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IntroductionIn immunocompromised patients, Epstein-Barr virus (EBV) infection or reactivation is associated with increased morbidity and mortality, including the development of B-cell lymphomas. The first-line treatment consists of reduction of immunosuppression and administration of rituximab (anti-CD20 antibody). Furthermore, the presence of EBV-specific T cells against latent EBV proteins is crucial for the control of EBV-associated diseases. Therefore, in addition to effective treatment strategies, appropriate monitoring of T cells of high-risk patients is of great importance for improving clinical outcome. In this study, we hypothesized that rituximab-mediated lysis of malignant EBV-infected B cells leads to the release and presentation of EBV-associated antigens and results in an augmentation of EBV-specific effector memory T-cell responses.MethodsEBV-infected B lymphoblastoid cell lines (B-LCLs) were used as a model for EBV-associated lymphomas, which are capable of expressing latency stage II and III EBV proteins present in all known EBV-positive malignant cells. Rituximab was administered to obtain cell lysates containing EBV antigens (ACEBV). Efficiency of cross-presentation of EBV-antigen by B-LCLs compared to cross-presentation by professional antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells was investigated by in vitro T-cell immunoassays. Deep T-cell profiling of the tumor-reactive EBV-specific T cells in terms of activation, exhaustion, target cell killing, and cytokine profile was performed, assessing the expression of T-cell differentiation and activation markers as well as regulatory and cytotoxic molecules by interferon-γ (IFN-γ) EliSpot assay, multicolor flow cytometry, and multiplex analyses.ResultsBy inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous ACEBV-derived antigens mainly through major histocompatibility complex (MHC) class I molecules. This mechanism is comparable to that for DCs and B cells and resulted in a strong EBV-specific CD8+ cytotoxic T-cell response. Stimulation with ACEBV-loaded APCs also led to the activation of CD4+ T helper cells, suggesting that longer peptide fragments are processed via the classical MHC class II pathway. In addition, B-LCLs were also found to be able to take up exogenous antigens from surrounding cells by endocytosis leading to induction of EBV-specific T-cell responses although to a much lesser extent than cross-presentation of ACEBV-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with ACEBV-loaded APCs, which showed high proliferative and cytotoxic capacity as indicated by enhanced EBV-specific frequencies and increased secretion levels of cytotoxic effector molecules (e.g. IFN-γ, granzyme B, perforin, and granulysin). Expression of the regulatory proteins PD-1 and Tim-3 was induced but had no negative impact on effector T-cell functions.ConclusionIn this study, we showed for the first time that rituximab-mediated lysis of EBV-infected tumor cells can efficiently boost EBV-specific endogenous effector memory T-cell responses through cross-presentation of EBV-derived antigens. This promotes the restoration of antiviral cellular immunity and presents an efficient mechanism to improve the treatment of CD20+ EBV-associated malignancies. This effect is also conceivable for other therapeutic antibodies or even for therapeutically applied unmodified or genetically modified T cells, which lead to the release of tumor antigens after specific cell lysis.

International audience; Ectomycorrhizal (ECM) fungi associate with the roots of woody plants in temperate and boreal forest, helping them to acquire water and nutrients. The molecular mechanisms responsible for these nutritional transfers are still poorly understood. The ECM fungus Hebeloma cylindrosporum expresses three H+:Pi symporters (HcPT1.1, HcPT1.2, and HcPT2) in extraradical and intraradical hyphae of ectomycorrhizae to transport phosphorus (P) from the soil to colonized roots. Since HcPT1.1 is only upregulated under low P conditions, we wondered whether an artificial overexpression of this transporter by a fungal Agrotransformation strategy could result in better P nutrition of the host plant Pinus pinaster. Surprisingly, we demonstrated that plants interacting with transgenic fungal lines overexpressing HcPT1.1 did not accumulate more P in their shoot compared to plants colonized with control isolates. Although the overexpression of HcPT1.1 did not affect the expression level of the other two P transporters in pure culture, a strong reduction of HcPT2 proteins in ectomycorrhizae, particularly in the hyphae internally colonizing roots, was observed by an immunolocalization approach. Our results revealed that the artificial overexpression of the P transporter HcPT1.1 strongly decreased the distribution of HcPT2 proteins in symbiotic structures, but still improved the P status of the host plant shoots compared to non-mycorrhizal plants. This suggests that a tight regulation and a functional redundancy between the H+:Pi symporters of H. cylindrosporum might exist to ensure a sustainable P delivery to P. pinaster roots.

