Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.     

Environmental relationships of the brush-tailed rabbit-rat, Conilurus penicillatus, and other small mammals on the Tiwi Islands, northern Australia

Aim To describe the habitat characteristics and status of the brush‐tailed rabbit‐rat, Conilurus penicillatus Gould, 1842, on the Tiwi Islands, northern Australia, as part of a broader programme aimed at the conservation management of this species. In addition, comparable environmental modelling is undertaken for other co‐occurring small native mammals, including the black‐footed tree‐rat, Mesembriomys gouldii Gray, 1843, a taxonomically and ecologically related species. These objectives relate to the significance for mammal conservation of islands generally in Australia, and the recent intensification of plantation forestry on these previously little‐disturbed islands. Location Melville and Bathurst islands (Tiwi Islands), respectively, Australia's second and fifth largest islands. Methods A systematic survey was conducted for mammals across Bathurst (115 sampled quadrats) and Melville Island (236 quadrats). A broad range of environmental variables was recorded for every quadrat. All quadrats were classified by their woody plant species composition. The relative occurrence of individual mammal species across the resulting vegetation groups was examined using Kruskal–Wallis anova . The habitat relationships of C. penicillatus and the most commonly recorded mammal species were described by generalized linear modelling, with separate models for each island, for both islands combined, for all habitats and for only those sites dominated by eucalypts. Results Twelve small mammal species (excluding bats, macropods and feral animals) were recorded in this study. The most notable feature of this survey was the lack of records of M. gouldii from Bathurst Island. In contrast, the proportion of quadrats with C. penicillatus was not significantly different between the two islands. There was no significant tendency for these two species to co‐occur in quadrats on Melville Island more or less commonly than by chance. Conilurus penicillatus was most abundant in eucalypt forest while M. gouldii showed a weak association with eucalypt forests and woodlands and shrub land. The five most commonly recorded species showed highly idiosyncratic relationships with environmental variables, with this relationship showing some variation between the two islands. None showed any significant association with floristic variation within the extensive eucalypt forests, but most showed significant associations with tree height, basal area (especially of large trees), landscape position (distance to watercourse) and fire history. Main conclusions Conilurus penicillatus was most likely to occur in tall eucalypt forest away from watercourses. This habitat is now being targeted for clearance for the development of plantations of the exotic Acacia mangium. Seven of the 12 mammal species examined in this study (C. penicillatus, M. gouldii, Rattus tunneyi Thomas, 1904, Melomys burtoni Ramsay, 1887, Sminthopsis butleri Archer, 1979, Phascogale tapoatafa Meyer, 1793 and Petaurus breviceps Gould, 1842) were not recorded at all in plantations, and these (and other) species are likely to be severely disadvantaged by plantation development. The study also demonstrated that the two medium to large arboreal rodent species (C. penicillatus and M. gouldii) vary in environmental associations and found no evidence that C. penicillatus increased in areas unoccupied by M. gouldii.     

Mortality of coho salmon (Oncorhynchus kisutch) associated with burdens of multiple parasite species

Multiple analytical techniques were used to evaluate the impact of multiple parasite species on the mortality of threatened juvenile coho salmon (Oncorhynchus kisutch) from the West Fork Smith River, Oregon, USA. We also proposed a novel parsimonious mathematical representation of macroparasite distribution, congestion rate, which (i) is easier to use than traditional models, and (ii) is based on Malthusian parameters rather than probability theory. Heavy infections of Myxobolus insidiosus (Myxozoa) and metacercariae of Nanophyetus salmincola and Apophallus sp. occurred in parr (subyearlings) from the lower mainstem of this river collected in 2007 and 2008. Smolts (yearlings) collected in 2007–2010 always harboured fewer Apophallus sp. with host mortality recognised as a function of intensity for this parasite. Mean intensity of Apophallus sp. in lower mainstem parr was 753 per fish in 2007 and 856 per fish in 2008, while parr from the tributaries had a mean of only 37 or 13 parasites per fish, respectively. Mean intensity of this parasite in smolts ranged between 47 and 251 parasites per fish. Over-dispersion (variance to mean ratios) of Apophallus sp. was always lower in smolts compared with all parr combined or lower mainstem parr. Retrospective analysis based on smolt data using both the traditional negative binomial truncation technique and our proposed congestion rate model showed identical results. The estimated threshold level for mortality involving Apophallus sp. was at 400–500 parasites per fish using both analytical methods. Unique to this study, we documented the actual existence of these heavy infections prior to the predicted mortality. Most of the lower mainstem parr (approximately 75%) had infections above this level. Heavy infections of Apophallus sp. metacercariae may be an important contributing factor to the high over-wintering mortality previously reported for these fish that grow and develop in this section of the river. Analyses using the same methods for M.insidiosus and N. salmincola generally pointed to minimal parasite-associated mortality.     

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.     

The SUN protein Mps3 is required for spindle pole body insertion into the nuclear membrane and nuclear envelope homeostasis

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.     

Integrating fish and parasite data as a holistic solution for identifying the elusive stock structure of Pacific sardines (Sardinops sagax)

There is an urgent need to clarify how different stocks, or subpopulations of fish species, are vulnerable to fishing pressure and unfavorable ocean conditions because of the increasing demand on fisheries for human consumption. For marine fishes, the potential for high gene flow increases the difficulty in determining the number of subpopulations managed in a specific fishery. Although the use of molecular data has become a common method in the past 15 years to identify fish subpopulations, no single technique or suite of techniques has been established for fish stock structure studies. We review the use of fish morphometrics, artificial tags, fish genetics, parasite genetics, and parasites as biological tags to identify subpopulations of marine fishes with a focus on the Pacific sardine (Sardinops sagax) fishery off the west coast of North America. We suggest an integration of fish- and parasite-based techniques for future stock structure studies, particularly for pelagic fish species whose stock structure can be elusive. An integration of techniques may also resolve fish stock structure over small geographic areas by increasing the number of spatial and temporal scales studied simultaneously leading to methods for successful management of marine fish species.     

Recent increase in Nybelinia surmenicola prevalence and intensity in Pacific hake (Merluccius productus) off the United States west coast

A larval marine cestode was found in 82.0% of 834 Pacific hake (Merluccius productus) stomachs collected from 341 trawl stations along the United States west coast during the summers of 2008 and 2009. Morphology and DNA sequencing was used to identify the cestode as Nybelinia surmenicola. In an examination of 131 Pacific hake stomachs collected from the same region in 1999, N. surmenicola prevalence was 35.1%. The results from a general linear model suggested that their prevalence is influenced by year and latitude, Pacific hake size, and sex. Mean intensity of N. surmenicola in 2008-2009 was 20.22 (±1.13 SE) and was positively related to Pacific hake length and the latitude of collection. Year-1 Pacific hake (<27 cm length) had significantly lower prevalence and intensity of N. surmenicola compared to older and larger fish. Pacific hake collected south of Point Conception, California (32.5 to 35°N) had lower prevalence and intensity of N. surmenicola compared to those collected in northern latitudes (35.1 to 48.4°N). Higher N. surmenicola prevalence in Pacific hake in recent years suggests food-web fluctuations in the northern California current ecosystem caused by changes in ocean transport of zooplankton or pelagic fish distributions and warrants future monitoring as a metric for ecosystem change.