Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

Levels of pneumococcal conjugate vaccine coverage and indirect protection against invasive pneumococcal disease and pneumonia hospitalisations in Australia: An observational study

BackgroundThere is limited empiric evidence on the coverage of pneumococcal conjugate vaccines (PCVs) required to generate substantial indirect protection. We investigate the association between population PCV coverage and indirect protection against invasive pneumococcal disease (IPD) and pneumonia hospitalisations among undervaccinated Australian children.Methods and findingsBirth and vaccination records, IPD notifications, and hospitalisations were individually linked for children aged <5 years, born between 2001 and 2012 in 2 Australian states (New South Wales and Western Australia; 1.37 million children). Using Poisson regression models, we examined the association between PCV coverage, in small geographical units, and the incidence of (1) 7-valent PCV (PCV7)-type IPD; (2) all-cause pneumonia; and (3) pneumococcal and lobar pneumonia hospitalisation in undervaccinated children. Undervaccinated children received <2 doses of PCV at <12 months of age and no doses at ≥12 months of age. Potential confounding variables were selected for adjustment a priori with the assistance of a directed acyclic graph.There were strong inverse associations between PCV coverage and the incidence of PCV7-type IPD (adjusted incidence rate ratio [aIRR] 0.967, 95% confidence interval [CI] 0.958 to 0.975, p-value < 0.001), and pneumonia hospitalisations (all-cause pneumonia: aIRR 0.991 95% CI 0.990 to 0.994, p-value < 0.001) among undervaccinated children. Subgroup analyses for children <4 months old, urban, rural, and Indigenous populations showed similar trends, although effects were smaller for rural and Indigenous populations. Approximately 50% coverage of PCV7 among children <5 years of age was estimated to prevent up to 72.5% (95% CI 51.6 to 84.4) of PCV7-type IPD among undervaccinated children, while 90% coverage was estimated to prevent 95.2% (95% CI 89.4 to 97.8). The main limitations of this study include the potential for differential loss to follow-up, geographical misclassification of children (based on residential address at birth only), and unmeasured confounders.ConclusionsIn this study, we observed substantial indirect protection at lower levels of PCV coverage than previously described—challenging assumptions that high levels of PCV coverage (i.e., greater than 90%) are required. Understanding the association between PCV coverage and indirect protection is a priority since the control of vaccine-type pneumococcal disease is a prerequisite for reducing the number of PCV doses (from 3 to 2). Reduced dose schedules have the potential to substantially reduce program costs while maintaining vaccine impact.

In an observational study, Jocelyn Chan and colleagues investigate associations between pneumococcal conjugate vaccine coverage and incidence of invasive pneumococcal disease and pneumonia among children under 5 years in Australia.     

Reproductive skew and relatedness in social groups of European badgers, Meles meles

Reproductive skew is a measure of the proportion of individuals of each sex that breed in a group and is a valuable measure for understanding the evolution and maintenance of sociality. Here, we provide the first quantification of reproductive skew within social groups of European badgers Meles meles , throughout an 18-year study in a high-density population. We used 22 microsatellite loci to analyse within-group relatedness and demonstrated that badger groups contained relatives. The average within-group relatedness was high ( R =  0.20) and approximately one-third of within-group dyads were more likely to represent first-order kin than unrelated pairs. Adult females within groups had higher pairwise relatedness than adult males, due to the high frequency of extra-group paternities, rather than permanent physical dispersal. Spatial clustering of relatives occurred among neighbouring groups, which we suggest was due to the majority of extra-group paternities being attributable to neighbouring males. Reproductive skew was found among within-group candidate fathers ( B  = 0.26) and candidate mothers ( B  = 0.07), but not among breeding individuals; our power to detect skew in the latter was low. We use these results to evaluate reproductive skew models. Although badger society best fits the assumptions of the incomplete-control models, our results were not consistent with their predictions. We suggest that this may be due to female control of paternity, female–female reproductive suppression occurring only in years with high food availability resulting in competition over access to breeding sites, extra-group paternity masking the benefits of natal philopatry, and/or the inconsistent occurrence of hierarchies that are linear when established.     

Defences against oxidative stress in vibrios associated with corals

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.     

Functional interplay between the Yersinia pseudotuberculosis YpsRI and YtbRI quorum sensing systems modulates swimming motility by controlling expression of flhDC and fliA

Quorum sensing (QS) in Yersinia pseudotuberculosis involves two pairs of LuxRI orthologues (YpsRI and YtbRI) and multiple N -acylhomoserine lactones (AHLs). In a ypsI/ytbI mutant, AHL synthesis was abolished, unaffected in a ypsR/ytbR double mutant and substantially reduced in a ypsI / ytbR mutant, indicating that neither YpsR nor YtbR is essential for AHL synthesis. To determine the interrelationship between YpsRI and YtbRI we constructed chromosomal lux –promoter fusions to ypsR , ypsI , ytbR and ytbI and examined their expression in each of the QS mutant backgrounds. The YpsRI system negatively autoregulates itself but positively regulates the expression of the ytbRI system whereas the ytbRI system is positively autoregulated but only at the level of ytbI expression. YtbRI does not control expression of ypsR or ypsI. This hierarchical QS system controls swimming motility via regulation of flhDC and fliA . The AHLs synthesized via YtbI play a dual role, activating flhDC , in conjunction with YpsR but repressing fliA in conjunction with YtbR and YpsR. In liquid and plate assays, the early onset of motility observed in ypsR and ypsI mutants was abolished in ytbI , ytbR ypsI/ytbI , ypsR/ytbR mutants, indicating that QS regulates motility both positively (via YtbRI) and negatively (via YpsRI).     

Biocompatible wound dressings based on chemically degradable triblock copolymer hydrogels

The synthesis of a series of thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) and the central B block is poly(2-(methacryloyloxy)ethyl phosphorylcholine) is achieved using atom transfer radical polymerization. These novel triblock copolymers form thermo-reversible physical gels with critical gelation temperatures and mechanical properties that are highly dependent on the copolymer composition and concentration. TEM studies on dried dilute copolymer solutions indicate the presence of colloidal aggregates, which is consistent with micellar gel structures. This hypothesis is consistent with the observation that incorporating a central disulfide bond within the B block leads to thermo-responsive gels that can be efficiently degraded using mild reductants such as dithiothreitol (DTT) over time scales of minutes at 37 degrees C. Moreover, the rate of gel dissolution increases at higher DTT/disulfide molar ratios. Finally, these copolymer gels are shown to be highly biocompatible. Only a modest reduction in proliferation was observed for monolayers of primary human dermal fibroblasts, with no evidence for cytotoxicity. Moreover, when placed directly on 3D tissue-engineered skin, these gels had no significant effect on cell viability. Thus, we suggest that these thermo-responsive biodegradable copolymer gels may have potential applications as wound dressings.     

Enhancement of tumor-TRAIL susceptibility by modulation of autophagy

Autophagy has recently been recognized as an important cellular response to stress. However, the prospect of manipulating the autophagic process for the enhancement of cancer therapy remains unresolved. This lack of resolution stems from the current controversy regarding the fundamental function of autophagy in tumor stress response: Does it have a positive or negative impact on tumor survival capability? Our studies were designed to investigate the role of autophagy in the response to TRAIL of tumor cells with various apoptotic defects. Based on our findings, we propose that divergent mechanisms of resistance to TRAIL can be reversed by a common approach of targeting specific components of the autophagic process for inhibition. This concept may have significant implications for the development of new strategies to circumvent TRAIL resistance in tumors.