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Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi, is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo, that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T. cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy., Competing Interests: The authors have declared that no competing interests exist.

RNA-modifying enzymes are difficult to assay due to the absence of fluorometric substrates. Here we show that the Broccoli, a previously reported fluorescent RNA-dye complex, can be modified to contain N(6)-methyladenosine, a prevalent mRNA base modification. Methylated Broccoli is nonfluorescent but, upon demethylation by the RNA demethylases fat mass and obesity-associated protein (FTO) or ALKBH5, it binds and activates the fluorescence of its cognate fluorophore. We describe a high-throughput screen (HTS) for FTO inhibitors using the fluorogenic methylated Broccoli substrate HTS assay, which performs robustly with a Z' factor >0.8 in the LOPAC1280 library. This allowed the identification of novel high-affinity FTO inhibitors. Several of these compounds were selective for FTO over the related demethylase, ALKBH5, and increase methylation of endogenous FTO target mRNAs in cells. Lastly, we show that Broccoli can be modified to contain other base modifications, suggesting that this approach could be generally applicable for assaying diverse RNA-modifying enzymes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The ability to screen and identify new ligands for cell surface receptors has been a long-standing goal as it might allow targeting of pharmaceutically relevant receptors, such as integrins or G protein coupled receptors. Here, we present a method to amplify hits from a library of PNA-tagged peptides. To this end, human cells, overexpressing either integrins or the CCR6 receptor, were treated with a 10,000 member PNA-encoded peptide library. Extraction of the PNA tags from the surface of the cells was followed by a PNA-tag to DNA translation and amplification enabling decoding of the tags via microarray hybridization. This approach to ligand discovery facilitates screening for differences in surface-receptor ligands and/or receptor expression between different cell types, and opens up a practical approach to PNA-tag amplification., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The ability to efficiently and economically generate libraries of defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. In this manuscript DNA microarrays were used to allow the linear amplification of immobilized DNA sequences from the array followed by PCR amplification. Arrays of increasing sophistication (1, 10, 3,875, 10,000 defined sequences) were used to validate the process, with sequences verified by selective hybridization to a complementary DNA microarray and DNA sequencing, which demonstrated a PCR error rate of 9.7×10(-3)/site/duplication. This technique offers an economical and efficient way of producing specific DNA libraries of hundreds to thousands of members with the DNA-arrays being used as "factories" allowing specific DNA oligonucleotide pools to be generated. We also found substantial variance observed between the sequence frequencies found via Solexa sequencing and microarray analysis, highlighting the care needed in the interpretation of profiling data.