Intramolecular Epistasis and the Evolution of a New Enzymatic Function

Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine

N. elongata Produces Type IV Pili That Mediate Interspecies Gene Transfer with N. gonorrhoeae

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species

Recombination-induced suppression of cell division following P1-mediated generalized transduction in Klebsiella aerogenes

Klebsiella aerogenes recombinants resulting from bacteriophage P1-mediated generalized transduction failed to increase in number for approximately six generations after transduction. Nevertheless these recombinants continued to grow and became sensitive to penicillin after a transient resistance, suggesting that the cells were growing as long, non-dividing filaments. When filamentous cells were isolated from transduced cultures by gradient centrifugation, recombinants were 1000-fold more frequent among the filaments than among the normal-sized cells. The suppression of cell-division lasted for six generations whether markers near the origin ( gln, ilv ) or terminus ( his, trp ) of chromosome replication were used, despite a 50-fold difference in transduction frequencies for these markers. The suppression of cell division was a host response to recombination rather than to P1 invasion since cells lysogenized by P1 in these same experiments showed only a short (two generation) suppression of cell division. We speculate that the suppression of cell-division is an SOS response triggered by the degraded DNA not incorporated in the final recombinant. We demonstrate that both the filamentation and the transient penicillin resistance of recombinant cells can be exploited to enrich greatly for recombinants, raising transduction frequencies to as high as 10 -3 .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47551/1/438_2004_Article_BF00337815.pd

Making effective use of healthcare data using data-to-text technology

Healthcare organizations are in a continuous effort to improve health outcomes, reduce costs and enhance patient experience of care. Data is essential to measure and help achieving these improvements in healthcare delivery. Consequently, a data influx from various clinical, financial and operational sources is now overtaking healthcare organizations and their patients. The effective use of this data, however, is a major challenge. Clearly, text is an important medium to make data accessible. Financial reports are produced to assess healthcare organizations on some key performance indicators to steer their healthcare delivery. Similarly, at a clinical level, data on patient status is conveyed by means of textual descriptions to facilitate patient review, shift handover and care transitions. Likewise, patients are informed about data on their health status and treatments via text, in the form of reports or via ehealth platforms by their doctors. Unfortunately, such text is the outcome of a highly labour-intensive process if it is done by healthcare professionals. It is also prone to incompleteness, subjectivity and hard to scale up to different domains, wider audiences and varying communication purposes. Data-to-text is a recent breakthrough technology in artificial intelligence which automatically generates natural language in the form of text or speech from data. This chapter provides a survey of data-to-text technology, with a focus on how it can be deployed in a healthcare setting. It will (1) give an up-to-date synthesis of data-to-text approaches, (2) give a categorized overview of use cases in healthcare, (3) seek to make a strong case for evaluating and implementing data-to-text in a healthcare setting, and (4) highlight recent research challenges.Comment: 27 pages, 2 figures, book chapte

Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

Biofilm Development on Caenorhabditis elegans by Yersinia Is Facilitated by Quorum Sensing-Dependent Repression of Type III Secretion

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS

Constitutive Activation of PrfA Tilts the Balance of Listeria monocytogenes Fitness Towards Life within the Host versus Environmental Survival

PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells

Competitive Tendering In The Netherlands: Central Planning Or Functional Specifications?

Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior