Laboratory Animal Management Assistant (LAMA): a LIMS for active research colonies

Laboratory Animal Management Assistant (LAMA) is an internet-based system for tracking large laboratory mouse colonies. It has a user-friendly interface with powerful search capabilities that ease day-to-day tasks such as tracking breeding cages and weaning litters. LAMA was originally developed to manage hundreds of new mouse strains generated by a large functional genomics program, the Pleiades Promoter Project (http://www.pleiades.org). The software system has proven to be highly flexible, suitable for diverse management approaches to mouse colonies. It allows custom tagging and grouping of animals, simplifying project-specific handling and access to data. Finally, LAMA was developed in close collaboration with mouse technicians to ease the transition from paper- or Excel-based management systems to computerized tracking, allowing data export in a popular spreadsheet format and automatic printing of cage cards. LAMA is an open-access software tool, freely available to the research community at http://launchpad.net/mousedb.     

Divergence genetics analysis reveals historical population genetic processes leading to contrasting phylogeographic patterns in co-distributed species

Coalescent samplers are computational time machines for inferring the historical demographic genetic processes that have given rise to observable patterns of spatial genetic variation among contemporary populations. We have used traditional characterizations of population structure and coalescent‐based inferences about demographic processes to reconstruct the population histories of two co‐distributed marine species, the frilled dog whelk, Nucella lamellosa, and the bat star, Patiria miniata. Analyses of population structure were consistent with previous work in both species except that additional samples of N. lamellosa showed a larger regional genetic break on Vancouver Island (VI) rather than between the southern Alexander Archipelago as in P. miniata. Our understanding of the causes, rather than just the patterns, of spatial genetic variation was dramatically improved by coalescent analyses that emphasized variation in population divergence times. Overall, gene flow was greater in bat stars (planktonic development) than snails (benthic development) but spatially homogeneous within species. In both species, these large phylogeographic breaks corresponded to relatively ancient divergence times between populations rather than regionally restricted gene flow. Although only N. lamellosa shows a large break on VI, population separation times on VI are congruent between species, suggesting a similar response to late Pleistocene ice sheet expansion. The absence of a phylogeographic break in P. miniata on VI can be attributed to greater gene flow and larger effective population size in this species. Such insights put the relative significance of gene flow into a more comprehensive historical biogeographic context and have important implications for conservation and landscape genetic studies that emphasize the role of contemporary gene flow and connectivity in shaping patterns of population differentiation.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Encapsulation of recombinant <Emphasis Type="Italic">E. coli</Emphasis> expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂ and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells. Both encapsulated and free cells reached the end point of the Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.     

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer’s disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1^−/− cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of lipid rafts in these processes.     

Explicit reformulations of time-dependent solution for a Michaelis-Menten enzyme reaction model

The exact closed-form solution to the Michaelis-Menten equation is expressed in terms of the Lambert W(x) function. However, the utility of this solution is limited because the W(x) function is not widely available in curve-fitting software. Based on various approximations to the W(x) function, different explicit equations expressed in terms of the elementary functions are proposed here as useful shortcuts to fit time depletion of substrate concentration directly to progress curves using commonly available nonlinear regression computer programs. The results are compared with those obtained by fitting other algebraic equations that have been proposed previously in the literature.     

Rapid sympatric ecological differentiation of crater lake cichlid fishes within historic times

Background  

After a volcano erupts, a lake may form in the cooled crater and become an isolated aquatic ecosystem. This makes fishes in crater lakes informative for understanding sympatric evolution and ecological diversification in barren environments. From a geological and limnological perspective, such research offers insight about the process of crater lake ecosystem establishment and speciation. In the present study we use genetic and coalescence approaches to infer the colonization history of Midas cichlid fishes (Amphilophus cf. citrinellus) that inhabit a very young crater lake in Nicaragua-the ca. 1800 year-old Lake Apoyeque. This lake holds two sympatric, endemic morphs of Midas cichlid: one with large, hypertrophied lips (~20% of the total population) and another with thin lips. Here we test the associated ecological, morphological and genetic diversification of these two morphs and their potential to represent incipient speciation.     

Fruit,flies and filamentous fungi – experimental analysis of animal–microbe competition using Drosophila melanogaster and Aspergillus mould as a model system

In addition to their fundamental role in nutrient recycling, saprobiotic microorganisms may be considered as typical consumers of food‐limited ephemeral resource patches. As such, they may be engaged in inter‐specific competition with saprophagous animals feeding from the same resource. Bacteria and filamentous fungi are known to synthesise secondary metabolites, some of which are toxic and have been proposed to deter or harm animals. The microorganisms may, however, also be negatively affected if saprophagous animals do not avoid microbe‐laden resources but feed in the presence of microbial competitors. We hypothesised that filamentous fungi compete with saprophagous insects, whereby secondary metabolites provide a chemical shield against the insect competitors. For testing this, we developed a new ecological model system representing a case of animal–microbe competition between saprobiotic organisms, comprising Drosophila melanogaster and species of the fungus Aspergillus (A. nidulans, A. fumigatus, A. flavus). Infestation of Drosophila breeding substrate with proliferating fungal colonies caused graduated larval mortality that strongly depended on mould species and colony age. Confrontation with conidiospores only, did not result in significant changes in larval survival, suggesting that insect death may not be ascribed to pathogenic effects. When confronted with colonies of transgenic fungi that lack the ability to express the global secondary metabolite regulator LaeA (ΔlaeA), larval mortality was significantly reduced compared to the impact of the wild type strains. Yet, also in the ΔlaeA strains, inter‐specific variation in the influence on insect growth occurred. Competition with Drosophila larvae impaired fungal growth, however, wild type colonies of A. nidulans and A. flavus recovered more rapidly from insect competition than the corresponding ΔlaeA mutants (not in A. fumigatus). Our findings provide genetic evidence that toxic secondary metabolites synthesised by saprotrophic fungi may serve as a means to combat insect competitors. Variation in the ability of LaeA to control expression of various secondary metabolite gene clusters might explain the observed species‐specific variation in Drosophila–Aspergillus competition.     

Patterning of mutually interacting bacterial bodies: close contacts and airborne signals

Background  

Bacterial bodies (colonies) can develop complex patterns of color and structure. These patterns may arise as a result of both colony-autonomous developmental and regulatory processes (self-patterning) and environmental influences, including those generated by neighbor bodies. We have studied the interplay of intra-colony signaling (self-patterning) and inter-colony influences in related clones of Serratia rubidaea grown on rich media.