Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.     

Limnological Studies on the River Tigris,Iraq. I. Environmental Characteristics

Regional and seasonal variations of some environmental characteristics in the 630 km stretch of the lower Tigris were studied and discussed. The lowest air and average water temperatures were recorded in January. The highest average pH values, generally found in October and July, are due mainly to the abundance of phytoplankton. The remarkably low average chlorosity values found in the periods of high water were correlated with the high discharge. The highest average values for dissolved oxygen concentration were generally recorded in January and April, and the lowest ones were observed in October and July.     

40S hnRNP particles are a novel class of nuclear biomolecular condensates

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.     

Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA’s ATPase rate. Since UvrA’s ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA’s ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity.     

Bacteria as computers making computers

Various efforts to integrate biological knowledge into networks of interactions have produced a lively microbial systems biology. Putting molecular biology and computer sciences in perspective, we review another trend in systems biology, in which recursivity and information replace the usual concepts of differential equations, feedback and feedforward loops and the like. Noting that the processes of gene expression separate the genome from the cell machinery, we analyse the role of the separation between machine and program in computers. However, computers do not make computers. For cells to make cells requires a specific organization of the genetic program, which we investigate using available knowledge. Microbial genomes are organized into a paleome (the name emphasizes the role of the corresponding functions from the time of the origin of life), comprising a constructor and a replicator, and a cenome (emphasizing community-relevant genes), made up of genes that permit life in a particular context. The cell duplication process supposes rejuvenation of the machine and replication of the program. The paleome also possesses genes that enable information to accumulate in a ratchet-like process down the generations. The systems biology must include the dynamics of information creation in its future developments.     

Proton NMR quantitative profiling for quality assessment of greenhouse-grown tomato fruit

Tomato is an essential crop in terms of economic importance and nutritional quality. In France, the third most important region for tomato (Solanum lycopersicum L.) production is Aquitaine where the major part of production is now grown soilless under greenhouse conditions with harvest from March to November. Tomato fruit quality at harvest is a direct function of its metabolite content at that time. The aim of this work was to use a global approach to characterize changes in the fruit organoleptic quality at harvest under commercial culture conditions during an entire season for two varieties and two different fertilization practices (with or without recycling of the nutrient solution) for one variety. Absolute quantification data of 32 major compounds in fruit without seeds were obtained through untargeted (proton nuclear magnetic resonance, ¹H-NMR) quantitative profiling. These data were complemented by colorimetric analysis of ascorbate and total phenolics. They were analyzed with chemometric approaches. Principal component analysis (PCA) or partial least square analyses (PLS) revealed more discriminant metabolites for season than for variety and showed that nutrient solution recycling had very little effect on fruit composition. These tendencies were confirmed with univariate analyses. ¹H-NMR profiling complemented with colorimetric analyses therefore provided a diagnostic tool to follow the changes in organoleptic and nutritional quality of tomato. In addition the quantitative information generated will help to increase our knowledge on the mechanisms of plant response to environmental modifications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prion Fibrils of Ure2p Assembled under Physiological Conditions Contain Highly Ordered, Natively Folded Modules

The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra. Prion fibrils formed under physiological conditions are therefore well-ordered objects on the molecular level. Comparing the full-length NMR spectra with the corresponding spectra of the prion and globular domains in isolation reveals that the globular part in particular shows almost perfect structural order. The NMR linewidths in these spectra are as narrow as the ones observed in crystals of the isolated globular domain. For the prion domain, the spectra reflect partial disorder, suggesting structural heterogeneity, both in isolation and in full-length Ure2p fibrils, although to different extents. The spectral quality is surprising in the light of existing structural models for Ure2p and in comparison to the corresponding spectra of the only other full-length prion fibrils (HET-s) investigated so far. This opens the exciting perspective of an atomic-resolution structure determination of the fibrillar form of a prion whose assembly is not accompanied by significant conformational changes and documents the structural diversity underlying prion propagation.     

Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.