This dissertation examines depictions of refugees in twenty-first-century German-language literature by Elfriede Jelinek, Jenny Erpenbeck, Navid Kermani, Maxi Obexer and Firas Alshater. Their selected works challenge stereotypical narratives about refugees as ‘undesirables,’ ‘invasive others,’ and ‘problems’ that threaten liberal nation-states in Europe. This study aims to illustrate how their texts convey that refugees are persons with many complex social identities rather than ‘invaders.’ My interdisciplinary methodology is based on three pillars: a mobility paradigm taken from human geography, a philosophical discussion of personhood, and notions of place derived from human geography and philosophy. I will show how interconnections between mobility, personhood, and place are relevant because refugees are adaptable persons in need of a new place (at least temporarily), but they experience a differential mobility compared to privileged migrants. First, the mobility paradigm enables a reading of literature that shows how current European Union asylum rules attempt to prevent displaced persons from accessing individual member country jurisdictions. As a result, refugees’ ability to physically move towards and within Europe is restricted. Second, literature can evoke philosophical ideas of personhood that illustrate detrimental effects when the label ‘refugee’ is repeatedly affixed to a displaced person’s life story. This label overrides all their past, present, and future experiences, and it unjustly portrays them as ‘problems’ or ‘undesirables’ rather than resourceful and capable individuals. Third, literature can reflect how places are shaped, maintained, and continually changed because social relations, experiences, and understandings intersect in persons’ life narratives. The findings of my interdisciplinary study are relevant for literary discourses on the categorization of migrants and on current European Union politics that support the free flow of goods and money but undermine the arrival of those in need of refuge.

Additional file 3. Changes on standard blood counts and the cellular immune status after continuous and interval exercise. Peripheral blood samples of healthy donors (n = 12) were analysed by flow cytometry at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Determined frequencies of CD45+ leukocytes, CD14+ monocytes, CD19+ B cells, CD3+/CD56+ NKT cells, and CD3−/CD56+ NK cells with their bright and dim subsets are displayed as normalized frequencies. Data are mean ± SD.

Additional file 4. Changes in standard blood counts after continuous and interval exercise differentiating between moderately and highly fit donors. Peripheral blood samples of healthy donors (n = 12) were analysed using a haemocytometer at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Donors were grouped according to their fitness level (moderate, n = 8, and high, n = 4, tested with the International Physical Activity Questionnaire, IPAQ). (A) Lymphocyte, granulocyte, and monocyte frequency is shown as percentage of the total leukocyte count. (B) White blood cell (WBC), lymphocyte, granulocyte, and monocyte counts are shown as absolute counts (×103/µl blood). Data are shown as mean ± SD.

Background: Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B∗35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact. Methods: TÜ165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B∗35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells. Results: After co-cultivation with EBV target cells, TÜ165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-gand tumor necrosis factor-a). Moreover, TÜ165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines. Conclusions: Our results demonstrate that TÜ165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. TÜ165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation.

Additional file 7. Effects of a single continuous and interval exercise on CD8+ T-cell cholesterol and activation levels in a donor-related setting. Peripheral blood samples of healthy donors (n = 5) were analysed at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise) to (A) quantify the level of cholesterol (µM)/1 × 106 cells (Amplex Red Cholesterol Assay Kit) and (B) to obtain the expression of CD69 on CD8+ T cells (flow cytometry). Data are shown in a donor-related setting as mean ± SD. Statistically significant difference is indicated by (*p

Additional file 1. Representative flow cytometric plots. Representative flow cytometry plots illustrating the gating strategy used to analyse (A) the cellular immune status in whole blood sample on a single-cell platform using TruCount tubes, (B) the enrichment efficiency of IFN-γ+ VSTs selected from PBMCs using the IFN-γ cytokine secretion assay, and (C) the activation level of isolated CD8+ T cells using CD69 as specific marker before and after exercise.

