Phenotypic Pattern-Based Assay for Dynamically Monitoring Host Cellular Responses to Salmonella Infections

The interaction between mammalian host cells and bacteria is a dynamic process, and the underlying pathologic mechanisms are poorly characterized. Limited information describing the host-bacterial interaction is based mainly on studies using label-based endpoint assays that detect changes in cell behavior at a given time point, yielding incomplete information. In this paper, a novel, label-free, real-time cell-detection system based on electronic impedance sensor technology was adapted to dynamically monitor the entire process of intestinal epithelial cells response to Salmonella infection. Changes in cell morphology and attachment were quantitatively and continuously recorded following infection. The resulting impedance-based time-dependent cell response profiles (TCRPs) were compared to standard assays and showed good correlation and sensitivity. Biochemical assays further suggested that TCRPs were correlated with cytoskeleton-associated morphological dynamics, which can be largely attenuated by inhibitions of actin and microtubule polymerization. Collectively, our data indicate that cell-electrode impedance measurements not only provide a novel, real-time, label-free method for investigating bacterial infection but also help advance our understanding of host responses in a more physiological and continuous manner that is beyond the scope of current endpoint assays

A Conserved Mechanism for Control of Human and Mouse Embryonic Stem Cell Pluripotency and Differentiation by Shp2 Tyrosine Phosphatase

Recent studies have suggested distinctive biological properties and signaling mechanisms between human and mouse embryonic stem cells (hESCs and mESCs). Herein we report that Shp2, a protein tyrosine phosphatase with two SH2 domains, has a conserved role in orchestration of intracellular signaling cascades resulting in initiation of differentiation in both hESCs and mESCs. Homozygous deletion of Shp2 in mESCs inhibited differentiation into all three germ layers, and siRNA-mediated knockdown of Shp2 expression in hESCs led to a similar phenotype of impaired differentiation. A small molecule inhibitor of Shp2 enzyme suppressed both hESC and mESC differentiation capacity. Shp2 modulates Erk, Stat3 and Smad pathways in ES cells and, in particular, Shp2 regulates BMP4-Smad pathway bi-directionally in mESCs and hESCs. These results reveal a common signaling mechanism shared by human and mouse ESCs via Shp2 modulation of overlapping and divergent pathways

Deletion of Shp2 in the Brain Leads to Defective Proliferation and Differentiation in Neural Stem Cells and Early Postnatal Lethality▿ †

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation

Riok3 inhibits the antiviral immune response by facilitating TRIM40-mediated RIG-I and MDA5 degradation.

The type I interferon (IFN) pathway is a key component of innate immune response upon invasion of foreign pathogens. It is also under precise control to prevent excessive upregulation and undesired inflammation cascade. In the present study, we report that Riok3, an atypical kinase, negatively regulates retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) sensing-induced type I IFN signaling. Riok3 deficiency selectively inhibits RNA viral replication in vitro, resulting from an upregulated type I IFN pathway. Mice with myeloid-specific Riok3 knockout also show a more robust induction of type I IFN upon RNA virus infection and are more resistant to RNA virus-induced pathogenesis. Mechanistically, Riok3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to the degradation of RIG-I and melanoma differentiation-associated gene-5 (MDA5) via K48- and K27-linked ubiquitination. Collectively, our data reveal the mechanism that Riok3 employs to be a negative regulator of antiviral innate immunity