The Interfacial Interactions of Glycine and Short Glycine Peptides in Model Membrane Systems.

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via 1H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.

Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes.

AIMS/HYPOTHESIS: Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development. METHODS: MIN6 and EndoC-ßH1 beta cell lines and human islets were treated with IL-1ß, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed. RESULTS: Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants. CONCLUSIONS/INTERPRETATION: We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.

Expanding the genetic toolkit of Tribolium castaneum.

The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. However, many standard genetic tools are lacking or underdeveloped in this system. Here, we present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that labels cell membranes and nuclei with different fluorophores for the analysis of cellular morphology. This approach also demonstrates the functionality of the viral T2A peptide for bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve as a blueprint for the further development of this powerful model's genetic toolkit.

Selective speciation improves efficacy and lowers toxicity of platinum anticancer and vanadium antidiabetic drugs.

Improving efficacy and lowering resistance to metal-based drugs can be addressed by consideration of the coordination complex speciation and key reactions important to vanadium antidiabetic drugs or platinum anticancer drugs under biological conditions. The methods of analyses vary depending on the specific metal ion chemistry. The vanadium compounds interconvert readily, whereas the reactions of the platinum compounds are much slower and thus much easier to study. However, the vanadium species are readily differentiated due to vanadium complexes differing in color. For both vanadium and platinum systems, understanding the processes as the compounds, Lipoplatin and Satraplatin, enter cells is needed to better combat the disease; there are many cellular metabolites, which may affect processing and thus the efficacy of the drugs. Examples of two formulations of platinum compounds illustrate how changing the chemistry of the platinum will result in less toxic and better tolerated drugs. The consequence of the much lower toxicity of the drug, can be readily realized because cisplatin administration requires hospital stay whereas Lipoplatin can be done in an outpatient manner. Similarly, the properties of Satraplatin allow for development of an oral drug. These forms of platinum demonstrate that the direct consequence of more selective speciation is lower side effects and cheaper administration of the anticancer agent. Therefore we urge that as the community goes forward in development of new drugs, control of speciation chemistry will be considered as one of the key strategies in the future development of anticancer drugs.