The fatty acid amide hydrolase (FAAH) gene variant rs324420 AA/AC is not associated with weight loss in a 1-year lifestyle intervention for obese children and adolescents

Adult obese carriers of the A allele of SNP rs324420 in the fatty acid amide hydrolase (FAAH) gene lose more weight and improve associated phenotypes better than non-carriers during an intervention. We aimed to replicate this finding in obese children and adolescents undergoing a one year lifestyle intervention (Obeldicks program). A total of 453 overweight and obese children and adolescents (10.8±2.6 years, BMI-SDS 2.4±0.5; 55% girls) were genotyped for rs324420 (C/A) by restriction fragment length polymorphism (RFLP) analysis. Participants were prescribed a balanced diet, containing 55 En% carbohydrates, 30 En% fat, and 15 En% proteins. Moreover, they took part in an exercise therapy once a week. Blood was taken at baseline and after 1 year of intervention. Anthropometric (height, weight, BMI, and BMI-SDS) and plasma parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol, triacylglycerides, glucose, insulin, and HOMA) as well as blood pressure were measured. Both mean BMI and BMI-SDS improved significantly. The mean systolic blood pressure was also lowered and concentrations of HDL-cholesterol increased significantly. However, none of the measured changes were associated with FAAH rs324420 AA/AC genotype. We did not detect evidence for an association of FAAH genotypes with weight reduction in overweight and obese children and adolescents. Hence, the previous finding in adults could not be confirmed. As the length (1 year as compared to 3 months) and mode of treatment (hypocaloric diet in adults vs. physical activity plus balanced meals) of the interventions varied, these parameters might have influenced the inconsistent results.     

Universal Kinetics of the Onset of Cell Spreading on Substrates of Different Stiffness

When plated onto substrates, cell morphology and even stem-cell differentiation are influenced by the stiffness of their environment. Stiffer substrates give strongly spread (eventually polarized) cells with strong focal adhesions and stress fibers; very soft substrates give a less developed cytoskeleton and much lower cell spreading. The kinetics of this process of cell spreading is studied extensively, and important universal relationships are established on how the cell area grows with time. Here, we study the population dynamics of spreading cells, investigating the characteristic processes involved in the cell response to the substrate. We show that unlike the individual cell morphology, this population dynamics does not depend on the substrate stiffness. Instead, a strong activation temperature dependence is observed. Different cell lines on different substrates all have long-time statistics controlled by the thermal activation over a single energy barrier ΔG ≈ 18 kcal/mol, whereas the early-time kinetics follows a power law ～t⁵. This implies that the rate of spreading depends on an internal process of adhesion complex assembly and activation; the operational complex must have five component proteins, and the last process in the sequence (which we believe is the activation of focal adhesion kinase) is controlled by the binding energy ΔG.     

Exposure to apoptotic activated CD4+ T cells induces maturation and APOBEC3G-mediated inhibition of HIV-1 infection in dendritic cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+) T cells (ApoInf) or apoptotic uninfected activated CD4(+) T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+) T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+) T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.     

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica: identification of a novel glycosylated lipoprotein required for virulence

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.     

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6^?/? mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6^?/? mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6^?/? mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6^?/? mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.     

Enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate. A study on the bioactive conformation and chiral recognition of a moth sex pheromone component

The enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate, which are conformationally constrained analogues of (Z)-5-decenyl acetate (1), a sex pheromone component of the turnip moth, Agrotis segetum, have been synthesized and tested using the electrophysiological single-sensillum technique. The analogues mimic a cisoid and transoid conformation of 1, respectively. In addition, the enantiomers of each of the cis- and trans-isomers are conformationally constrained analogues of enantiomeric cisoid and transoid conformations of 1. Thus, the compounds prepared and tested are well suited to investigate the nature of the bioactive conformation of the natural pheromone component 1 and the chiral sense of its interaction with the receptor. Electrophysiological single-sensillum recordings show that the activity of the most active cis-isomer, which has a (1S,4R)-configuration, is more than two orders of magnitude higher than that of the most active trans-isomer. Furthermore, the (1S,4R)-isomer is at least 100 times more active than its enantiomer. These results strongly support a previously proposed cisoid bioactive conformation of 1. Furthermore, the (1S,4R)-configuration of most active stereoisomer identifies the chiral sense of the interaction between the natural pheromone component 1 and its receptor.     

Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.Subject terms: Ubiquitylation, Screening