MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.     

Bacterial luciferase reporters: the Swiss army knife of molecular biology

Bioluminescence is a process during which light in the visible spectrum is emitted as a consequence of an enzymatic reaction catalyzed by luciferases. Luciferases have been identified mainly in marine organisms and are used for several biological purposes include camouflage, repulsion, attraction, communication and illumination. Some of the currently known luciferases have become indispensible tools in modern molecular biology and are used for diverse applications such as autoinducer-1 activity assays, promoter test assays in both prokaryotes and eukaryotes, imaging of bacterial infections in live animals, in vivo activity assays genes involved in host response and disease and monitoring of bacterial contaminations of food products. With the present review, the authors intend to give an overview on the currently used bacterial luciferase reporter systems, their methodologies and applications and compare them to other reporter systems.     

A recombinant α-dioxygenase from rice to produce fatty aldehydes using <Emphasis Type="Italic">E. coli</Emphasis>

Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an α-dioxygenase (αDOX) from Oryza sativa (rice) is presented. α-Dioxygenases are the initial enzymes of α-oxidation in plants and oxidize long and medium-chain C _n fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C _n − 1 fatty aldehydes by spontaneous decarboxylation. Successful expression of αDOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The usage of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.     

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.     

CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.     

Decisions for Others Become Less Impulsive the Further Away They Are on the Family Tree

Background

People tend to prefer a smaller immediate reward to a larger but delayed reward. Although this discounting of future rewards is often associated with impulsivity, it is not necessarily irrational. Instead it has been suggested that it reflects the decision maker’s greater interest in the ‘me now’ than the ‘me in 10 years’, such that the concern for our future self is about the same as for someone else who is close to us.

Methodology/Principal Findings

To investigate this we used a delay-discounting task to compare discount functions for choices that people would make for themselves against decisions that they think that other people should make, e.g. to accept $500 now or $1000 next week. The psychological distance of the hypothetical beneficiaries was manipulated in terms of the genetic coefficient of relatedness ranging from zero (e.g. a stranger, or unrelated close friend), .125 (e.g. a cousin), .25 (e.g. a nephew or niece), to .5 (parent or sibling).

Conclusions/Significance

The observed discount functions were steeper (i.e. more impulsive) for choices in which the decision-maker was the beneficiary than for all other beneficiaries. Impulsiveness of decisions declined systematically with the distance of the beneficiary from the decision-maker. The data are discussed with reference to the implusivity and interpersonal empathy gaps in decision-making.     

Characterisation of particle dynamics and turnover in the gastrointestinal tract of Holstein cows fed forage diets differing in fibre and protein contents

An improved understanding of the role of forage quality on the processes of particle dynamics and turnover is important for the development of healthier and cost-effective feeding strategies that aim at lowering the proportions of concentrates in the diets of cattle. The aim of this study was to evaluate the effects of feeding hays of different qualities on particle dynamics, digestion kinetics and turnover in the gastrointestinal tract (GIT). Three non-lactating, rumen fistulated Holstein cows were fed diets consisting exclusively of hay with either low quality [Group LH; 605 ± 12.4 g/kg neutral detergent fibre (NDF) and 63 ± 4.7 g/kg crude protein (CP)] or good quality (Group GH; 551 ± 20.1 g/kg NDF and 116 ± 3.6 g/kg CP). Data showed that in situ dry matter (DM) disappearance of the soluble fraction was greater for Group GH (p < 0.05). Feeding good quality hay also lowered the proportion of particles >1.18 mm particularly during the eating process (p < 0.05). Changes in the particle size occurring afterwards were greater for Group GH as well (p < 0.05); approximately 30% in the comminution in the particle size occurred postruminally. Feeding hay of good quality lowered DM content of solid rumen digesta (p < 0.05), accelerated (p < 0.05) the turnover rate of DM and NDF in the GIT and increased DM intake (p < 0.05). In conclusion, feeding forages of better quality significantly promoted degradation processes and kinetics in the GIT with positive effects on turnover rate of digesta and feed intake in Holstein cows.     

Oxidation of fatty aldehydes to fatty acids by Escherichia coli cells expressing the Vibrio harveyi fatty aldehyde dehydrogenase (FALDH)

Fatty acids represent an important renewable feedstock for the chemical industry. To enable biotechnological one carbon truncations of fatty acids, the enzymes α-dioxygenase and fatty aldehyde dehydrogenase (FALDH) have to be combined in a two-step process. We expressed an FALDH from V. harveyi in E. coli and characterized its substrate spectrum with a focus on the number and position of double bonds in the fatty aldehyde molecules. Synthesis of the expected fatty acid products was proven by analysis of whole cell biotransformation products. Coexpression of a H₂O-forming NADPH oxidase (NOX) from Lactobacillus sanfranciscensis led to the implementation of a cofactor regeneration cycle in in vitro oxidation experiments. The presence of NOX in whole cell biotransformations improved reaction velocity but did not result in higher product yields. We could further demonstrate that at least part of the endogenous NAD(P)⁺ regeneration capacity in the resting cells results from the respiratory chain. The whole cell catalyst with the high broad range FALDH activity described here is an important biotechnological module for lipid biotransformation processes, especially the shortening of fatty acids.     

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.