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// Maroua Manai 1, 5, 6, 7 , Jeanne Thomassin-Piana 2, * , Amor Gamoudi 6, * , Pascal Finetti 1, * , Marc Lopez 1 , Radhia Eghozzi 6 , Sinda Ayadi 6 , Olfa Ben Lamine 6 , Mohamed Manai 5 , Khaled Rahal 6 , Emmanuelle Charafe-Jauffret 2, 3 , Jocelyne Jacquemier 2 , Patrice Viens 3, 4 , Daniel Birnbaum 1 , Hamouda Boussen 5, 7 , Max Chaffanet 1 , Francois Bertucci 1, 3, 4 1 Departement d’Oncologie Moleculaire, Centre de Recherche en Cancerologie de Marseille, Aix Marseille Universite, Marseille, France 2 Departement de Bio-Pathologie, Institut Paoli-Calmettes, Marseille, France 3 UFR de Medecine, Aix Marseille Universite, Marseille, France 4 Departement d’Oncologie Medicale, Institut Paoli-Calmettes, Marseille, France 5 Departement de Biologie, Unite de Biochimie et Biologie Moleculaire, Faculte des Sciences de Tunis, Universite de Tunis El Manar, Tunisie 6 Departement d’Oncologie Medicale, Institut Salah Azaiez, Tunis, Tunisie 7 Service d’Oncologie Medicale, Hopital l’Ariana, Tunis, Tunisie * These authors contributed equally to second authors Correspondence to: Francois Bertucci, email: bertuccif@ipc.unicancer.fr Keywords: expression, immunohistochemistry, inflammatory breast cancer, MARCKS, survival Received: July 29, 2016 Accepted: December 14, 2016 Published: December 21, 2016 ABSTRACT Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. Results: Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E−09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. Materials and Methods: We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). Conclusions: MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC.

International audience; To report epidemiological and anatomo-clinical features within a retrospective series of inflammatory breast cancer and to evaluate prognostic factors. This retrospective study included 210 Tunisian patients presenting a clinically diagnosed IBC, treated at the Institute Salah Azaiez (ISA) of Tunis, Tunisia, from 2008 to 2013. We collected data on epidemiology, anatomo-clinical and biological features and histologic response to neoadjuvant therapy. Overall and disease-free survivals were calculated by Kaplan-Meier method and compared by log-rank tests and Cox's models were used to identify prognostic factors impacting survival. The 210 IBC patients had a median age of 42 years (24-62) and 15% of them were aged less than 35 years. Mean age at menarche was 13 years and 45% had their 1st childbirth before 23 years. On histology, grades III represented 42% of cases, hormone receptors were negative in 59%, HER2 over-expressed in 32, 25% of our IBC cases had a triple negative profile and Ki-67 was >20% in 53% of cases. High pathological grade III was significantly correlated to TN subtype (58%) (Fisher's exact test, P=7.5×10-3). Further, high Ki-67 expression (>20%) was evident in the TN subtype (84%) (Fisher's exact test, P=3.7×10-4). After neoadjuvant therapy (and trastuzumab in 88 and 69% of HER2+ patients, respectively), we observed 49% of objective clinical responses and 35% of pathological complete response (pCR) and >3 axillary lymph nodes were invaded on a resected tumor in 55% of cases. Overall survival (OS) was associated with age at menarche (Wald-test, P=2.2×10-2) and metastases at diagnosis (Wald-test, P=2.4×10-2). Reaching a pCR was correlated with a better metastasis-free survival (MFS), (Fisher's exact test, P=3.6×10-2).

