Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.     

Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.     

Expression, purification and characterization of full-length RNA-free hepatitis B core particles

The nucleocapsid or core particle of the hepatitis B virus has become one of the favourite recombinant vaccine carriers for foreign peptides, proteins and stimulatory oligonucleotides. The core protein consists of three regions: an N-terminal, a central and a C-terminal region that can accommodate the addition or insertion of the foreign sequences. The protamine-like C-terminal region that binds host RNA randomly during recombinant particle formation is often truncated. It is commonly thought that these truncations do not affect particle assembly. Recent studies have demonstrated that the C-terminal domains mediate a glycosaminoglycan-dependent attachment of nucleocapsids to the plasma membranes of host cells. This interaction might well contribute to the immunogenicity of nucleocapsids. Testing the hypothesis that full-length particles might be safer and superior for the induction of an immune response against the nucleocapsids and inserted sequences, requires the availability of purified particles. In this report, we detail a novel method for the synthesis and purification of full-length core particles essentially free of RNA from Escherichia coli.     

Quantitative characterization of agglomerates and aggregates of pyrogenic and precipitated amorphous silica nanomaterials by transmission electron microscopy

ABSTRACT: BACKGROUND: The interaction of a nanomaterial (NM) with a biological system depends not only on the sizeof its primary particles but also on the size, shape and surface topology of its aggregates andagglomerates. A method based on transmission electron microscopy (TEM), to visualize theNM and on image analysis, to measure detected features quantitatively, was assessed for itscapacity to characterize the aggregates and agglomerates of precipitated and pyrogenicsynthetic amorphous silicon dioxide (SAS), or silica, NM. RESULTS: Bright field (BF) TEM combined with systematic random imaging and semi-automatic imageanalysis allows measuring the properties of SAS NM quantitatively. Automation allows measuring multiple and arithmetically complex parameters simultaneously on high numbersof detected particles. This reduces operator-induced bias and assures a statistically relevantnumber of measurements, avoiding the tedious repetitive task of manual measurements.Access to multiple parameters further allows selecting the optimal parameter in function of aspecific purpose.Using principle component analysis (PCA), twenty-three measured parameters wereclassified into three classes containing measures for size, shape and surface topology of theNM. CONCLUSION: The presented method allows a detailed quantitative characterization of NM, like dispersionsof precipitated and pyrogenic SAS based on the number-based distributions of their meandiameter, sphericity and shape factor.     

Positional gene enrichment analysis of gene sets for high-resolution identification of overrepresented chromosomal regions

The search for feature enrichment is a widely used method to characterize a set of genes. While several tools have been designed for nominal features such as Gene Ontology annotations or KEGG Pathways, very little has been proposed to tackle numerical features such as the chromosomal positions of genes. For instance, microarray studies typically generate gene lists that are differentially expressed in the sample subgroups under investigation, and when studying diseases caused by genome alterations, it is of great interest to delineate the chromosomal regions that are significantly enriched in these lists. In this article, we present a positional gene enrichment analysis method (PGE) for the identification of chromosomal regions that are significantly enriched in a given set of genes. The strength of our method relies on an original query optimization approach that allows to virtually consider all the possible chromosomal regions for enrichment, and on the multiple testing correction which discriminates truly enriched regions versus those that can occur by chance. We have developed a Web tool implementing this method applied to the human genome (http://www.esat.kuleuven.be/~bioiuser/pge). We validated PGE on published lists of differentially expressed genes. These analyses showed significant overrepresentation of known aberrant chromosomal regions.     

Sex and sugar in yeast: two distinct GPCR systems

Although eukaryotic G-protein coupled receptor (GPCR) systems are well known for their ability to detect and mediate rapid responses to extracellular signals, the full range of stimuli to which they respond may not yet have been identified. Activation of GPCRs by hormones, pheromones, odorants, neurotransmitters, light and different taste compounds is well established. However, the recent discovery of a glucose-sensing GPCR system in Saccharomyces cerevisiae has unexpectedly added common nutrients to this list of stimuli. This GPCR system mediates glucose activation of adenylate cyclase during the switch from respirative/gluconeogenic metabolism to fermentation. The GPCR system involved in pheromone signalling in S. cerevisiae has already served as an important model and tool for the study of GPCR systems in higher eukaryotic cell types. Here, we highlight the similarities and differences between these two signalling systems. We also indicate how the new glucose-sensing system can serve as a model for GPCR function and as a tool with which to screen for heterologous components of signalling pathways as well as for novel ligands in high-throughput assays.     

Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Genotype-dependent interactions among sympatric Microcystis strains mediated by Daphnia grazing

Natural populations of the bloom forming cyanobacterium Microcystis are typically composed of several distinct genotypes. Using Microcystis strains that differ in growth rate, microcystin production and colony formation, we conducted a laboratory experiment in the presence and absence of a grazer, the water flea Daphnia, to investigate whether interactions among strains can be predicted from functional traits, and whether the outcome of competition between strains is influenced by a grazer. Two toxic and two non‐toxic Microcystis strains, isolated from a single lake, were grown during four weeks as single strains, in all possible combinations of two strains and all together, in the presence and absence of Daphnia magna. The relative abundance of strains in the populations was assessed using denaturing gradient gel electrophoresis, and the growth rate of each strain in mixed populations was compared to its growth rate in monoculture to determine interactions between strains. The observed interactions were strain‐specific, and the relative abundances of strains in mixed populations could be partially explained by taking toxicity and colony formation into account. Importantly, some of the interactions were strongly altered by the presence of Daphnia. Daphnia induced colony formation in one strain, which then became a better competitor. Daphnia grazing also caused a higher evenness in the populations, both through a weakening of competitive interactions as well as by facilitation effects. Strong facilitation effects were due to non‐toxic strains benefiting from the protection offered by toxic strains in the presence of predation. Overall, our results emphasize the presence of strong competitive interactions between Microcystis strains in the absence of grazing, whereas indirect positive interactions are prevalent in the presence of a generalist grazer. Our results suggest that differences in functional traits and grazer‐mediated facilitation effects may enhance coexistence of Microcystis strains, including toxic and non‐toxic strains.