Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome

Fungal iron availability during deep seated candidiasis is defined by a complex interplay involving systemic and local events

Peer reviewedPublisher PD

How does one become spiritual? The Spiritual Modeling Inventory of Life Environments (SMILE)

We report psychometric properties, correlates and underlying theory of the Spiritual Modeling Index of Life Environments (SMILE), a measure of perceptions of spiritual models, defined as everyday and prominent people who have functioned for respondents as exemplars of spiritual qualities, such as compassion, self-control, or faith. Demographic, spiritual, and personality correlates were examined in an ethnically diverse sample of college students from California, Connecticut, and Tennessee (N=1010). A summary measure of model influence was constructed from perceived models within family, school, religious organization, and among prominent individuals from both tradition and media. The SMILE, based on concepts from Bandura\u27s (1986) Social Cognitive Theory, was well-received by respondents. The summary measure demonstrated good 7-week test/retest reliability (r=.83); patterns of correlation supporting convergent, divergent, and criterion-related validity; demographic differences in expected directions; and substantial individual heterogeneity. Implications are discussed for further research and for pastoral, educational, and health-focused interventions

Spectroscopic and biochemical correlations during the course of human lens aging

BACKGROUND: With age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract. METHODS: Spectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years). RESULTS: Our experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ(210 nm)-λ(470 nm). A relative increase in the absorptivity at λ((511–520 nm)), with age, was also observed. In addition, the ratio of absorptivity at λ((511–520 nm)) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λ(Ex312 nm)) and UV-A (λ(Ex365 nm)) were found to be positively correlated (r(2 )= 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years. CONCLUSION: Based on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years

The living microarray: a high-throughput platform for measuring transcription dynamics in single cells

<p>Abstract</p> <p>Background</p> <p>Current methods of measuring transcription in high-throughput have led to significant improvements in our knowledge of transcriptional regulation and Systems Biology. However, endpoint measurements obtained from methods that pool populations of cells are not amenable to studying time-dependent processes that show cell heterogeneity.</p> <p>Results</p> <p>Here we describe a high-throughput platform for measuring transcriptional changes in real time in single mammalian cells. By using reverse transfection microarrays we are able to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a single microscope slide and image these clusters every 20 minutes. We use a fast-maturing, destabilized and nuclear-localized reporter that is suitable for automated segmentation to accurately measure promoter activity in single cells. We tested this platform with synthetic drug-inducible promoters that showed robust induction over 24 hours. Automated segmentation and tracking of over 11 million cell images during this period revealed that cells display substantial heterogeneity in their responses to the applied treatment, including a large proportion of transfected cells that do not respond at all.</p> <p>Conclusions</p> <p>The results from our single-cell analysis suggest that methods that measure average cellular responses, such as DNA microarrays, RT-PCR and chromatin immunoprecipitation, characterize a response skewed by a subset of cells in the population. Our method is scalable and readily adaptable to studying complex systems, including cell proliferation, differentiation and apoptosis.</p

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

Evidence for the ‘Good Genes’ Model: Association of MHC Class II DRB Alleles with Ectoparasitism and Reproductive State in the Neotropical Lesser Bulldog Bat, Noctilio albiventris

The adaptive immune system has a major impact on parasite resistance and life history strategies. Immunological defence is costly both in terms of immediate activation and long-term maintenance. The ‘good genes’ model predicts that males with genotypes that promote a good disease resistance have the ability to allocate more resources to reproductive effort which favours the transmission of good alleles into future generations. Our study shows a correlation between immune gene constitution (Major Histocompatibility Complex, MHC class II DRB), ectoparasite loads (ticks and bat flies) and the reproductive state in a neotropical bat, Noctilio albiventris. Infestation rates with ectoparasites were linked to specific Noal-DRB alleles, differed among roosts, increased with body size and co-varied with reproductive state particularly in males. Non-reproductive adult males were more infested with ectoparasites than reproductively active males, and they had more often an allele (Noal-DRB*02) associated with a higher tick infestation than reproductively active males or subadults. We conclude that the individual immune gene constitution affects ectoparasite susceptibility, and contributes to fitness relevant trade-offs in male N. albiventris as suggested by the ‘good genes’ model

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface

Evolution of Mutational Robustness in the Yeast Genome: A Link to Essential Genes and Meiotic Recombination Hotspots

Deleterious mutations inevitably emerge in any evolutionary process and are speculated to decisively influence the structure of the genome. Meiosis, which is thought to play a major role in handling mutations on the population level, recombines chromosomes via non-randomly distributed hot spots for meiotic recombination. In many genomes, various types of genetic elements are distributed in patterns that are currently not well understood. In particular, important (essential) genes are arranged in clusters, which often cannot be explained by a functional relationship of the involved genes. Here we show by computer simulation that essential gene (EG) clustering provides a fitness benefit in handling deleterious mutations in sexual populations with variable levels of inbreeding and outbreeding. We find that recessive lethal mutations enforce a selective pressure towards clustered genome architectures. Our simulations correctly predict (i) the evolution of non-random distributions of meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers and EG clustering, (iii) the evolution of EG enrichment in pericentromeric regions and (iv) the associated absence of meiotic crossovers (cold centromeres). Our results furthermore predict optimal crossover rates for yeast chromosomes, which match the experimentally determined rates. Using a Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal phenotypes result predominantly from mutation of single loci and generally do not impair mating, which leads to an accumulation of mutational load following meiosis and mating. We hypothesize that purging of deleterious mutations in essential genes constitutes an important factor driving meiotic crossover. Therefore, the increased robustness of populations to deleterious mutations, which arises from clustered genome architectures, may provide a significant selective force shaping crossover distribution. Our analysis reveals a new aspect of the evolution of genome architectures that complements insights about molecular constraints, such as the interference of pericentromeric crossovers with chromosome segregation