Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

. incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction.Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs

Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers

Genome-wide localization of histone variants in Toxoplasma gondii implicates variant exchange in stage-specific gene expression.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. RESULTS: H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5'UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. CONCLUSIONS: Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation

Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933

Extrasynaptic Neurotransmission in the Modulation of Brain Function. Focus on the Striatal Neuronal–Glial Networks

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks

Toxoplasma gondii Hsp90: potential roles in essential cellular processes of the parasite

Hsp90 is a widely distributed and highly conserved molecular chaperone that is ubiquitously expressed throughout nature, being one of the most abundant proteins within non-stressed cells. This chaperone is up-regulated following stressful events and has been involved in many cellular processes. In Toxoplasma gondii, Hsp90 could be linked with many essential processes of the parasite such as host cell invasion, replication and tachyzoite-bradyzoite interconversion. A Protein-Protein Interaction (PPI) network approach of TgHsp90 has allowed inferring how these processes may be altered. In addition, data mining of T. gondii phosphoproteome and acetylome has allowed the generation of the phosphorylation and acetylation map of TgHsp90. This review focuses on the potential roles of TgHsp90 in parasite biology and the analysis of experimental data in comparison with its counterparts in yeast and human

Molecular Approach for Engineering Interfacial Interactions in Magnetic/Topological Insulator Heterostructures

Altres ajuts: Interreg V-A España-Francia-Andorra (EFA 194/16 TNSI)Controlling interfacial interactions in magnetic/topological insulator heterostructures is a major challenge for the emergence of novel spin-dependent electronic phenomena. As for any rational design of heterostructures that rely on proximity effects, one should ideally retain the overall properties of each component while tuning interactions at the interface. However, in most inorganic interfaces, interactions are too strong, consequently perturbing, and even quenching, both the magnetic moment and the topological surface states at each side of the interface. Here, we show that these properties can be preserved using ligand chemistry to tune the interaction of magnetic ions with the surface states. By depositing Co-based porphyrin and phthalocyanine monolayers on the surface of BiTe thin films, robust interfaces are formed that preserve undoped topological surface states as well as the pristine magnetic moment of the divalent Co ions. The selected ligands allow us to tune the interfacial hybridization within this weak interaction regime. These results, which are in stark contrast with the observed suppression of the surface state at the first quintuple layer of BiSe induced by the interaction with Co phthalocyanines, demonstrate the capability of planar metal-organic molecules to span interactions from the strong to the weak limit

Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.Facultad de Ciencias Veterinaria

Experimental demonstration of a magnetically induced warping transition in a topological insulator mediated by rare-earth surface dopants

Magnetic topological insulators (MTI) constitute a novel class of materials where the topologically protected band structure coexists with long-range ferromagnetic order, which can lead to the breaking of time-reversal symmetry (TRS), introducing a bandgap in the Dirac cone-shaped topological surface state (TSS). The gap opening in MITs has been predicted to be accompanied by a distortion in the TSS, evolving its warped shape from hexagonal to trigonal. In this work, we demonstrate such a transition by means of angle-resolved photoemission spectroscopy after the deposition of low concentrations of magnetic rare earths, namely Er and Dy, on the ternary three-dimensional prototypical topological insulator Bi$_2$Se$_2$Te. Signatures of the gap opening occurring as a consequence of the TRS breaking have also been observed, whose existence is supported by the observation of the aforementioned transition. Moreover, increasing the Er coverage results in a tunable p-type doping of the TSS. As a consequence, the Fermi level (E$_{\textrm{F}}$) of our Bi$_2$Se$_2$Te crystals can be gradually tuned towards the TSS Dirac point, and therefore to the magnetically induced bandgap; thus fulfilling two of the necessary prerequisites for the realization of the quantum anomalous Hall effect (QAHE) in this system. The experimental results are rationalized by a theoretical model where a magnetic Zeeman out-of-plane term is introduced in the hamiltonian governing the TSS band dispersion. Our results offer new strategies to control magnetic interactions with TSSs based on a simple approach and open up viable routes for the realization of the QAHE

An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy