Fluorescence-based detection of<i>lacZ</i>reporter gene expression in intact and viable bacteria including<i>Mycobacterium</i>species

Rowland, Belinda; Purkayastha, Anjan; Monserrat, Catherine; Casart, Yveth; Takiff, Howard; McDonough, Kathleen A.

doi:10.1111/j.1574-6968.1999.tb08744.x

Cited by 34 publications

(10 citation statements)

References 21 publications

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“…FACS-gal analysis was performed using the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside (FDG) [15]. The FluoReporter® lacZ Flow Cytometry Kit (Invitrogen F-1930) was used according to manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Zhang

Adileh

Martin

et al. 2017

Cellular Signalling

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Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5− basal cells and c-Kit+ and c-Kit− luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5− cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a “mini-breast” organoid.

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Section: Methodsmentioning

confidence: 99%

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Zhang

Adileh

Martin

et al. 2017

Cellular Signalling

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“…Promoter activity of dnaK was measured in Msm::pSD5B- dnaK by fluorescent based detection of lacZ expression, as described earlier [51]. Briefly, Msm::pSD5B- dnaK cultures were grown in 7H9 broth medium containing kanamycin (25 mg/L) to an OD 600 of 1.0, washed twice with 7H9 medium and resuspended in 7H9 medium.…”

Section: Methodsmentioning

confidence: 99%

Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP

2014

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Mycobacterium tuberculosis (Mtb) secretes excess of a second messenger molecule, 3',5'-cyclic AMP (cAMP), which plays a critical role in the survival of Mtb in host macrophages. Although Mtb produces cAMP in abundance, its exact role in the physiology of mycobacteria is elusive. In this study we have analyzed the expression of 16 adenylate cyclases (ACs) and kinetics of intracellular cAMP levels in Mtb during in vitro growth under the regular culture conditions, and after exposure to different stress agents. We observed a distinct expression pattern of these ACs which is correlated with intracellular cAMP levels. Interestingly cAMP levels are significantly elevated in Mtb following heat stress, whereas other stress conditions such as oxidative, nitrosative or low pH do not affect intracellular cAMP pool in vitro. A significant increase in expression by >2-fold of five ACs namely Rv1647, Rv2212, Rv1625c, Rv2488c and Rv0386 after heat stress further suggested that cAMP plays an important role in controlling Mtb response to heat stress. In the light of these observations, effect of exogenous cAMP on global gene expression profile was examined by using microarrays. The microarray gene expression analysis demonstrated that cAMP regulates expression of a subset of heat stress-induced genes comprising of dnaK, grpE, dnaJ, and Rv2025c. Further we performed electrophoretic mobility shift assay by using cAMP-receptor protein of Mtb (CRPM), which demonstrated that CRPM specifically recognizes a sequence −301 AGCGACCGTCAGCACG −286 in 5'-untranslated region of dnaK.

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“…The screen employed a β-galactosidase reporter gene in wild-type M. smegmatis, using the β-galactosidase substrate 5-acetylamino fluorescein diβ- d -galactopyranoside (C2FDG), which is active in live cells and produces a fluorescence signal upon cleavage by the β-galactosidase enzyme. 14 This screening approach allowed sensitive detection of growth arrest in high throughput without cell lysis.…”

Section: Results and Discussionmentioning

confidence: 99%

Novel Imidazoline Antimicrobial Scaffold That Inhibits DNA Replication with Activity against Mycobacteria and Drug Resistant Gram-Positive Cocci

Harris

Fay²,

Yan³

et al. 2014

ACS Chem. Biol.

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Bacterial antimicrobial resistance is an escalating public health threat, yet the current antimicrobial pipeline remains alarmingly depleted, making the development of new antimicrobials an urgent need. Here, we identify a novel, potent, imidazoline antimicrobial compound, SKI-356313, with bactericidal activity against Mycobacterium tuberculosis and Gram-positive cocci, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). SKI-356313 is active in murine models of Streptococcus pneumoniae and MRSA infection and is potently bactericidal for both replicating and nonreplicating M. tuberculosis. Using a combination of genetics, whole genome sequencing, and a novel target ID approach using real time imaging of core macromolecular biosynthesis, we show that SKI-356313 inhibits DNA replication and displaces the replisome from the bacterial nucleoid. These results identify a new antimicrobial scaffold with a novel mechanism of action and potential therapeutic utility against nonreplicating M. tuberculosis and antibiotic resistant Gram-positive cocci.

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Fluorescence-based detection oflacZreporter gene expression in intact and viable bacteria includingMycobacteriumspecies

Cited by 34 publications

References 21 publications

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP

Novel Imidazoline Antimicrobial Scaffold That Inhibits DNA Replication with Activity against Mycobacteria and Drug Resistant Gram-Positive Cocci

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