Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

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.     

Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H ^attP, reintroduced a wild type H genomic and a cDNA-construct (H ^gwt, H ^cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H ^LD, H ^iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N ⁵⁴¹⁹) and Delta (Dl ^B2) mutants were addressed. Overall, phenotypes were largely as expected: both H ^LD and H ^iD were similar to the H ^attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H ^gwt and H ^cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H ^cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H ^gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Infection of Coscinodiscus spp. by the parasitoid nanoflagellate Pirsonia diadema: II. Selective infection behaviour for host species and individual host cells

The parasitoid nanoflagellate (PNF) Pirsonia diadema is hostspecific for the marine centric diatom Coscinodiscus spp. Experimentsshowed that flagellates significantly prefer C. wailesii overC.granii as host species (interspecific selectivity). This preferencewas independent of light conditions (dark, irradiance of 10and 70 µmol m^–2 s^–1) and temperature (10 and15C). Among unicellular host diatoms, the infection behaviourwas selective for individual cells: already infected C.graniicells were more attractive for further flagellate attachmentthan non-infected cells (intraspecific selectivity). Individualcells (     

Occurrence and distribution of muscle spindles in masticatory and suprahyoid muscles of the rat.

The occurrence and distribution of muscle spindles was studied in histochemically and conventionally stained serial cross sections of 6-week-old and adult rat masticatory and suprahyoid muscles. Spindles were present in moderate to large numbers in jaw closers, but they were absent in jaw openers and two of four muscles of an accessory suprahyoid group. In jaw closers, 67% or more of the total spindle population was concentrated relatively distant from the temporomandibular joint, in muscle portions which contained large numbers of extrafusal fibers reacting strongly for oxidative enzymes. Because of their location, spindles in these portions should be stretched more and, subsequently, should respond with a greater afferent discharge at any given muscle length than spindles situated nearer to the joint. Spindles in jaw closers, especially the medial pterygoid and deep masseter, often occurred in clusters and complex forms near the terminal branching of intramuscular nerve trunks. No such concentrations were seen in the two muscles of the accessory suprahyoid group that had spindles. The association in jaw closers of spindles with extrafusal fibers high in oxidative enzyme activity is consistent with the view that spindles are the sensory component of a reflex system that recruits these fibers for finely-graded contractions in response to small internal length-changes of the muscle (Botterman et al., '78); however, in jaw openers and two muscles of the accessory suprahyoid group, the absence of spindles, coupled with the presence of large populations of extrafusal fibers high in oxidative enzyme activity, is not easily reconciled with this concept.     

Measuring ecological impact of water consumption by bioethanol using life cycle impact assessment

Purpose  

Though the development of biofuel has attracted numerous studies for quantifying potential water demand applying life cycle thinking, the impacts of biofuel water consumption still remain unknown. In this study, we aimed to quantify ecological impact associated with corn-based bioethanol water consumption in Minnesota in responding to different refinery expansion scenarios by applying a life cycle impact assessment method.