International audience; Apoptosis is the distinctive form of programmed cell death that complements cell proliferation in maintaining normal tissue homeostasis. The significance of constitutive apoptosis in the recurrence of Non Muscle Invasive Bladder Cancer has yet to be investigated. The aim of this study is to investigate the prognostic significance of Bax and Bcl-2 in terms of recurrence after BCG immunotherapy. Immunohistochemical analysis was performed on frozen biopsies to evaluate bcl-2 and Bax proteins expression in 28 cases of NMIBC. All patients with confirmed NMIBC were treated with intravesical BCG-immunotherapy. The follow up was performed for 26 months. The correlation between clinicopathological, immunohistochemical data and the response to BCG therapy was performed. Univariate analysis showed that, PT1 stage, High grade and Bax expression increased significantly the risk of recurrence (P = 0.015, P = 0.015 and P= 0.034 respectively). In addition, multivariate analysis selected the model involving stage, age, Bax and Bcl-2 expression as the best independent variables of recurrence. In conclusion, the expression of Bcl-2 and Bax in NMIBC could have a prognostic value in assessing the risk of recurrence after BCG immunotherapy. These findings require further investigations on larger cohort in order to ascertain new molecular markers of the response to BCG immunotherapy.

Modifications of mechanical properties of wheat dough during thermal treatments depend mainly on the capacity of wheat gluten proteins to establish intra- and intermolecular interactions when subjected to high-temperature processing. The present study investigates the effect of thermal treatments on the mechanical properties and protein solubility of wheat gluten-based network. The increase in treatment temperatures (from 80 to 135 C) induces an increase in mechanical resistance of the gluten network (tensile strength increases from 0.26 to 2.04 MPa) and a decrease in deformability (elongation decreases from 468 to 236%). The increase in temperature (from 80 to 135 C) also induces a very strong reduction of protein solubility in 2% SDS (from 68 to 0%) that could be correlated to the mechanical changes observed. It was concluded that the modifications of the wheat gluten network properties seem to depend mainly on the temperature level, as temperatures >108-116 C allow activation of thermosetting reactions.

Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid-maturation seed cDNA libraries. The T. aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T. durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5 % similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment, anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13 391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5′-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP : thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed α-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S-S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the alpha and beta fractions were most potent, followed by the gamma and omega types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the alpha, beta, and gamma types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP-thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

International audience; Gelsolin and calponin are cytoskeletal and signalling proteins that form a tight 1:1 complex (GCC). We show that calponin within the GCC inhibits the rate of gelsolin mediated nucleation of actin polymerization. The actin-binding function of calponin is ablated within the GCC as the actin-binding site overlaps with one of the gelsolin binding sites. The structure of filaments that result from nucleation by GCC are different to those nucleated by gelsolin alone in that they are longer, loosely bundled and stain heterogeneously with phalloidin. GCC nucleated filaments appear contorted and wrap around each to form the loose bundles.

In order to develop QTL applications, eight new loci were mapped on barley chromosome 7 using 124 doubled haploid lines of the North American Barley Genome Mapping Project (NABGMP) progeny ('Steptoe' x 'Morex'). These loci involve six genomic DNA restriction fragment length polymorphisms (RFLPs) and two cDNA-RFLPs including a puroindoline gene. The distribution of these markers on barley chromosome 7 was compared with that of homoeologous wheat counterparts, i.e. wheat group 5. One locus on chromosome 7 was associated with a QTL for β-glucanase activity measured in green and finished barley malt.

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International audience; Knowledge on physiological mechanisms and plant metabolism can be used to enhance metal uptake. The capacity to uptake metals of transgenic tobaccos overexpressing ferritin in plastids (P6) or in cytoplasm (C5) and a control tobacco (A) is assessed in three polluted soils from the same soil series, with a similar Cd content, but displaying pH from 5.8 to 7 (8b2, 8b3, S11). Differences in dry leave weight were not significant between the three tobaccos growing on each soil. Iron concentration in ferritin overexpression either in P6 or in C5 tobaccos increased only on the S11 soil, which had a soil pH 7, in comparison to A tobacco. In both 8b2 and 8b3 soils at pH lower than 7, the leaf Fe content was not different between the three tobaccos. Only the P6 tobacco growing in the S11 soil accumulated more Cd, Zn and Mn compared with the A and C5 tobaccos. This increase represents, compared to A tobacco, 88% for Fe, 120% for Mn, 30% for Cd, 80% for Zn. In soils with a pH higher than 7, the ferritin synthesis is over-expressed in the P6 tobacco cells inducing a iron deficiency. These metal accumulations were increased, owing to a rhizosphere modification. The A and P6 tobaccos uptake Cd in the same soil pool. The Cd totality in the 8b2 soil is labile and in the S11 soil, the labile Cd represents 86–88% of the total Cd

