Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.     

Exploring Protein Space: From Hydrolase to Ligase by Substitution

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence–function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.     

Movement patterns of temperate wrasses (Labridae) within a small marine protected area

The movement patterns of three commercially important wrasse (Labridae) species inside a small marine protected area (~ 0.15 km²) on the west coast of Norway were analysed over a period of 21 months. The mean distance between capture and recapture locations varied between 10 and 187 m, and was species and season specific. The extent of movement was not related to body size or sex. These results imply that a network of small strategically located marine protected areas can be used as management tools to protect wrasses from size- and sex-selective fishing mortality.     

South–South Comparisons: A Syntegrated Approach to the Teaching of the Arts for Primary School Teacher Preparation in South Africa and Australia

In light of the tendency to present the arts in an integrated fashion in many education systems worldwide, this article examines the consequences of integration for discrete art forms. In particular, we investigate the advantages of adopting a syntegrated approach to the facilitation of arts in teacher preparation. A specific comparison between the implementation of arts curricula in South Africa and Australia is made. The disjuncture between policy and practice in arts education that has been reported internationally needs constant monitoring. We conclude that the heart of curriculum transfer and transformation lies in the classroom.     

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice

Introduction

The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).

Methods

Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.

Results

Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5 × 10⁶ purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

Conclusions

Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.     

Sustained tumour eradication after induced caspase-3 activation and synchronous tumour apoptosis requires an intact host immune response

Effective anticancer treatments often result in the induction of large amounts of tumour cell death. In vivo, such dying tumour cells are a potential source of antigens for T-cell stimulation. Although apoptosis is generally considered nonimmunogenic, recent evidence suggests that some anticancer therapies that induce apoptosis can elicit antitumour immune responses. Here, a doxycycline-inducible, constitutively active caspase-3 (‘death switch'') was constructed in a murine tumour model to explore the impact of the host immune response to rapid, synchronous and substantial tumour cell apoptosis. In vitro, up to 80% of tumour cells underwent apoptotic cell death within 24 h and death was accompanied by the release of potential ‘danger signal'' molecules HMGB1 and HSP90. In vivo, death switch induction provoked rapid, pronounced tumour regression in immune-competent and immune-deficient mice, but sustained tumour eradication was observed only in immune-competent mice. Moreover, the majority of mice that were tumour free after death switch induction were protected from further tumour rechallenge. In addition, long-term remission after induction of the death switch was completely abrogated following depletion of CD8 T cells. These data suggest that sustained tumour eradication after substantial tumour apoptosis requires an antitumour host immune response that prevents tumour relapse. In many patients, cancer therapies produce encouraging initial responses that are only short lived. These results provide new insights that may have important implications for further development of strategies that result in long-term tumour clearance after initially effective anticancer treatment.     

Prolongation of Life in Anephric Rats following de novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.