During chronic infection, the single celled parasite, Toxoplasma gondii, can migrate to the brain where it has been associated with altered dopamine function and the capacity to modulate host behavior, increasing risk of neurocognitive disorders. Here we explore alterations in dopamine-related behavior in a new mouse model based on stimulant (cocaine)-induced hyperactivity. In combination with cocaine, infection resulted in heightened sensorimotor deficits and impairment in prepulse inhibition response, which are commonly disrupted in neuropsychiatric conditions. To identify molecular pathways in the brain affected by chronic T. gondii infection, we investigated patterns of gene expression. As expected, infection was associated with an enrichment of genes associated with general immune response pathways, that otherwise limits statistical power to identify more informative pathways. To overcome this limitation and focus on pathways of neurological relevance, we developed a novel context enrichment approach that relies on a customized ontology. Applying this approach, we identified genes that exhibited unexpected patterns of expression arising from the combination of cocaine exposure and infection. These include sets of genes which exhibited dampened response to cocaine in infected mice, suggesting a possible mechanism for some observed behaviors and a neuroprotective effect that may be advantageous to parasite persistence. This model offers a powerful new approach to dissect the molecular pathways by which T. gondii infection contributes to neurocognitive disorders. 相似文献
The biomolecular recognition of D-mannose-binding lectin from Artocarpus heterophyllus (ArtinM) by Horseradish Peroxidase (HRP) mediated by glycosylation allows their application in a multitude of biological systems. The present work describes the use of molecular dynamics (MD) to assess the Gibbs free energy associated with the formation of a ArtinM-HRP conjugate mediated by a glycosylation molecule. For the enthalpy term, we applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method and for the vibrational entropy term, we use the quasi-harmonic approximation. Our results show that, even without glycosylation, the binding free energy between ArtinM and HRP is − 196.154 kJmol− 1, an extremely high affinity with low selectivity, originated mainly through the van der Waals energy terms. The binding free energy between ArtinM and the glycosylated HRP (gHRP) was calculated at − 66.156 kJmol− 1, an absolute and considerably lower value, however, originated from electrostatic energy terms, which increases the selectivity of molecular recognition. Our work has shown that the HRP active site region has a high affinity and low selectivity for other biomolecules. The presence of glycosylation plays a role in increasing this selectivity for this region. Thus, we conclude that performing mutagenesis of amino acid residues near the entrance of the catalytic site, can improve the activity of non-glycosylated HRPs. This illustrates new insights that can be applied to carbohydrate-based immunochemistry.
Matrix metalloproteinase 9 (MMP9) is involved in the proteolysis of extracellular proteins and plays a critical role in pancreatic ductal adenocarcinoma (PDAC) progression, invasion and metastasis. The therapeutic potential of an anti‐MMP9 antibody (αMMP9) was evaluated in combination with nab‐paclitaxel (NPT)‐based standard cytotoxic therapy in pre‐clinical models of PDAC. Tumour progression and survival studies were performed in NOD/SCID mice. The mechanistic evaluation involved RNA‐Seq, Luminex, IHC and Immunoblot analyses of tumour samples. Median animal survival compared to controls was significantly increased after 2‐week therapy with NPT (59%), Gem (29%) and NPT+Gem (76%). Addition of αMMP9 antibody exhibited further extension in survival: NPT+αMMP9 (76%), Gem+αMMP9 (47%) and NPT+Gem+αMMP9 (94%). Six‐week maintenance therapy revealed that median animal survival was significantly increased after NPT+Gem (186%) and further improved by the addition of αMMP9 antibody (218%). Qualitative assessment of mice exhibited that αMMP9 therapy led to a reduction in jaundice, bloody ascites and metastatic burden. Anti‐MMP9 antibody increased the levels of tumour‐associated IL‐28 (1.5‐fold) and decreased stromal markers (collagen I, αSMA) and the EMT marker vimentin. Subcutaneous tumours revealed low but detectable levels of MMP9 in all therapy groups but no difference in MMP9 expression. Anti‐MMP9 antibody monotherapy resulted in more gene expression changes in the mouse stroma compared to the human tumour compartment. These findings suggest that anti‐MMP9 antibody can exert specific stroma‐directed effects that could be exploited in combination with currently used cytotoxics to improve clinical PDAC therapy. 相似文献
The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate. 相似文献