Additional file 5. Effects of a single continuous and interval exercise on stress, proliferation markers, T-cell cytotoxicity and parameters of cellular age. Peripheral blood samples of healthy donors (n = 12) were analysed at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise) using real-time PCR for quantification of (A) HSP70- and Ki67-, IFN-γ-, and granzyme B-mRNA levels. Constitutively expressed GAPDH gene was used as the reference standard for normalization of mRNA levels. RQ values were calculated by the delta–delta CT method. (B) Blood samples were further analysed regarding p16 levels, telomere ratio and telomere base pairs. Results are displayed as RQ, with the value from before exercise being the base line, and shown as mean ± SD.

Additional file 6. Impact of a single continuous and interval exercise on antigen-specific T-cell responses. Peripheral blood samples of healthy donors (n = 12) were analysed separately with the two-sample t approach adjusting for period at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Isolated PBMCs were stimulated overnight with CMV-, EBV- and AdV-specific peptide pools (CMV pp65, CMV IE1, EBV EBNA1, and EBV Consensus, AdV5 Hexon, and AdV5 Penton) and frequencies of functional-active virus-specific T cells were determined by IFN-γ EliSpot assay as spots per 1000 CD3+ T cells

Post-transplant lymphoproliferative disease (PTLD) represents a serious complication following allogeneic hematopoietic stem cell transplantation (alloHSCT). Previously, survival rates of PTLD have improved due to the introduction of rituximab. However, reports on curative management of refractory PTLD are scarce. Today, there is no consensus how to treat rituximab-refractory PTLD, especially in highly aggressive disease. Here, we describe successful management of refractory EBV-associated PTLD, specifically DLBCL, with combined brentuximab vedotin and third-party EBV-specific T-cells in a multidisciplinary treatment approach.

Post-transplant lymphoproliferative disease (PTLD) represents a serious complication following allogeneic hematopoietic stem cell transplantation (alloHSCT). Previously, survival rates of PTLD have improved due to the introduction of rituximab. However, reports on curative management of refractory PTLD are scarce. Today, there is no consensus how to treat rituximab-refractory PTLD, especially in highly aggressive disease. Here, we describe successful management of refractory EBV-associated PTLD, specifically DLBCL, with combined brentuximab vedotin and third-party EBV-specific T-cells in a multidisciplinary treatment approach. CA extern

International audience

Arbuscular mycorrhizal fungi (AMF) establish a symbiotic association with the roots of 80% of terrestrial plants and form complex tree-shaped feeding structures called arbuscules in colonized root cells. This association with AMF not only provides more efficient uptake of nutrients for the plant, but also confers protection against pathogens and increased tolerance to environmental stress such as salt stress [1]. Rice (Oryza sativa), the most salt-sensitive crop species amongst cereals, has a productivity strongly reduced around the world due to soil salinity/salinization and increased sea level (in deltas). High Na+ concentrations (salt stress conditions) impairs K+ uptake and inhibits many K+-activated enzymes. Rice exhibits molecular mechanisms to alleviate salt stress such as maintaining a high cellular K+/Na+ ratio, e.g. by a more efficient K+ uptake, which was recently shown to occur upon root/AMF interaction [2]. Knowledge on the role of root/AMF interaction on K+/Na+ transport upon salt stress is sparse [2, 3]. The aim of the research is to understand the mechanisms by which AMF improve plant K+ nutrition upon salt stress, taking Rhizophagus irregularis (model AMF)-rice interaction as a model. We will investigate the mechanisms by which AMF mediate K+ uptake and translocation towards root cells. Rice loss-of-function mutants for each of the three major K+ uptake systems of the plant, OsAKT1, OsHAK5 and OsHAK1, were inoculated with R. irregularis and submitted to salt stress, and their K+ contents and K+/Na+ ratios were monitored to identify key player(s) for AMF-mediated improved rice K+ nutrition and salt tolerance.

