Structured DVD Education: Impact on Teaching Satisfaction and Anxiety Among Patients With Leukemia and Lymphoma and Their Family Members.

BACKGROUND: Many factors can interfere with a patient's ability to cope with a new cancer diagnosis. The method of education delivery may improve satisfaction with teaching and reduce anxiety. Structured DVD education combined with other teaching methods has shown positive results. However, few such studies have included family members. OBJECTIVES: The purpose of this study was to evaluate the impact of structured DVD education versus standard one-on-one education on satisfaction with teaching and on anxiety among patients newly diagnosed with leukemia and lymphoma and their families. METHODS: A post-test randomized controlled trial study design evaluated the effects of structured DVD education compared to standard one-on-one education. FINDINGS: Family members in the intervention group had higher satisfaction with teaching than those in the control group; this difference was found to be statistically significant.

Characterization of the cytolethal distending toxin (typhoid toxin) in non-typhoidal Salmonella serovars.

BACKGROUND: For many putative Salmonella enterica subsp. enterica virulence genes, functional characterization across serovars has been limited. Cytolethal distending toxin B (CdtB) is an incompletely characterized virulence factor that is found not only in Salmonella enterica subsp. enterica serovar Typhi (Salmonella Typhi) and dozens of Gram negative bacterial pathogens, but also in non-typhoidal Salmonella (NTS) serovars. METHODS: A comparative genomics approach was performed to characterize sequence conservation of the typhoid toxin (TT), encoded in the CdtB-islet, between Salmonella Typhi and NTS serovars. The cytotoxic activity of representative Salmonella enterica subsp. enterica serovars Javiana, Montevideo and Schwarzengrund strains and their respective isogenic cdtB mutants was determined in human intestinal epithelial Henle-407 cells by assessment of cell cycle progression of infected cells using fluorescence-activated cell sorting (FACS). Two-way analysis of variance (ANOVA) was used to determine whether cdtB deletion had a significant (p < 0.05) effect on the percentage of Henle-407 cells at each stage of the cell cycle. RESULTS: Here we show that a CdtB-islet encoding the cytolethal distending toxin B (CdtB), pertussis-like toxin A (PltA), and pertussis-like toxin B (PltB) is present in a dozen NTS serovars and that these proteins have a high level of sequence conservation and each form monophyletic clades with corresponding Salmonella Typhi genes. Human epithelial Henle-407 cells infected with three representative CdtB-encoding NTS serovars displayed G2/M phase cell cycle arrest that was absent in cells infected with corresponding isogenic cdtB null mutants (p < 0.0001 for the factor ∆cdtB deletion). CONCLUSION: Our results show that CdtB encoded by NTS serovars has a genomic organization, amino acid sequence conservation and biological activity similar to the TT, and thus, may contribute to disease pathogenesis.

Listeria monocytogenes shows temperature-dependent and -independent responses to salt stress, including responses that induce cross-protection against other stresses.

The food-borne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. During transmission, L. monocytogenes is likely to experience osmotic stress at different temperatures and may adapt to osmotic stress in a temperature-dependent manner. To understand the impact of temperature on the responses this pathogen uses to adapt to osmotic stress, we assessed genome-wide changes in the L. monocytogenes H7858 transcriptome during short-term and long-term adaptation to salt stress at 7°C and 37°C. At both temperatures, the short-term response to salt stress included increased transcript levels of sigB and SigB-regulated genes, as well as mrpABCDEFG, encoding a sodium/proton antiporter. This antiporter was found to play a role in adaptation to salt stress at both temperatures; ΔmrpABCDEFG had a significantly longer lag phase than the parent strain in BHI plus 6% NaCl at 7°C and 37°C. The short-term adaptation to salt stress at 7°C included increased transcript levels of two genes encoding carboxypeptidases that modify peptidoglycan. These carboxypeptidases play a role in the short-term adaptation to salt stress only at 7°C, where the deletion mutants had significantly different lag phases than the parent strain. Changes in the transcriptome at both temperatures suggested that exposure to salt stress could provide cross-protection to other stresses, including peroxide stress. Short-term exposure to salt stress significantly increased H(2)O(2) resistance at both temperatures. These results provide information for the development of knowledge-based intervention methods against this pathogen, as well as provide insight into potential mechanisms of cross-protection.

FSL J1-208, a virulent uncommon phylogenetic lineage IV Listeria monocytogenes strain with a small chromosome size and a putative virulence plasmid carrying internalin-like genes.

