Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities.

Mechanistic elements and critical factors of cellular reprogramming revealed by stepwise global gene expression analyses.

A better understanding of the cellular and molecular mechanisms involved in the reprogramming of somatic cells is essential for further improvement of induced pluripotent stem (iPS) cell technology. In this study, we enriched for cells actively undergoing reprogramming at different time points by sorting the cells stained with a stem cell-selective fluorescent chemical probe CDy1 for their global gene expression analysis. Day-to-day comparison of differentially expressed genes showed highly dynamic and transient gene expressions during reprogramming, which were largely distinct from those of fully-reprogrammed cells. An unbiased analysis of functional regulation indicated robust modulation of concurrent programs at critical junctures. Globally, transcriptional programs involved in cell proliferation, morphology and signal transduction were instantly triggered as early as 3 days-post-infection to prepare the cell for reprogramming but became somewhat muted in the final iPS cells. On the other hand, the highly coordinated metabolic reprogramming process was more gradually activated. Subsequent network analysis of differentially expressed genes indicated PDGF-BB as a core player in reprogramming which was verified by our gain- and loss-of-function experiments. As such, our study has revealed previously-unknown insights into the mechanisms of cellular reprogramming.

Development of a fluorescent chalcone library and its application in the discovery of a mouse embryonic stem cell probe.

We report the first fluorescent diamino-chalcone library and its application in the discovery of a mouse embryonic stem cell (mESC) probe. CDg4, a novel green fluorescent mESC probe was discovered through a high-content image based screening of 160 members of the chalcone library. Interestingly, the molecular binding target of CDg4 was identified as the glycogen of the stem cell colony surface, rather than a conventional protein target from an intracellular source.

A fluorescent screening platform for the rapid evaluation of chemicals in cellular reprogramming.

Current strategies to monitor reprogramming into induced pluripotent stem cells (iPSCs) are limited in that they rely on the recognition of advanced stage biomarkers or they involve the transduction of genetically-modified cells. These limitations are particularly problematic in high-throughput screenings where cell availability, low cost and a rapid experimental protocol are critical issues. Herein we report the application of a pluripotent stem cell fluorescent probe (i.e. CDy1) as a reporter for the rapid screening of chemicals in reprogramming iPSCs. CDy1 stains early-stage iPSCs at 7dpi as well as matured iPSCs; hence it can partially overcome the slow kinetics of the reprogramming process. As a proof of concept, we employed a CDy1-based screening in 384 well-plates to examine the effect of newly synthesized hydroxamic acid derivatives in reprogramming mouse fibroblasts transduced with Oct4, Sox2 and Klf-4 without c-Myc. One compound (1-26) was identified as a reprogramming enhancer by 2.5-fold and we confirmed that 1-26 behaves as a histone deacetylase (HDAC) inhibitor. The successful identification of novel small molecules enhancing the generation of iPSCs by means of a rapid and simple protocol demonstrates the suitability of this CDy1-based screening platform for the large scale and high-throughput evaluation of iPSC modulators.

Solid phase combinatorial synthesis of a xanthone library using click chemistry and its application to an embryonic stem cell probe.

We report the first solid phase synthesis of a xanthone library CX and its application to embryonic stem cell probe development. The CX library was further derivatised with an activated ester resin to provide an acetylated CX (CXAC) library. Screening of these libraries led to the discovery of a novel fluorescent mESC probe, CDb8.