Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand

Bacteria of the genera Photorhabdus and Xenorhabdus produce a plethora of natural products to support their similar symbiotic life cycles. For many of these compounds, the specific bioactivities are unknown. One common challenge in natural product research when trying to prioritize research efforts is the rediscovery of identical (or highly similar) compounds from different strains. Linking genome sequence to metabolite production can help in overcoming this problem. However, sequences are typically not available for entire collections of organisms. Here, we perform a comprehensive metabolic screening using HPLC-MS data associated with a 114-strain collection (58 Photorhabdus and 56 Xenorhabdus) across Thailand and explore the metabolic variation among the strains, matched with several abiotic factors. We utilize machine learning in order to rank the importance of individual metabolites in determining all given metadata. With this approach, we were able to prioritize metabolites in the context of natural product investigations, leading to the identification of previously unknown compounds. The top three highest ranking features were associated with Xenorhabdus and attributed to the same chemical entity, cyclo(tetrahydroxybutyrate). This work also addresses the need for prioritization in high-throughput metabolomic studies and demonstrates the viability of such an approach in future research.     

L1CAM: A major driver for tumor cell invasion and motility

The L1 cell adhesion molecule (L1CAM) plays a major role in the development of the nervous system and in the malignancy of human tumors. In terms of biological function, L1CAM comes along in two different flavors: (1) a static function as a cell adhesion molecule that acts as a glue between cells; (2) a motility promoting function that drives cell migration during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are: (1) the cleavage from the cell surface by membrane proximal proteolysis and (2) the ability to change binding partners and engage in L1CAM-integrin binding. Recent studies have shown that the cleavage of L1CAM by metalloproteinases and the binding of L1CAM to integrins via its RGD-motif in the sixth Ig-domain activate signaling pathways distinct from the ones elicited by homophilic binding. Here we highlight important features of L1CAM proteolysis and the signaling of L1CAM via integrin engagement. The novel insights into L1CAM downstream signaling and its regulation during tumor progression and epithelial-mesenchymal transition (EMT) will lead to a better understanding of the dualistic role of L1CAM as a cell adhesion and/or motility promoting cell surface molecule.     

Signatures of T cells as correlates of immunity to Francisella tularensis

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4⁺ and/or CD8⁺ T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naïve). Significant differences were detected between either of the immune donor groups and naïve individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, MCP-1, and MIP-1β. Expression of IFN-γ, MIP-1β, and CD107a by CD4⁺CD45RO⁺ or CD8⁺CD45RO⁺ T cells correlated to antigen concentrations. In particular, IFN-γ and MIP-1β strongly discriminated between immune and naïve individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-α-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.     

Optimizations of Pulsed Plated p and n‐type Bi2Te3‐Based Ternary Compounds by Annealing in Different Ambient Atmospheres

This work presents a comprehensive study of the fabrication and optimization of electrodeposited p‐ and n‐type thermoelectric films. The films are deposited on Au and stainless steel substrates over a wide range of deposition potentials. The influence of the preparative parameters such as the composition of the electrolyte bath and the deposition potential are investigated. Furthermore, the p‐doped (Bi_xSb_1‐x)₂Te₃ and the n‐doped Bi₂(Te_xSe_1‐x)₃ films are annealed for a period of about 1 h under helium and under tellurium atmosphere at 250 °C for 60h. Annealing in He already leads to significant improvements in the thermoelectric performance. Furthermore, due to the equilibrium conditions during the process, annealing in Te atmosphere leads to a strongly improved film composition, charge carrier density and mobility. The Seebeck coefficients increase to values up to +182 μV K^?1 for p‐doped and–130 μV K^?1 for n‐doped materials at room temperature. The power factors also exhibit improvements with 1320 μW m^?1 K^?2 and 820 μW m^?1 K^?2 for p‐doped and n‐doped films, respectively. Additionally, in‐situ XRD measurements performed during annealing of the films up to 600K under He atmosphere show stepwise improvements of the crystal structure leading to the improvements in thermoelectric parameters. The thermal conductivity is between 1.2 W m^?1 K^?1 and 1.0 W m^?1 K^?1.     

Improved quality of 1H NMR spectroscopic data for enhanced metabolic profiling of low molecular weight metabolites in human serum

NMR based metabolic profiling of blood samples in epidemiological studies can be used for molecular phenotyping and biomarker discovery. Often metabolic changes in blood are more subtle and demand a high quality spectrum especially when looking at low molecular weight compounds. In order to improve ¹H NMR spectroscopic data we compared different serum sample preparation methods. Application of phosphate buffer reduces chemical shift variation, enhances resolution of signal multiplicity, facilitates visual inspection of NMR spectra and annotation of signals compared to traditionally used saline. For analysis of low molecular weight compounds we found that standard 1D spectra of ultrafiltrated serum samples show enhanced spectral quality of small metabolites as compared to transverse relaxation edited spectra (also called Carr–Purcell–Meiboom–Gill, CPMG) spectra of unfiltered serum samples due to improved signal-to-noise ratio. Thus, NMR signals attributable to different amino acids and other small metabolites could readily be detected in spectra of ultrafiltrated serum, but remained invisible in the corresponding CPMG spectra. An OPLS model of fasting blood glucose showed an increase of Q² when using spectra from ultrafiltrated serum (Q² = 0.261) compared to using CPMG spectra (Q² = 0.173). Similar results were observed for OPLS models of BMI (Q² = 0.253 and Q² = 0.216, respectively). Furthermore, a reduction in model dimensionality was observed when using ultrafiltrated serum data. In conclusion we recommend sample preparation of serum samples in phosphate buffer instead of saline. Ultrafiltration of serum samples prior to NMR analysis is beneficial especially for low concentrated small metabolites.     

Recombinant Synthesis of Human ABCG2 Expressed in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: an Experimental Methodological Study

Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS–PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.     

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.     

Characterization of the Src/Abl hybrid kinase SmTK6 of Schistosoma mansoni

Cellular protein-tyrosine kinases play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner, the cellular protein-tyrosine kinase SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in this study confirmed the first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (herbimycin A) and Abl (imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners, we identified among others the known Src kinase SmTK3 and the Venus kinase receptor SmVKR1 of S. mansoni by yeast two-hybrid analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes, it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a receptor tyrosine kinase, Syk and Src kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.