Unravelling nutrient exchange in ectomycorrhizal symbiosis. 3rd international Molecular Mycorrhiza Meeting

»Ins Freie« entwirft Grundzüge einer philosophischen Ästhetik vor dem Hintergrund künstlerischer Praxis. Brennpunkt ist dabei ein Vergleich von künstlerischen und philosophischen Tätigkeiten, die als »Praktiken des Öffnens« beschrieben werden. Der Redesinn dieser Formulierung speist sich aus zwei Quellen. Zum einen spricht Theodor W. Adorno in seiner Negativen Dialektik vom Ziel, dem »Telos der Philosophie« als »das Offene und Ungedeckte«. In einer solchen Ausrichtung entzieht Denken sich unaufhörlich einem abschließenden Urteil und hinterfragt beständig die bestehenden Verhältnisse. Zum anderen wurde die Formulierung »Öffnen« von Untersuchungen inspiriert, die im Kontext von Performance-Kunst unternommen werden. Dort unterbrechen Experimentelle Live-Interventionen Handlungsmuster und Wahrnehmungserwartungen und produzieren Unvorhersehbarkeiten im Kommunikationsverlauf mit einem Publikum. Die Erfahrung solcher Impulse kann dazu motivieren, bestehende Identifikationen plötzlich in Zweifel zu ziehen. Sie kann bestehende Wahrnehmungs- und Denk-Gewohnheiten für eine andere Selbst- und Weltwahrnehmung öffnen. Woran kann man einen Öffnungs-vorgang erkennen? Im Kontext dieser Frage formuliert sich die Ausgangslage und der Grundgedanke der vorliegenden Arbeit: Zeitgenössische Kunst bildet nicht einfach ab, sondern hat sich in gewisser Weise darauf spezialisiert, gewohnte Wahrnehmungs- und Denkmuster zu unterbrechen. Der heutige Künstler scheint ein »Spezialist für Ent-selbstverständlichung« zu sein. In dieser Perspektivierung stellt sich »Ins Freie« der Herausforderung, die künstlerische Reflexion von verlaufsoffenen Prozessen beschreibbar zu machen. Die zugrunde liegenden systematischen Fragen lauten • Sind Philosophie und Kunst in ihren Tätigkeiten vergleichbar? • In welchen Medien bewegen sie sich? • Was heißt Reflexion in dem einen wie dem anderen Fall? • Gibt es in der Philosophie etwas einem »verlaufsoffenen Prozess« vergleichbares? • Welche Positionen von »Künstler und Philosoph« bieten sich im Hinblick auf einen Dialog an und warum? • Ist es überhaupt möglich, verlaufsoffene Prozesse im Zusammenhang künstlerischer und philosophischer Tätigkeiten zu beschreiben und welche Aspekte können dabei als Koordinaten dienen? Ausgehend von der anfänglichen Beobachtung, dass Kunst im musealen Kontext von einer zunehmenden Flut von kommentierenden Texten begleitet wird, entwickelt die vorliegende Arbeit einen philosophischen Vergleich von Werken des amerikanischen Künstlers Richard Serra mit Werken des französischen Philosophen Maurice Merleau-Ponty und exemplifiziert dabei die Annahme, dass man künstlerisch reflektieren kann, ohne einem Kunstwerk eine Aussage zu unterstellen.