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A type 2 non-specific lipid transfer protein (ns-LTP2) was isolated from wheat ( Triticum durum Desf.) endosperm and the primary structure of the protein was fully determined. The protein is basic with a predicted p I of 8.4 and a molecular mass of 6980 Da. It contains 67 amino acids including eight cysteine residues. Oligonucleotide probes covering part of the protein sequence were designed to isolate the corresponding cDNA that contains an open reading frame of 96 amino acids, including a 29 amino acid signal peptide. The T. durum ns-LTP2 showed 37.3–77.6% identity with other ns-LTP2 and is most closely related to the barley and rice sequences. PCR carried out on wheat genomic DNA indicated that the gene encoding this ns-LTP2 does not contain introns. Within developing seeds, the level of ltp7.1 gene transcripts detected by RT-PCR remained broadly similar from the cellular division phase to dry seed. This contrast with the expression pattern of the ltp9.1 gene, the transcripts of which started to accumulate during the filling phase. During germination, the level of ltp7.1 gene transcripts decreased more rapidly than those of the ltp9.1 gene.

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Background: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. Objective: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. Methods: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. Results: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. Conclusion: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

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The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to #Triticum aestivum# puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to - 1068, -388, -210 or - 124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the #uidA# reporter gene encoding beta-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the #uidA# reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the #puroindoline-b# promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5' promoter deletions identified the region between -388 and -210 as essential for endosperm expression, and the region between -210 and -124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes. (Author's abstract)

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International audience; Swards of cocksfoot (cvs KM2, Lutetia) and perennial ryegrass (cvs Aurora, Vigor) were grown under full irrigation or severe (80 d) drought in a field experiment in the South of France. Responses of the bases of immature leaves plus enclosed tissues were made during the drought period and after rewatering. By the end of the drought, water content had fallen from 3.0 to 0.8 g(water)g(dm)(-1), and osmotic potential from -1.0 to -4.5 MPa in all cvs. Measured minerals and water-soluble carbohydrates contributed, respectively, c 19 and 44% to osmotic potential in droughted leaf bases. The drought-sensitive cocksfoot cv. Lutetia was characterized by a large proportion of fructans having a low degree of polymerization (DP = 3, 4). As drought progressed, accumulation of dehydrin transcripts and ABA were higher in leaf bases of the sensitive cv. Lutetia than in the resistant cv. KM2. After rewatering, the water status of immature leaf bases returned to control levels in 1-2 d, and then increased further as leaves began to grow and new tissue was produced. High-DP-fructans remained unchanged in leaf bases of 'Lutetia' but were depleted by over 55%, and therefore remobilized, in leaf bases of other cvs after 8 d. It is concluded that enclosed immature leaf bases survive drought by tolerating a low water status and that changes conventionally associated with desiccation tolerance are expressed most strongly in susceptible plants least able to maintain their water supply.

*INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA) Diffusion du document : INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA); International audience

*INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA) Diffusion du document : INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA); International audience

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International audience; Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.

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We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

A molecular-marker linkage map of wheat (Triticum aestivum L. em. Thell) provides a powerful tool for identifying genomic regions influencing breadmaking quality. A variance analysis for kernel hardness was conducted using 114 recombinant inbred lines (F7) from a cross between a synthetic and a cultivated wheat. The major gene involved in kernel hardness, ha (hard), known to be on chromosome arm 5DS, was found to be closely linked with the locus Xmta9 corresponding to the gene of puroindoline-a. This locus explained around 63% of the phenotypic variability but there was no evidence that puroindoline-a is the product of Ha (soft). Four additional regions located on chromosomes 2A, 2D, 5B, and 6D were shown to have single-factor effects on hardness, while three others situated on chromosomes 5A, 6D and 7A had interaction effects. Positive alleles were contributed by both parents. A three-marker model explains about 75% of the variation for this trait.

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International audience; Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca(2+) mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation-contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.