Endogenous NUCB2/Nesfatin-1 Regulates Energy Homeostasis Under Physiological Conditions in Male Rats.

Nesfatin-1 is the proteolytic cleavage product of Nucleobindin 2, which is expressed both in a number of brain nuclei (e. g., the paraventricular nucleus of the hypothalamus) and peripheral tissues. While Nucleobindin 2 acts as a calcium binding protein, nesfatin-1 was shown to affect energy homeostasis upon central nervous administration by decreasing food intake and increasing thermogenesis. In turn, Nucleobindin 2 mRNA expression is downregulated in starvation and upregulated in the satiated state. Still, knowledge about the physiological role of endogenous Nucleobindin 2/nesfatin-1 in the control of energy homeostasis is limited and since its receptor has not yet been identified, rendering pharmacological blockade impossible. To overcome this obstacle, we tested and successfully established an antibody-based experimental model to antagonize the action of nesfatin-1. This model was then employed to investigate the physiological role of endogenous Nucleobindin 2/nesfatin-1. To this end, we applied nesfatin-1 antibody into the paraventricular nucleus of satiated rats to antagonize the presumably high endogenous Nucleobindin 2/nesfatin-1 levels in this feeding condition. In these animals, nesfatin-1 antibody administration led to a significant decrease in thermogenesis, demonstrating the important role of endogenous Nucleobindin 2/nesfatin-1in the regulation of energy expenditure. Additionally, food and water intake were significantly increased, confirming and complementing previous findings. Moreover, neuropeptide Y was identified as a major downstream target of endogenous Nucleobindin 2/nesfatin-1.

Enoyl-Coenzyme A Respiration via Formate Cycling in Syntrophic Bacteria.

Syntrophic bacteria play a key role in the anaerobic conversion of biological matter to methane. They convert short-chain fatty acids or alcohols to H2, formate, and acetate that serve as substrates for methanogenic archaea. Many syntrophic bacteria can also grow with unsaturated fatty acids such as crotonate without a syntrophic partner, and the reducing equivalents derived from the oxidation of one crotonate to two acetate are regenerated by the reduction of a second crotonate. However, it has remained unresolved how the oxidative and reductive catabolic branches are interconnected and how energy may be conserved in the reductive branch. Here, we provide evidence that during axenic growth of the syntrophic model organism Syntrophus aciditrophicus with crotonate, the NAD+-dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA is coupled to the reduction of crotonyl-CoA via formate cycling. In this process, the intracellular formate generated by a NAD+-regenerating CO2 reductase is taken up by a periplasmic, membrane-bound formate dehydrogenase that in concert with a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone oxidoreductase, ETF, and an acyl-CoA dehydrogenase reduces intracellular enoyl-CoA to acyl-CoA. This novel type of energy metabolism, referred to as enoyl-CoA respiration, generates a proton motive force via a methylmenaquinone-dependent redox-loop. As a result, the beneficial syntrophic cooperation of fermenting bacteria and methanogenic archaea during growth with saturated fatty acids appears to turn into a competition for formate and/or H2 during growth with unsaturated fatty acids. IMPORTANCE The syntrophic interaction of fermenting bacteria and methanogenic archaea is important for the global carbon cycle. As an example, it accomplishes the conversion of biomass-derived saturated fatty acid fermentation intermediates into methane. In contrast, unsaturated fatty acid intermediates such as crotonate may serve as growth substrate for the fermenting partner alone. Thereby, the reducing equivalents generated during the oxidation of one crotonate to two acetate are regenerated by reduction of a second crotonate to butyrate. Here, we show that the oxidative and reductive branches of this pathway are connected via formate cycling involving an energy-conserving redox-loop. We refer to this previously unknown type of energy metabolism as to enoyl-CoA respiration with acyl-CoA dehydrogenases serving as cytoplasmic terminal reductases.

Functional diversity of prokaryotic HdrA(BC) modules: Role in flavin-based electron bifurcation processes and beyond.

In methanogenic archaea, the archetypical complex of heterodisulfide reductase (HdrABC) and hydrogenase (MvhAGD) couples the endergonic reduction of CO2 by H2 to the exergonic reduction of the CoB-S-S-CoM heterodisulfide by H2 via flavin-based electron bifurcation. Presently known enzymes containing HdrA(BC)-like components play key roles in methanogenesis, acetogenesis, respiratory sulfate reduction, lithotrophic reduced sulfur compound oxidation, aromatic compound degradation, fermentations, and probably many further processes. This functional diversity is achieved by a modular architecture of HdrA(BC) enzymes, where a big variety of electron input/output modules may be connected either directly or via adaptor modules to the HdrA(BC) components. Many, but not all HdrA(BC) complexes are proposed to catalyse a flavin-based electron bifurcation/confurcation. Despite the availability of HdrA(BC) crystal structures, fundamental questions of electron transfer and energy coupling processes remain. Here, we address the common properties and functional diversity of HdrA(BC) core modules integrated into electron-transfer machineries of outstanding complexity.

The Outcome of Ex Vivo TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic In Vitro Growth Capacity between T-Cell Clones.

PURPOSE: During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded in vitro and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the in vitro expansion. EXPERIMENTAL DESIGN: We compared the clonal composition of TIL preparations before and after in vitro expansion using T-cell receptor (TCR) deep sequencing. Our findings for PDA were benchmarked to experiments with melanoma TILs. RESULTS: We found that the TIL TCR repertoire changes dramatically during in vitro expansion, leading to loss of tumor- dominant T-cell clones and overgrowth by newly emerging T-cell clones that are barely detectable in the tumor. These changes are primarily driven by differences in the intrinsic in vitro expansion capacity of T-cell clones. Single-cell experiments showed an association between poor proliferative capacity and expression of markers related to antigen experience and dysfunction. Furthermore, we found that spatial heterogeneity of the TIL repertoire resulted in TCR repertoires that are greatly divergent between TIL cultures derived from distant tumor samples of the same patient. CONCLUSIONS: Culture-induced changes in clonal composition are likely to affect tumor reactivity of TIL preparations. TCR deep sequencing provides important insights into the factors that govern the outcome of in vitro TIL expansion and thereby a path toward optimization of the production of TIL preparations with high therapeutic efficacy.See related commentary by Lozano-Rabella and Gros, p. 4177.