Ectomycorrhizal (ECM) symbiosis is defined as a mutual association between fungal symbionts and roots of woody plants. This symbiosis is strongly represented in temperate and boreal forests and is considered to be the most widespread means of increasing P acquisition by trees. Despite the importance of the ECM association, current knowledge about molecular mechanisms involved in membrane transport between the two partners is still limited. Therefore, the identification of the mechanisms essential for the symbiosis is a major issue. To better understand these mechanisms, we used a model association between the ECM basidiomycete Hebeloma cylindrosporum and the maritime pine Pinus pinaster. The fungal genome contains three transporters of Pi (HcPT1.1, HcPT1.2, and HcPT2), which have been already characterized as H+ Pi carriers. We hypothesized that one of these carriers could be responsible for both influx and efflux of Pi from the soil to the fungus and from the fungus to the plant. Our results suggest that HcPT2 would be the best candidate to fill both roles. An in vitro ectomycorrhizal system was used to investigate phosphate flux from H. cylindrosporum toward a liquid compartment containing pine roots. Interestingly, the expression pattern of HcPT2 was correlated to fungal phosphate efflux. Moreover, immuno-localization experiments showed that the protein is located (i) in the extraradical hyphae and the fungal sheath, compatible with Pi uptake, and (ii) within the Hartig net, which is the place of exchange between fungal and host cells. In order to study more specifically the role of HcPT2 at the fungus-plant interface, overexpressing and knock-down transgenic H. cylindrosporum strains were produced. Fungal strains with up regulated expression of HcPT2 increased phosphate efflux within the interaction medium, supporting a role of HcPT2 in phosphate transfer to the plant. In addition, the modification of HcPT2 expression strongly affects Pi nutrition of P. pinaster.

Extracellular proteins play crucial roles in the interaction between mycorrhizal fungi and their environment. Computational prediction and experimental detection allowed identification of 869 proteins constituting the exoproteome of Hebeloma cylindrosporum. Small secreted proteins (SSPs) and carbohydrate-active enzymes (CAZymes) were the two major classes of extracellular proteins. Twenty-eight per cent of the SSPs were secreted by free-living mycelia and five of the 10 most abundant extracellular proteins were SSPs. By contrast, 63-75% of enzymes involved in nutrient acquisition were secreted. A total of 150 extracellular protein-coding genes were differentially expressed between mycorrhizas and free-living mycelia. SSPs were the most affected. External environmental conditions also affected expression of 199 exoproteome genes in mycorrhizas. SSPs displayed different patterns of regulation in response to presence of a host plant or other environmental signals. Several of the genes most overexpressed in the presence of organic matter encoded oxidoreductases. Hebeloma cylindrosporum has not fully lost its ancestral saprotrophic capacities but rather adapted them not to harm its hosts and to use soil organic nitrogen. The complex and divergent patterns of regulation of SSPs in response to a symbiotic partner and/or organic matter suggest various roles in the biology of mycorrhizal fungi.

In forest ecosystems, the availability of phosphorus as inorganic P (Pi) limits tree growth. To overcome this deficiency, trees are associated with fungi through new organs called ectomycorrhizae (ECM). Despite the importance of phosphate nutrition, the mechanisms involved in the unidirectional transfer of P between the fungus and the plant are still unknown. Therefore, the identification of these mechanisms essential for symbiosis is a major issue. To understand better these mechanisms, we used a model association between the ECM basidiomycete Hebeloma cylindrosporum and the maritime Pine, Pinus pinaster. The fungal genome contains three transporters of Pi (HcPT1.1, HcPT1.2 and HcPT2) which have been already characterized as H+:Pi carriers.We hypothesized that one of these carriers could be responsible for both influx and efflux of Pi respectively from the soil to the fungus and from the fungus to the plant. Our first results suggest that HcPT2 would be the best candidate to fill both roles. Immunolocalization showed that the protein is located in (i) the extraradical hyphae and the fungal sheath involved in Pi uptake and (ii) the Hartig net, ie the interface between fungal and host cells. We also produced overexpressing and knock-down transgenic H. cylindrosporum strains to study specifically the role of HcPT2 in (i) the P efflux from the fungus measured in vitro and (ii) the P nutrition of the host plant grown at low and high P levels. Finally, if our hypothesis is confirmed, the main question will be to define how this transporter is regulated to provide both influx and efflux of phosphate in ECM symbiosis.

