A Highly Selective Fluorescent Probe for Direct Detection and Isolation of Mouse Embryonic Stem Cells

AbstractStem 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

Characterisation of the putative cysteine protease effectors OspD2 and OspD3 from Shigella species

© 2020 Yogeswari ChandranDiarrheal disease caused by bacterial pathogens continues to be a major public health concern worldwide due to significant increases in mortality annually. Members of the Shigella genus contribute significantly to bacterial diarrheal incidences worldwide. Shigella is a Gram negative facultative anaerobe that belongs to the family Enterobacteriaceae. They are considered highly infectious as only 10-100 organisms are required to cause disease. Like many other Gram-negative gut pathogens, Shigella utilizes a type III secretion system (T3SS) during infection to translocate bacterial effector proteins into host cells which interfere with host signaling pathways to benefit their survival. The exact function of many T3 effector proteins remains unknown. However recently, the T3SS effector EspL from enteropathogenic Escherichia coli (EPEC), was shown to contain a cysteine protease catalytic motif that targets and degrades the host RHIM domain containing proteins, RIPK1, RIPK3, TRIF and DAI, hence blocking inflammation and necroptotic cell death during infection. Homologues of EspL are also found in Shigella, namely: OspD2 and OspD3. Although previously labelled as Shigella toxins, the exact function of these effectors is yet to be elucidated. The primary aim of my study is to characterize the role of OspD2 and OspD3 and to determine their host cell targets. Overexpression experiments with OspD2 or OspD3 and the RHIM family of proteins suggest that OspD3, but not OspD2 targets and cleaves the RHIM family of proteins. We also showed OspD3 blocking both inflammation and necroptotic cell death in NF-kB dependent luciferase assays and cell viability assays respectively. However, this was not seen upon infection of HeLa 299s with wildtype and mutant Shigella strains. This therefore led to further investigations for the identification of other host cell targets of OspD2 and OspD3 via mass spectrometry. Several unique host cell targets for OspD2 and OspD3 were identified from mass spectrometry and an enrichment analysis of the hits, suggest the involvement of these proteins in anti-viral defense, particularly the Type I IFN signaling pathway. Elucidating the roles of these effectors in the interferon signaling pathways will be important in understanding the roles between bacteria pathogens and interferon as unlike viral infections, these are severely understudied.Further analysis via overexpression studies showed that OspD2 and OspD3 may be working cooperatively in cleaving IRF3/IRF7 and IRF9 during Type I IFN signaling and hence blocking the pathway. Furthermore, we also demonstrated by infection of HeLa 299s that OspD2 cleaves IRF3, however cleavage of IRF9 by OspD3 was not seen. In summary, we have performed characterization of two cysteine protease effectors of Shigella and their host cell targets. This work creates the platform for understanding how these effectors function and how this knowledge may be used as valuable tools for subsequent investigation of host cell defense mechanism

HIGHLY SPECIFIC FLUORESCENT PROBE FOR MOUSE PLURIPOTENT STEM CELLS

Master'sMASTER OF SCIENC

Glucagon-secreting alpha cell selective two-photon fluorescent probe TP-α: For live pancreatic islet imaging

10.1021/ja5115776Journal of American Chemical Society137165355-536

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-alpha: 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-alpha (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-alpha from alpha TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-alpha have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-alpha was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-alpha can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.1123sciescopu

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

10.1016/j.scr.2012.06.006Stem Cell Research93185-19

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

10.1016/j.scr.2014.03.002Stem Cell Research123730-74

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.Originally included in thesis in manuscript form. </p