Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease,depending on steroid response

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.     

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant–pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air–liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.     

Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.     

Assessing transpiration in the tussock grass Stipa tenacissima L.: the crucial role of the interplay between morphology and physiology

Plant transpiration has a key role on both plant performance and ecosystem functioning in arid zones, but realistic estimates at appropriate spatial-temporal scales are scarce. Leaf and tiller morphology and crown architecture were studied together with leaf physiology and whole plant water balance in four individual plants of Stipa tenacissima of different sizes to determine the relative influence of processes taking place at different spatial and temporal scales on whole plant transpiration. Transpiration was estimated in potted plants by leaf-level gas exchange techniques (infrared gas analyzer and porometer), by sap flow measurements, and by integrating leaf physiology and crown architecture with the 3-D computer model Yplant. Daily transpiration of each individual plant was monitored using a gravimetric method, which rendered the reference values. Leaves on each individual plant significantly varied in their physiological status. Young and green parts of the leaves showed five times higher chlorophyll concentration and greater photosynthetic capacity than the senescent parts of the foliage. Instantaneous leaf-level transpiration measurements should not be used to estimate plant transpiration, owing to the fact that extrapolations overestimated individual transpiration by more than 100%. Considering leaf age effects and scaling the estimates according to the relative amount of each foliage category reduced this difference to 46% though it was still significantly higher than gravimetric measurements. Sap flow calculations also overestimated tussock transpiration. However, 3-D reconstruction of plants with Yplant and transpiration estimates, considering both the physiological status and the daily pattern of radiation experienced by each individual leaf section within the crown, matched the gravimetric measurements (differences were only 4.4%). The complex interplay of leaf physiology and crown structure must be taken into account in scaling up plant transpiration from instantaneous, leaf-level measurements, and our study indicates that transpiration of complex crowns is easily overestimated.     

The effect of guanosine nucleotides on the multiple forms of protein synthesis elongation factor 1 from wheat embryos

At least two species of elongation factor 1 from wheat embryo have been detected by sucrose gradient analysis and Sephadex gel filtration. The heavy species (EF1_h) which has a molecular weight of 200,000 can be converted into the light species (EF1_l) with a molecular weight of approximately 50,000 by addition of GTP or GDP. The conversion of EF1_h to EF1_l is more rapid in the presence of GDP than in the presence of GTP. Aminoacyl-tRNA which reacts preferentially with EF1_l favors the conversion of EF1_h to EF1_l with GTP. Both GTP and GDP promote inactivation of EF1_h, but the addition of aminoacyl-tRNA counteracts the effect of the guanosine triphosphate. These reactions are discussed with respect to the function of the various forms of EF1 in aminoacyl-tRNA binding to ribosomes.     

Segregation of elongation potential inEscherichia coli mediated by thewee genetic system

Under normal conditions, the two cells within a pair ofEscherichia coli siblings elongate at a similar rate. Genetic impairment of thewee genetic system leads to significant variations in the rate of elongation of each cell within a given pair of siblings. This result is in accordance with previous reports that showed the need of active cell division for expression of the Wee phenotype. The genetic location of one element of the system,weeA, has been determined to be at min 67 of theE. coli map; this does not coincide with the previously reported genetic location. The inability to reproduce the Wee phenotype in a wild-type background by transduction ofweeA suggests that more genetic elements should participate in the segregation of elongation zones at cell division.     

Siderophore cooperation of the bacterium Pseudomonas fluorescens in soil

While social interactions play an important role for the evolution of bacterial siderophore production in vitro, the extent to which siderophore production is a social trait in natural populations is less clear. Here, we demonstrate that siderophores act as public goods in a natural physical environment of Pseudomonas fluorescens: soil-based compost. We show that monocultures of siderophore producers grow better than non-producers in soil, but non-producers can exploit others'' siderophores, as shown by non-producers'' ability to invade populations of producers when rare. Despite this rare advantage, non-producers were unable to outcompete producers, suggesting that producers and non-producers may stably coexist in soil. Such coexistence is predicted to arise from the spatial structure associated with soil, and this is supported by increased fitness of non-producers when grown in a shaken soil–water mix. Our results suggest that both producers and non-producers should be observed in soil, as has been observed in marine environments and in clinical populations.     

Mechanism of mate detection in parasitoid wasps: sound and vibratory cues change with the developmental progress of future mates inside host pupal cases

Insects including parasitoid wasps use acoustic and vibratory signals in the context of sexual communication, mate recognition, courtship and mating. Males of the parasitoid wasp Pimpla disparis Viereck (Hymenoptera: Ichneumonidae) detect insect host pupae parasitized by a conspecific female, learn their location, visit them repeatedly and remain on or near them when the prospective mate nears emergence. In the present study, the acoustic and vibratory cues that males exploit to detect the presence and track the developmental progress of a future mate inside a host pupal case are investigated. Responses are acquired from developing parasitoids (DePa) by airborne sound and laser Doppler vibrometer recordings, after gently stimulating each of 20 wax moth host pupae with a paintbrush on days 1–23 post parasitism. Sound and vibratory cues produced by DePa are detectable from day 7 onward and relate mostly to spinning movements. Parameters of sound and vibratory cues (amplitude, dominant frequency, upper limit of frequency band) change significantly over time and thus could ‘inform’ a visiting adult male about the stage of development of DePa. Adult males antennating a parasitized pupa and flying around it also induce vibrations, which in turn may inform DePa about the presence of a male. There is no experimental evidence for true signalling and rapid information exchange between DePa and adult males. Delaying reply signals may help DePa avoid attacks by illicit receivers of such signals, including female (hyper)parasitoids and invertebrate predators.