Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor

The Sir Jimmy Savile Scandal: Child Sexual Abuse and Institutional Denial at the BBC

This study advances research on scandal through an empirical examination of one of the most extraordinary UK institutional child sexual abuse (CSA) scandals in the post-war period. Sir Jimmy Savile (1926–2011) was a BBC celebrity, showbiz friend of the establishment and philanthropist. In October 2012, one year after his death, an ITV documentary alleged that Savile was also a prolific sexual predator who for decades had exploited his BBC status to abuse teenage girls. As we demonstrate, this incendiary documentary triggered a news media feeding frenzy that in less than one week destroyed Savile’s reputation and thrust the BBC – the institution that made him a star – into a multi-faceted, globally reported CSA scandal. This study has four purposes. First, we propose a model of institutional CSA scandals that can account for critical transitions between key phases in the scandal process. Second, we apply this model to analyse the transition between the ‘latent’ and ‘activated’ phases of the Savile scandal. This transition corresponded with a dramatic transformation in the inferential structuring of Savile from ‘national treasure’, who had devoted decades to working with children, to ‘prolific sexual predator’, who spent decades abusing them. Third, we demonstrate how the BBC’s denial of responsibility for Savile’s sexual offending and its subsequent institutional cover-up triggered a ‘trial by media’ which in turn initiated the next phase in the scandal’s development – ‘amplification’. Finally, we consider the significance of our analysis of the Sir Jimmy Savile scandal for understanding the activation and development of scandals more generally

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

The Environmental Dependence of Inbreeding Depression in a Wild Bird Population

BACKGROUND: Inbreeding depression occurs when the offspring produced as a result of matings between relatives show reduced fitness, and is generally understood as a consequence of the elevated expression of deleterious recessive alleles. How inbreeding depression varies across environments is of importance for the evolution of inbreeding avoidance behaviour, and for understanding extinction risks in small populations. However, inbreeding-by-environment (IxE) interactions have rarely been investigated in wild populations. METHODOLOGY/PRINCIPAL FINDINGS: We analysed 41 years of breeding events from a wild great tit (Parus major) population and used 11 measures of the environment to categorise environments as relatively good or poor, testing whether these measures influenced inbreeding depression. Although inbreeding always, and environmental quality often, significantly affected reproductive success, there was little evidence for statistically significant I x E interactions at the level of individual analyses. However, point estimates of the effect of the environment on inbreeding depression were sometimes considerable, and we show that variation in the magnitude of the I x E interaction across environments is consistent with the expectation that this interaction is more marked across environmental axes with a closer link to overall fitness, with the environmental dependence of inbreeding depression being elevated under such conditions. Hence, our analyses provide evidence for an environmental dependence of the inbreeding x environment interaction: effectively an I x E x E. CONCLUSIONS/SIGNIFICANCE: Overall, our analyses suggest that I x E interactions may be substantial in wild populations, when measured across relevant environmental contrasts, although their detection for single traits may require very large samples, or high rates of inbreeding

New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

A Customer Perspective on Product Eliminations: How the Removal of Products Affects Customers and Business Relationships

Regardless of the apparent need for product eliminations, many managers hesitate to act as they fear deleterious effects on customer satisfaction and loyalty. Other managers do carry out product eliminations, but often fail to consider the consequences for customers and business relationships. Given the relevance and problems of product eliminations, research on this topic in general and on the consequences for customers and business relationships in particular is surprisingly scarce. Therefore, this empirical study explores how and to what extent the elimination of a product negatively affects customers and business relationships. Results indicate that eliminating a product may result in severe economic and psychological costs to customers, thereby seriously decreasing customer satisfaction and loyalty. This paper also shows that these costs are not exogenous in nature. Instead, depending on the characteristics of the eliminated product these costs are found to be more or less strongly driven by a company’s behavior when implementing the elimination at the customer interface

Inhibition of Toxic Shock by Human Monoclonal Antibodies against Staphylococcal Enterotoxin B

BACKGROUND: Staphylococcus aureus is implicated in many opportunistic bacterial infections around the world. Rising antibiotic resistance and few alternative methods of treatment are just two looming problems associated with clinical management of S. aureus. Among numerous virulence factors produced by S. aureus, staphylococcal enterotoxin (SE) B is a secreted protein that binds T-cell receptor and major histocompatibility complex class II, potentially causing toxic shock mediated by pathological activation of T cells. Recombinant monoclonal antibodies that target SEB and block receptor interactions can be of therapeutic value. METHODOLOGY/PRINCIPAL FINDINGS: The inhibitory and biophysical properties of ten human monoclonal antibodies, isolated from a recombinant library by panning against SEB vaccine (STEBVax), were examined as bivalent Fabs and native full-length IgG (Mab). The best performing Fabs had binding affinities equal to polyclonal IgG, low nanomolar IC(50)s against SEB in cell culture assays, and protected mice from SEB-induced toxic shock. The orthologous staphylococcal proteins, SEC1 and SEC2, as well as streptococcal pyrogenic exotoxin C were recognized by several Fabs. Four Fabs against SEB, with the lowest IC(50)s, were converted into native full-length Mabs. Although SEB-binding kinetics were identical between each Fab and respective Mab, a 250-fold greater inhibition of SEB-induced T-cell activation was observed with two Mabs. CONCLUSIONS/SIGNIFICANCE: Results suggest that these human monoclonal antibodies possess high affinity, target specificity, and toxin neutralization qualities essential for any therapeutic agent