Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase ( PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of approximate to 86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions

Radiological characterisation in view of nuclear reactor decommissioning: On-site benchmarking exercise of a biological shield

Nearly all decommissioning and dismantling (D&D) projects are steered by the characterisation of the plant being dismantled. This radiological characterisation is a complex process that is updated and modified during the course of the D&D. One of the tools for carrying out this characterisation is the performance of in-situ measurements. There is a wide variety of equipment and methodologies used to carry out on-site measurements, depending on the environment in which they are to be carried out and also on the specific objectives of the measurements and the financial and personnel resources available. The extent to which measurements carried out with different types of equipment or methodologies providing comparable results can be crucial in view of the D&D strategy development and the decision-making process. This paper concerns an on-site benchmarking exercise carried out at the activated biological shield of Belgian Reactor 3 (BR3). This activity allows comparison and validation of characterisation methodologies and different equipment used as well as future interpretation of final results in terms of uncertainties and sensitivities. This paper describes the measurements and results from the analysis of this exercise. Other aspects of this exercise will be reported in separate papers. This paper provides an overview of the on-site benchmarking exercise, outlines the participating organisations and the measurement equipment used for total gamma, dose rate and gamma spectrometry measurements and finally, results obtained and their interpretations are discussed for each type of measurement as a function of detector type. Regarding the dose measurements, results obtained by using a large variety of equipment are very consistent. In view of mapping the inner surface of the biological shield the most appropriate equipment tested might be the organic scintillator, the BGO or even the ionisation chamber. In addition, for mapping this surface, the most appropriate total gamma equipment tested might be the LaBr$_{3}$(Ce), the thick organic scintillator or the BGO. These measurements can only be used as a secondary parameter in a relative way. Results for the gamma spectrometry are very consistent for all the equipment used and the main parameters to be determined

Transmissibility of Atypical Scrapie in Ovine Transgenic Mice: Major Effects of Host Prion Protein Expression and Donor Prion Genotype

Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ∼0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ∼6× the normal PrPc level died after a survival periods of 400 days, those with ∼1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein

Nutrient sensing modulates malaria parasite virulence

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence

Effective Gene Therapy in a Mouse Model of Prion Diseases

Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases

Policy Issues in NEG Models: Established Results and Open Questions

This paper provides a non-technical overview of NEG models dealing with policy issues. Considered policy measures include alternative categories of public expenditure, international tax competition, unilateral actions of protection/liberalisation, and trade agreements. The implications of public intervention in two-region NEG models are discussed by unfolding the impact of policy measures on agglomeration/dispersion forces. Results are described in contrast with those obtained in standard non-NEG theoretical models. The high degree of abstraction limits the applicability of NEG models to real world policy issues. We discuss in some detail two extensions of NEG models to reduce this applicability gap: the cases of multi-regional frameworks and firm heterogeneity

PrP Expression, PrPSc Accumulation and Innervation of Splenic Compartments in Sheep Experimentally Infected with Scrapie

BACKGROUND: In prion disease, the peripheral expression of PrP(C) is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP(Sc) accumulation, localisation of nerve fibres and PrP(C) expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep. METHODOLOGY/PRINCIPAL FINDINGS: Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP(C) and PrP(Sc) in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP(Sc) in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP(Sc) and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP(Sc) and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP(Sc)-laden germinal centres. However, the close association between nerves and PrP(Sc) was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres. CONCLUSIONS/SIGNIFICANCE: The findings suggest that the degree of PrP(Sc) accumulation does not depend on the expression level of PrP(C). Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP(Sc)