Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria

Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.     

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 10⁶ compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10⁶ compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.     

Detection of gunshot residues on cadaveric skin using sodium rhodizonate and a counterstain.

We report a staining method for cadaveric tissue using sodium rhodizonate as a skin marker for gunshot residues and a counterstain for the surrounding connective tissue. We studied six well preserved subjects who had died of close range gunshot injury. Skin fragments were removed from the bullet entrance hole including both the disrupted area and adjacent macroscopically intact tissue. Because microscopic examination of postmortem material is difficult after histomorphologic alterations already have occurred as a consequence of postmortem tissue changes, it is necessary to use a staining method that, while detecting gunshot residues, can also make skin cell constituents recognizable from both qualitative and quantitative perspectives. Triphenylmethane dyes (acid fuchsin, aniline blue WS, light green SF yellowish, brilliant green and ethyl green) have proven appropriate for the purpose.     

Identification and characterisation of a novel adhesin Ifp in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Background  

In order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

3-(Imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers: a novel series of mGluR2 positive allosteric modulators

The synthesis and structure-activity relationship (SAR) of a novel series of 3-(imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers, derived from a high throughput screening (HTS), are described. Subsequent optimization led to identification of potent, metabolically stable and orally available mGluR2 positive allosteric modulators (PAMs).     

Enzymatic production of 18-β-glycyrrhetinic acid from Glycyrrhiza glabra L.

Enzymatic conversion of glycyrrhizinic acid (GL), from root extract of Glycyrrhiza glabra L., into 18--glycyrrhetinic acid (GA) was achieved by using a commercial preparation of -glucuronidase from Aspergillus niger. Two different processes were tested: a batch process and a continuous process. The continuous process was the better one, giving 100% conversion of GL after 5 d and 80% recovery of enzyme activity. The process yielded a product containing 40% GA.