The bacterial genus Listeria contains both saprotrophic and facultative pathogenic species. A small genome size has been suggested to be associated with the loss of pathogenic potential of L. welshimeri and L. seeligeri. In this paper we present data on the genome of L. monocytogenes strain FSL J1-208, a representative of phylogenetic lineage IV. Although this strain was isolated from a clinical case in a caprine host and has no decreased invasiveness in human intestinal epithelial cells, our analyses show that this strain has one of the smallest Listeria chromosomes reported to date (2.78 Mb). The chromosome contains 2,772 protein-coding genes, including well-characterized virulence-associated genes, such as inlA, inlB, and inlC and the full prfA gene cluster. The small genome size is mainly caused by the absence of prophages in the genome of L. monocytogenes FSL J1-208, and further analyses showed that the total size of prophage-related regions is highly correlated to chromosome size in the genus Listeria. L. monocytogenes FSL J1-208 carries a unique type of plasmid of approximately 80 kbp that does not carry genes annotated as being involved in resistance to antibiotics or heavy metals. The accessory genes in this plasmid belong to the internalin family, a family of virulence-associated proteins, and therefore this is the first report of a potential virulence plasmid in the genus Listeria.

Growth temperature-dependent contributions of response regulators, σB, PrfA, and motility factors to Listeria monocytogenes invasion of Caco-2 cells.

Foodborne pathogens encounter rapidly changing environmental conditions during transmission, including exposure to temperatures below 37°C. The goal of this study was to develop a better understanding of the effects of growth temperatures and temperature shifts on regulation of invasion phenotypes and invasion-associated genes in Listeria monocytogenes. We specifically characterized the effects of L. monocytogenes growth at different temperatures (30°C vs. 37°C) on (i) the contributions to Caco-2 invasion of different regulators (including σ(B), PrfA, and 14 response regulators [RRs]) and invasion proteins (i.e., InlA and FlaA), and on (ii) gadA, plcA, inlA, and flaA transcript levels and their regulation. Overall, Caco-2 invasion efficiency was higher for L. monocytogenes grown at 30°C than for bacteria grown at 37°C (p = 0.0051 for the effect of temperature on invasion efficiency; analysis of variance); the increased invasion efficiency of the parent strain 10403S (serotype 1/2a) observed after growth at 30°C persisted for 2.5 h exposure to 37°C. For L. monocytogenes grown at 30°C, the motility RRs DegU and CheY and σ(B), but not PrfA, significantly contributed to Caco-2 invasion efficiency. For L. monocytogenes grown at 37°C, none of the 14 RRs tested significantly contributed to Caco-2 invasion, whereas σ(B) and PrfA contributed synergistically to invasion efficiency. At both growth temperatures there was significant synergism between the contributions to invasion of FlaA and InlA; this synergism was more pronounced after growth at 30°C than at 37°C. Our data show that growth temperature affects invasion efficiency and regulation of virulence-associated genes in L. monocytogenes. These data support increasing evidence that a number of environmental conditions can modulate virulence-associated phenotypes of foodborne bacterial pathogens, including L. monocytogenes.

Homopolymeric tracts represent a general regulatory mechanism in prokaryotes.

BACKGROUND: While, traditionally, regulation of gene expression can be grouped into transcriptional, translational, and post-translational mechanisms, some mechanisms of rapid genetic variation can also contribute to regulation of gene expression, e.g., phase variation. RESULTS: We show here that prokaryotes evolved to include homopolymeric tracts (HTs) within coding genes as a system that allows for efficient gene inactivation. Analyses of 81 bacterial and 18 archaeal genomes showed that poly(A) and poly(T) HTs are overrepresented in these genomes and preferentially located at the 5' end of coding genes. Location of HTs at the 5' end is not driven by a preferential placement of aminoacids encoded by the AAA and TTT codons at the N-terminal of proteins. The inlA gene of the pathogen L. monocytogenes was used as a model to further study the role of HTs in reversible gene inactivation. In a number of L. monocytogenes strains, inlA harbors a 5' poly(A) HT, which regularly shows frameshift mutation leading to expression of a truncated 8 aa InlA protein. Translational fusions of the inlA 5' end allowed us to estimate that the frequency of variation in this HT is about 1,000 fold higher than the estimated average point mutation frequency. CONCLUSIONS: As frameshift mutations in HTs can occur at high frequencies and enable efficient gene inactivation, hypermutable HTs appear to represent a universal system for regulation of gene expression in prokaryotes. Combined with other studies indicating that HTs also enable rapid diversification of both coding and regulatory genetic sequences in eukaryotes, our data suggest that hypermutable HTs represent a general and rapid evolutionary mechanism facilitating adaptation and gene regulation across diverse organisms.

Listeria monocytogenes sigmaB modulates PrfA-mediated virulence factor expression.

