Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system

The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl−-coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE- FPLC). A yield of 200–300 μg of the GAT1/GFP protein could be purified from 400–600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form

Transcriptome and proteome analysis of tyrosine Kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes.

Background Canine mast cell tumor proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumor type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumor cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect

Anti-PD-1 monoclonal antibody MEDI0680 in a phase I study of patients with advanced solid malignancies.

BACKGROUND: The safety, efficacy, pharmacokinetics, and pharmacodynamics of the anti-programmed cell death-1 antibody MEDI0680 were evaluated in a phase I, multicenter, dose-escalation study in advanced solid malignancies. METHODS: MEDI0680 was administered intravenously once every 2 weeks (Q2W) or once every 3 weeks at 0.1, 0.5, 2.5, 10 or 20 mg/kg. Two cohorts received 20 mg/kg once a week for 2 or 4 weeks, then 20 mg/kg Q2W. All were treated for 12 months or until progression. The primary endpoint was safety. Secondary endpoints were efficacy and pharmacokinetics. Exploratory endpoints included pharmacodynamics. RESULTS: Fifty-eight patients were treated. Median age was 62.5 years and 81% were male. Most had kidney cancer (n = 36) or melanoma (n = 9). There were no dose-limiting toxicities. Treatment-related adverse events occurred in 83% and were grade ≥ 3 in 21%. Objective clinical responses occurred in 8/58 patients (14%): 5 with kidney cancer, including 1 with a complete response, and 3 with melanoma. The relationship between dose and serum levels was predictable and linear, with apparent receptor saturation at 10 mg/kg Q2W and all 20 mg/kg cohorts. CONCLUSIONS: MEDI0680 induced peripheral T-cell proliferation and increased plasma IFNγ and associated chemokines regardless of clinical response. CD8+ T-cell tumor infiltration and tumoral gene expression of IFNG, CD8A, CXCL9, and granzyme K (GZMK) were also increased following MEDI0680 administration. TRIAL REGISTRATION: NCT02013804 ; date of registration December 12, 2013

Coupling microchip pressure regulators with chipHPLC as a step toward fully portable analysis system

Herein the coupling of a miniaturized, nanoliter scaled, pressure regulator (chipPR), and a chipHPLC device is introduced. The active temperature based flow control of the chipPR is able to generate rapid pressure changes and therefore enables on-chip pinched injection and flow gradients with reduced instrumental effort and minimal dead volumes. The functionality of the chipPR empowered chipHLPC device was demonstrated with high-speed HPLC-separations applying fluorescence and electrospray mass spectrometry (ESI-MS) detection. The system shows excellent long-term stability of chromatography integrity (retention times with RSD of 0.44-0.91%) due to the integration of a PID feedback regulation. This first chip-based HPLC device equipped with chipPRs enables precise flow control with significantly reduced technical effort compared to the state-of-the-art

Antimicrobial defence and persistent infection in insects revisited

Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus. The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitro

Development of an automated platform for monitoring microfluidic reactors through multi-reactor integration and online (chip-)LC/MS-detection

This work presents a novel microfluidic screening setup with real-time analytics for investigating reactions with immobilised biocatalysts. The setup combines microreactor technology, multi-reactor integration, and online (chip-)LC/MS analysis in a sequential automated workflow. We utilized in-house manufactured fused-silica glass chips as reusable packed-bed microreactors interconnected as individual tube reactors. The potential of this setup was showcased by conducting and optimising a biocatalytic aromatic bromination reaction as the first proof of concept using immobilised vanadium-dependent haloperoxidase from Curvularia inaequalis (CiVHPO). The fusion of a HaloTagTM to CiVHPO was used for efficient and mild covalent linkage of the enzyme onto chloroalkane-functionalized particles. Then, the biotransformation was continuously monitored with automated LC/MS data acquisition in a data-rich manner. By further developing the automation principle, it was possible to sequentially screen multiple different connected packed-bed microreactors for reaction optimization while using only miniature amounts of reactants and biocatalyst. Finally, we present a fast and modular chipHPLC solution for online analysis to reduce the overall solvent consumption by over 80%. We established a modern microfluidic platform for real-time reaction monitoring and evaluation of biocatalytic reactions through automation of the reactant feed integration, flexible microreactor selection, and online LC/MS analysis