production-of-haploid-zebrafish-embryos-by-in-vitro-fertilization

Production of Haploid Zebrafish Embryos by <em>In Vitro</em> Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

University of Notre Dame

When executives need to persuade an audience, most try to build a case with facts, statistics, and some quotes from authorities. In other words, they resort to "companyspeak," the tools of rhetoric they have been trained to use. In this conversation with HBR, Robert McKee, the world's best-known screenwriting lecturer, argues that executives can engage people in a much deeper--and ultimately more convincing--way if they toss out their Power-Point slides and memos and learn to tell good stories. As human beings, we make sense of our experiences through stories. But becoming a good storyteller is hard. It requires imagination and an understanding of what makes a story worth telling. All great stories deal with the conflict between subjective expectations and an uncooperative objective reality. They show a protagonist wrestling with antagonizing forces, not a rosy picture of results meeting expectations--which no one ends up believing. Consider the CEO of a biotech start-up that has discovered a chemical compound to prevent heart attacks. He could make a pitch to investors by offering up market projections, the business plan, and upbeat, hypothetical scenarios. Or he could captivate them by telling the story of his father, who died of a heart attack, and of the CEO's subsequent struggle against various antagonists--nature, the FDA, potential rivals--to bring to market the effective, low-cost test that might have prevented his father's death. Good storytellers are not necessarily good leaders, but they do share certain traits. Both are self-aware, and both are skeptics who realize that all people--and institutions--wear masks. Compelling stories can be found behind those masks.

Storytelling that moves people. A conversation with screenwriting coach Robert McKee.

Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection.

Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile.

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Robert McKee

Department of Biological Sciences

University of Notre Dame

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

Robert McKee

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

University of Notre Dame

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

Department of Biological Sciences