Efficiency,thermodynamic and kinetic stability of marketed gadolinium chelates and their possible clinical consequences: a critical review

Gadolinium-based contrast agents are widely used to enhance image contrast in magnetic resonance imaging (MRI) procedures. Over recent years, there has been a renewed interest in the physicochemical properties of gadolinium chelates used as contrast agents for MRI procedures, as it has been suggested that dechelation of these molecules could be involved in the mechanism of a recently described disease, namely nephrogenic systemic fibrosis (NSF). The aim of this paper is to discuss the structure-physicochemical properties relationships of marketed gadolinium chelates in regards to their biological consequences. Marketed gadolinium chelates can be classified according to key molecular design parameters: (a) nature of the chelating moiety: macrocyclic molecules in which Gd3+ is caged in the pre-organized cavity of the ligand, or linear open-chain molecules, (b) ionicity: the ionicity of the complex varies from neutral to tri-anionic agents, and (c) the presence or absence of an aromatic lipophilic residue responsible for protein binding. All these molecular characteristics have a profound impact on the physicochemical characteristics of the pharmaceutical solution such as osmolality, viscosity but also on their efficiency in relaxing water protons (relaxivity) and their biodistribution. These key molecular parameters can also explain why gadolinium chelates differ in terms of their thermodynamic stability constants and kinetic stability, as demonstrated by numerous in vitro and in vivo studies, resulting in various formulations of pharmaceutical solutions of marketed contrast agents. The concept of kinetic and thermodynamic stability is critically discussed as it remains a somewhat controversial topic, especially in predicting the amount of free gadolinium which may result from dechelation of chelates in physiological or pathological situations. A high kinetic stability provided by the macrocyclic structure combined with a high thermodynamic stability (reinforced by ionicity for macrocyclic chelates) will minimize the amount of free gadolinium released in tissue parenchymas.     

A typical browser, the roe deer, may consume substantial quantities of grasses in open landscapes

In open landscapes, grass leaves provide an abundant resource for ruminants, with potentially high nutritional value. However, their extensive digestion requires a long fermentation time, achieved through large rumen and the stratification of the rumen content. Due to anatomical and physiological differences, ruminants differ in their ability to process grass leaves. Particularly, the small roe deer, with its viscous saliva and unstratified rumen content, is generally classified as a strict browser. We hypothesised that roe deer may be able to use grass leaves in some circumstances, notably when the availability of other resources declines and when the quality of grass leaves is high. We expected that (1) grass leave consumption should be higher in open landscapes than in forest habitat because grasses are more widely available and more nutritious in open landscapes and (2) grass leave consumption should increase in winter when the availability of other resources declines. We assessed grass consumption by microscopic analysis of roe deer faecal pellets collected monthly both in forest habitat and in the surrounding open landscape. We found that both the occurrence and the proportion of grass leaves in the faeces were higher in the open landscape (predicted mean proportion 0.31) than in the forest (predicted mean proportion 0.05). In addition, the proportion of grass leaves in the faeces was higher in winter and lower in spring in both forest and open landscape. We suggest that roe deer are able to use grass leaves with unusually high nutritional quality in winter in this mild climate area. This involves a certain level of digestive plasticity to efficiently digest high quality grasses and may confer nutritional benefit to individuals feeding in an open landscape.     

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.     

Landscape fragmentation generates spatial variation of diet composition and quality in a generalist herbivore

Forest fragmentation may benefit generalist herbivores by increasing access to various substitutable food resources, with potential consequences for their population dynamics. We studied a European roe deer (Capreolus capreolus) population living in an agricultural mosaic of forest, woodlots, meadows and cultivated crops. We tested whether diet composition and quality varied spatially across the landscape using botanical analyses of rumen contents and chemical analyses of the plants consumed in relation to landscape metrics. In summer and non-mast winters, roe deer ate more cultivated seeds and less native forest browse with increasing availability of crops in the local landscape. This spatial variation resulted in contrasting diet quality, with more cell content and lower lignin and hemicellulose content (high quality) for individuals living in more open habitats. The pattern was less marked in the other seasons when diet composition, but not diet quality, was only weakly related to landscape structure. In mast autumns and winters, the consumption of acorns across the entire landscape resulted in a low level of differentiation in diet composition and quality. Our results reflect the ability of generalist species, such as roe deer, to adapt to the fragmentation of their forest habitat by exhibiting a plastic feeding behavior, enabling them to use supplementary resources available in the agricultural matrix. This flexibility confers nutritional advantages to individuals with access to cultivated fields when their native food resources are depleted or decline in quality (e.g. during non-mast years) and may explain local heterogeneities in individual phenotypic quality.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Theoretical study of mechano-chemical couplings in a compartmental enzyme system. I. Analytical treatment

This paper deals with theoretical aspects of the volume changes of a system in which diffusion, convection and reaction processes are coupled. This study involves a material able to swell in the presence of a chemical effector produced by an enzyme reaction. Three limiting factors of volume change rate were considered: fluid flow, diffusion or reaction limitations. Dimensionless diffusion-reaction and diffusion-convection parameters were introduced to allow quantitative predictions in limit cases. The steady states appear to be independent of convection processes; however, the transient states depend on diffusion, convection and reaction processes.     

CRISPR-Cas9 mediated gene deletions in lager yeast <Emphasis Type="Italic">Saccharomyces pastorianus</Emphasis>

Background

The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.

Results

This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.

Conclusions

These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.