International audience; Mycorrhizal associations are known to improve the hydro-mineral nutrition of their host plants. However, the importance of mycorrhizal symbiosis for plant potassium nutrition has so far been poorly studied. We therefore investigated the impact of the ectomycorrhizal fungus Hebeloma cylindrosporum on the potassium nutrition of Pinus pinaster and examined the involvement of the fungal potassium transporter HcTrk1. HcTrk1 transcripts and proteins were localized in ectomycorrhizas using in situ hybridization and EGFP translational fusion constructs. Importantly, an overexpression strategy was performed on a H. cylindrosporum endogenous gene in order to dissect the role of this transporter. The potassium nutrition of mycorrhizal pine plants was significantly improved under potassium-limiting conditions. Fungal strains overexpressing HcTrk1 reduced the translocation of potassium and phosphorus from the roots to the shoots of inoculated plants in mycorrhizal experiments. Furthermore, expression of HcTrk1 and the phosphate transporter HcPT1.1 were reciprocally linked to the external inorganic phosphate and potassium availability. The development of these approaches provides a deeper insight into the role of ectomycorrhizal symbiosis on host plant K(+) nutrition and in particular, the K(+) transporter HcTrk1. The work augments our knowledge of the link between potassium and phosphorus nutrition via the mycorrhizal pathway.

Shaker K(+) channels form the major K(+) conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K(+) channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K(+) rectifying channel1 (AtKC1). These two subunits contribute to K(+) transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K(+)) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.

International audience; Potassium (K+) is one of the most abundant element of soil composition but its very low availability limits plant growth and productivity of ecosystems. Because this cation participates in many biological processes, its constitutive uptake from soil solution is crucial for the plant cell machinery. Thus, the understanding of strategies responsible of K+ nutrition is a major issue in plant science. Mycorrhizal associations occurring between roots and hyphae of underground fungi improve hydro-mineral nutrition of the majority of terrestrial plants. The contribution of this mutualistic symbiosis to the enhancement of plant K+ nutrition is still underestimated and poorly studied so far. This mini-review examines the current knowledge about the impact of both arbuscular mycorrhizal and ectomycorrhizal symbioses on the transfer of K+ from the soil to the plants. A model summarizing plant and fungal transport systems identified and hypothetically involved in K+ transport is proposed. In addition, some data related to benefits for plants provided by the improvement of K+ nutrition thanks to mycorrhizal symbioses are presented.

Potassium (K+) is one of the most abundant element of soil composition but its very low availability limits plant growth and productivity of ecosystems. Because this cation participates in many biological processes, its constitutive uptake from soil solution is crucial for the plant cell machinery. Thus, the understanding of strategies responsible of K+ nutrition is a major issue in plant science. Mycorrhizal associations occurring between roots and hyphae of underground fungi improve hydro-mineral nutrition of the majority of terrestrial plants. The contribution of this mutualistic symbiosis to the enhancement of plant K+ nutrition is still underestimated and poorly studied so far. This mini-review examines the current knowledge about the impact of both arbuscular mycorrhizal and ectomycorrhizal symbioses on the transfer of K+ from the soil to the plants. A model summarizing plant and fungal transport systems identified and hypothetically involved in K+ transport is proposed. In addition, some data related to benefits for plants provided by the improvement of K+ nutrition thanks to mycorrhizal symbioses are presented.

