Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.     

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp^−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp^−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp^−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.     

Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery

High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.     

Nutritional assessment of genetically modified rapeseed synthesizing high amounts of mid-chain fatty acids including production responses of growing-finishing pigs

The nutritive value of genetically modified myristic acid-rich rapeseed, in which a acyl-thioesterase gene inserted, was studied. Crude nutrients, amino acid and fatty acid profiles as well as mineral and glucosinolate contents were determined and compared with those of the non-transgenic parental cultivar. The concentration of crude nutrients, minerals and amino acids were found to be within the range of natural variance. The myristic and palmitic acid content increased from 0.1 - 11.4% and from 3.6-20%, respectively, at the expense of oleic acid, which decreased from 68.6-42.6% of total fatty acids. The glucosinolate contents increased from 12.4 micromol/g in the parental plant to 19 micromol/g DM in the GM-plant. Full-fat rapeseed of both cultivars was incorporated in pig diets at a level of 15%, and the digestibility and the production efficiency were tested under ad libitum feeding conditions with ten pigs each over the growing finishing period from 32-105 kg BW. The experimental diets did not show significant differences in digestibility and energetic feeding value. However, feed intake and weight gain decreased presumably due to the increasing glucosinolate intake associated with the feeding of transgenic rapeseed. The dietary fatty acids profile influenced the fatty acid profile of body fat. Myristic acid accumulated in back fat and intramuscular fat while the oleic acid content decreased. The increased glucosinolate intake affected the weight of thyroid glands and their iodine concentration.     

Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake

Blood was collected from newborn calves before and after their first milk intake after birth; extracts of plasma were assayed by radioimmunoassay for the presence of beta-casomorphin-7 immunoreactive materials. No beta-casomorphin immunoreactivity was found in samples collected before milk ingestion; however, in samples collected after milk ingestion a beta-casomorphin-7 immunoreactive material was detected. Chromatographic characterization showed that this material was not identical with beta-casomorphin-7 but might rather represent a precursor thereof. The material proved resistant to enzymatic attack during a 30-min incubation period at 37 degrees C in the plasma of newborn calves, whereas beta-casomorphin-7 was degraded under these conditions. A physiological significance of beta-casomorphin-7 eventually cleaved from such a precursor material at any site in the newborn mammal is suggested.     

The Role of Pulse Shape in Motor Cortex Transcranial Magnetic Stimulation Using Full-Sine Stimuli

A full-sine (biphasic) pulse waveform is most commonly used for repetitive transcranial magnetic stimulation (TMS), but little is known about how variations in duration or amplitude of distinct pulse segments influence the effectiveness of a single TMS pulse to elicit a corticomotor response. Using a novel TMS device, we systematically varied the configuration of full-sine pulses to assess the impact of configuration changes on resting motor threshold (RMT) as measure of stimulation effectiveness with single-pulse TMS of the non-dominant motor hand area (M1). In young healthy volunteers, we (i) compared monophasic, half-sine, and full-sine pulses, (ii) applied two-segment pulses consisting of two identical half-sines, and (iii) manipulated amplitude, duration, and current direction of the first or second full-sine pulse half-segments. RMT was significantly higher using half-sine or monophasic pulses compared with full-sine. Pulses combining two half-sines of identical polarity and duration were also characterized by higher RMT than full-sine stimuli resulting. For full-sine stimuli, decreasing the amplitude of the half-segment inducing posterior-anterior oriented current in M1 resulted in considerably higher RMT, whereas varying the amplitude of the half-segment inducing anterior-posterior current had a smaller effect. These findings provide direct experimental evidence that the pulse segment inducing a posterior-anterior directed current in M1 contributes most to corticospinal pathway excitation. Preferential excitation of neuronal target cells in the posterior-anterior segment or targeting of different neuronal structures by the two half-segments can explain this result. Thus, our findings help understanding the mechanisms of neural stimulation by full-sine TMS.