Listeria monocytogenes sigma(B) and positive regulatory factor A (PrfA) are pleiotropic transcriptional regulators that coregulate a subset of virulence genes. A positive regulatory role for sigma(B) in prfA transcription has been well established; therefore, observations of increased virulence gene expression and hemolytic activity in a DeltasigB strain initially appeared paradoxical. To test the hypothesis that L. monocytogenes sigma(B) contributes to a regulatory network critical for appropriate repression as well as induction of virulence gene expression, genome-wide transcript profiling and follow-up quantitative reverse transcriptase PCR (qRT-PCR), reporter fusion, and phenotypic experiments were conducted using L. monocytogenes prfA*, prfA* DeltasigB, DeltaprfA, and DeltaprfA DeltasigB strains. Genome-wide transcript profiling and qRT-PCR showed that in the presence of active PrfA (PrfA*), sigma(B) is responsible for reduced expression of the PrfA regulon. sigma(B)-dependent modulation of PrfA regulon expression reduced the cytotoxic effects of a PrfA* strain in HepG2 cells, highlighting the functional importance of regulatory interactions between PrfA and sigma(B). The emerging model of the role of sigma(B) in regulating overall PrfA activity includes a switch from transcriptional activation at the P2(prfA) promoter (e.g., in extracellular bacteria when PrfA activity is low) to posttranscriptional downregulation of PrfA regulon expression (e.g., in intracellular bacteria when PrfA activity is high).

Contributions of two-component regulatory systems, alternative sigma factors, and negative regulators to Listeria monocytogenes cold adaptation and cold growth.

The ability of Listeria monocytogenes to grow at refrigeration temperatures is critical for transmission of this foodborne pathogen. We evaluated the contributions of different transcriptional regulators and two-component regulatory systems to L. monocytogenes cold adaptation and cold growth. L. monocytogenes parent strain 10403S and selected isogenic null mutants in genes encoding four alternative sigma factors (sigB, sigH, sigC, and sigL), two regulators of sigmaB (rsbT and rsbV), two negative regulators (ctsR and hrcA), and 15 two-component response regulators were grown in brain heart infusion broth at 4 degrees C with (i) a high-concentration starting inoculum (10(8) CFU/ml), (ii) a low-concentration starting inoculum (102 CFU/ml), and (iii) a high-concentration starting inoculum of cold-adapted cells. With a starting inoculum of 10(8) CFU/ml, null mutants in genes encoding selected alternative sigma factors (DeltasigH, DeltasigC, and DeltasigL), a negative regulator (DeltactsR), regulators of sigmaB (DeltarsbT and DeltarsbV), and selected two-component response regulators (DeltalisR, Deltalmo1172, and Deltalmo1060) had significantly reduced growth (P < 0.05) compared with the parent strain after 12 days at 4 degrees C. The growth defect for DeltasigL was limited and was not confirmed by optical density (OD600) measurement data. With a starting inoculum of 102 CFU/ml and after monitoring growth at 4 degrees C over 84 days, only the DeltactsR strain had a consistent but limited growth defect; the other mutant strains had either no growth defects or limited growth defects apparent at only one or two of the nine sampling points evaluated during the 84-day growth period (DeltasigB, DeltasigC, and Deltalmo1172). With a 10(8) CFU/ml starting inoculum of cold-adapted cells, none of the mutant strains that had a growth defect when inoculation was performed with cells pregrown at 37 degrees C had reduced growth as compared with the parent strain after 12 days at 4 degrees C, suggesting a specific defect in the ability of these mutant strains to adapt to 4 degrees C after growth at 37 degrees C. Our data indicate (i) selected sigma factors and two-component regulators may contribute to cold adaptation even though two-component regulatory systems, alternative sigma factors, and the negative regulators CtsR and HrcA appear to have limited contributions to L. monocytogenes growth at 4 degrees C in rich media, and (ii) inoculum concentration and pregrowth conditions affect the L. monocytogenes cold-growth phenotype.

The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner.

Listeria monocytogenes prfA, encoding positive regulatory factor A, is transcribed from three promoters (prfAP1, prfAP2, and PplcA). The prfAP2 promoter was previously proposed to be sigma B (sigma(B))-dependent. This hypothesis was tested by creating prfA promoter-gus transcriptional fusions in both L. monocytogenes wild-type (wt) and DeltasigB backgrounds and then measuring (i) beta-glucuronidase (GUS) activities; (ii) gus mRNA transcript levels; and (iii) the presence or absence of GUS in cells by immunofluorescence staining. prfAP2-directed expression increased as the wt L. monocytogenes strain entered stationary phase, whereas prfAP2-directed expression was greatly reduced in the DeltasigB strain, confirming both growth phase- and sigma(B)-dependent transcription of prfAP2. We conclude that prfAP2 is directly regulated by sigma(B).

Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes.

Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp(1) and prfAp(2)) or from the upstream plcA promoter. We demonstrate that prfAp(2) is a functional sigma(B)-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor sigma(B) contributes to virulence in L. monocytogenes.