Magnesiumphosphatschäume nehmen auf Grund ihrer guten Resorbierbarkeit, unter physiologischen Bedingungen, einen immer größeren Stellenwert als Knochenersatzmaterial ein. Ein weiterer Vorteil ist der neutrale pH-Wert den das entstehende Material besitzt. Magnesiumphosphatschäume besitzen eine hochporöse offenporige Struktur um zum einen den Knochen nachzuahmen und zum anderen die Steuerung und Bildung von Knochengewebe zu ermöglichen. In der vorliegenden Arbeit wurden die mechanischen Eigenschaften als auch die Zytokompatibilität der hergestellten Schäume untersucht. Es wurden unterschiedliche Herstellungsverfahren genutzt um Magnesiumphosphatschäume zu erhalten. Zum einen das Replika- Verfahren, die dabei entstandenen Farringtonit Schäume (Mg3(PO4)2, Farringtonit) wurden zu Struvit ((NH4)Mg(PO4)•6H2O) umgewandelt bzw. mit PLGA infiltriert und auf ihre mechanische Eigenschaften hin untersucht. Zum anderen wurde ein proteinbasierter Schaumbildner verwendet. Die Zytokompatibilitätsprüfung wurde mit der Osteosarkomzelllinie MG-63 durchgeführt. Es erfolgte die Untersuchung der Zellproliferation und der Zellaktivität (WST). Zudem wurden Proben mittels Licht- und Elektronenmikroskopie analysiert. Die Feststellung der Proteinexpression erfolgte nach gelelektrophoretischer Auftrennung mittels Western Blot und PCR Analyse., Mg-phosphate ceramics have gained growing interest as bone replacement materials due to their ability to degrade under physiological conditions. Another advantage of these materials is the setting at neutral pH value since the powder as well as the liquid component are non acidic. In order to mimic cancellous bone and to promote tissue repair mechanisms a highly macroporous structure with open cells is desired to allow cell ingrowth. The mechanical properties and the cytocompatibility of newly developed scaffolds were analyzed in this study. Open porous magnesiumphosphate scaffolds were produced by the Replika-technique and by a protein based foam generating agent. Farringtonite scaffolds (Mg3(PO4)2, farringtonite) were modified by transformation to struvite ((NH4)Mg(PO4)•6H2O) or infiltrated with PLGA. Physical sample characterization was done. Cytocompatibility was tested using osteoblasts like cell line MG63. Cell number and cell activity (WST) were tested. The surface dependent expression of osteoblast specific proteins as well as an indicator for adhesion were tested.

Control of K+ channel activity and transport in plants: the C-linker of Arabidopsis Shaker inward channels plays a crucial role in both surface expression and channel activity. Gordon Research Conferences - Salt & Water Stress in Plants

International audience; A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.

Durant une formation en biophysique, j'ai développé un grand intérêt pour les analyses et interprétations de phénomènes biologiques avec les moyens et lois de la physique. Les mouvements des ions à travers des membranes cellulaires étaient devenus quasiment visibles au niveau moléculaire par des enregistrements de petits courants de chaque protéine responsable du transport. Les progrès du développement de techniques spectaculaires comme le patch-clamp ont ouvert un domaine de la recherche complètement nouveau permettant dans une première période une description détaillée des flux ioniques à travers des membranes cellulaires chez les animaux comme chez les plantes. Dans une deuxième période, avec les progrès en biologie moléculaire, le séquençage de génomes, et analyses de mutants, l'identification de ces molécules responsables des courants observés et analysés a provoqué encore une diversification de la recherche dans ce domaine. Le challenge était et est toujours, de corréler les gènes et protéines avec les fonctions de transport ionique déjà décrites. Mes activités de recherche s'inscrivent dans ce contexte, après apprentissage de la technique du patch-clamp avec des moyens limités en Allemagne de l'Est (Thèse à Berlin) et ensuite, après la réunification en 1990, avec des moyens plus sophistiqués, j'ai participé à la description fonctionnelle des canaux ioniques au niveau des cellules végétales in vivo (initiation à Göttingen et post-doc à Gif-sur-Yvette). Ensuite, j'ai participé à l'identification des canaux au niveau moléculaire et à leur caractérisation par expression fonctionnelle dans les systèmes hétérologues comme les cellules d'insectes et les ovocytes de grenouille (post-doc à Golm). Mes présents projets sont en continuité avec ces travaux, mais concernent aussi un modèle physiologique encore plus complexe, la symbiose entre champignon et plante, spécifiquement l'analyse de mécanismes de transports ioniques entre les deux partenaires en analysant les canaux et transporteurs d'un champignon ectomycorhizien (CR à Montpellier).

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.