1 products using electrodes with immobilized enzymes. Enzymatic reduction reactions are well known from biological systems where CO2 is selectively reduced to formate, formaldehyde, or methanol at room temperature and ambient pressure. In the past, the use of such enzymatic reductions for CO2 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 CO2 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.

Lipid mediator release depends on the hydrolysis of triglyceride-rich lipid droplets mediated by ATGL, a potent regulator of inflammatory diseases., 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.

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.

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 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