Signaling by Eph receptors and cellsurface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact-dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5-induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/ nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors. IntroductionEph receptors and their membrane-bound ephrin ligands are developmental cell guidance cues that direct cell migration and orchestrate patterning processes by modulating adhesive or repulsive cell properties. 1 While their expression and function in normal adult tissues is limited, Eph and ephrin overexpression in human cancers often correlates with aggressive, invasive, and metastatic phenotypes. 2 Upon Eph/ephrin contact, heterotetrameric complexes between neighboring cells 3 assemble into large signaling clusters that are rapidly internalized. 4,5 Tyrosine autophosphorylation within the juxtamembrane region and kinase activation loop is required to fully activate the Eph kinase 6,7 and triggers signal pathways regulating cytoskeletal plasticity. 1,8 As a default, Eph activation leads to cytoskeletal contraction, loss of focal adhesions, cell rounding, and cell-cell repulsion or detachment. 8,9 Repulsion also requires the active Eph tyrosine kinase to mediate ephrin shedding by the ADAM10 metalloprotease, releasing the molecular tether connecting the Eph-and ephrin-expressing cells. 10,11 An alternative mechanism involving kinase-dependent "transendocytosis" of the whole EphB and ephrin-B signaling clusters into either cell has been proposed. 12 However, ephrin ligation of Ephs also results in cell-cell and cell-substrate adhesion 13,14 : interaction between kinasedeficient EphA7 splice variants and ephrin-A5 on opposing cell layers facilitates neural tube closure during mouse development, 15 and retinal axons respond to low ephrin-A2 density with adhesion. 14 Analysis of these disparate outcomes of Eph/ephrin signaling suggests...

Eph receptors and their membrane-bound ephrin ligands are developmental cell guidance cues that direct cell migration and orchestrate patterning processes by modulating adhesive or repulsive cell properties. During the past two decades, an exponentially growing interest in their function has resulted in a considerably advanced understanding of the cellular and molecular principles of Eph function in normal and oncogenic development. Ephs not only accurately guide the path of migrating cells, but also facilitate contact and communication between neighbouring cell populations, in particular at epithelial/mesenchymal boundaries. Precise cell positioning not only relies on accurately-graded expression of individual Eph/ephrin pairs, but on the sum of interactions within particular expression domains and their modulation through crosstalk with a range of other signalling systems. There is little doubt that Eph and ephrins provide exciting new targets for anti-cancer therapies, but in appreciation of the complexity of their signals and biological functions it is perhaps not surprising that the development of Eph-specific therapeutics is only emerging.

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP-binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence. IMPORTANCE The discovery of antibiotics such as penicillin and streptomycin marked a historic milestone in the 1940s and heralded a new era of antimicrobial therapy as the modern standard for medical treatment. Yet, even in those early days of discovery, it was noted that a small subset of cells (∼1 in 105) survived antibiotic treatment and continued to persist, leading to recurrence of chronic infection. These persisters are phenotypic variants that have modified their physiology to survive environmental stress. In this study, we have performed three transcriptomic screens to identify persistence genes that are common between three different stressor conditions. In particular, we identified genes that function in the synthesis of secondary metabolites, small molecules, and complex lipids, which are likely required to maintain the persistence state. Targeting universal persistence genes can lead to the development of clinically relevant antipersistence therapeutics for infectious disease management.

The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1β (interleukin 1β) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1β and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1β and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.

Volatile organic compound (VOC)-based diagnostics have great potential to be the next generation of screening tools for pathogen identification and infectious disease management. VOCs are low molecular weight metabolic compounds that have high vapor pressures and low boiling points, both of which facilitate evaporation at ambient temperatures. There is increasing evidence that particular VOCs, or profiles of VOCs, are unique to various disease states. Different pathogenic species have been found to produce characteristic profiles of VOCs by virtue of their distinct metabolisms. The detection of these metabolite profiles from patient samples could provide an effective means of rapid, non-invasive pathogen identification, thus enabling early diagnosis and treatment. In this review, we will discuss the potential of VOC profiles to be utilized as biomarkers of pathogenic infection, with a focus on bacterial pathogens. Herein we describe the common methods for clinical VOC sample collection, provide an overview of the various instruments and techniques used for VOC detection and analysis, and summarize the key findings of recent studies that have investigated VOC biomarkers in various infectious diseases. We will also discuss the challenges associated with translating VOC analysis into a clinical diagnostic tool.