A saltwater flotation technique to identify unincubated eggs

ABSTRACT.   Field studies on nesting birds sometimes involve questions related to nest initiation dates, length of the incubation period, or changes in parental incubation behavior during various stages of incubation. Some of this information can be best assessed when a nest is discovered before the eggs have undergone any incubation, and this has traditionally been assessed by floating eggs in freshwater. However, because the freshwater method is not particularly accurate in identifying unincubated eggs, we developed a more reliable saltwater flotation method. The saltwater method involves diluting a saturated saltwater solution with freshwater until a salt concentration is reached where unincubated eggs sink to the bottom and incubated eggs float to the surface. For Laughing Gulls ( Leucophaeus atricilla ), floating eggs in freshwater failed to identify 39.0% ( N = 251) of eggs that were subsequently found by candling to have undergone incubation prior to collection. By contrast, in a separate collection of gull eggs, no eggs that passed the saltwater test ( N = 225) were found by a later candling to have been incubated prior to collection. For Double-crested Cormorants ( Phalacrocorax auritus ), floating eggs in freshwater failed to identify 15.6% ( N = 250) of eggs that had undergone incubation prior to collection, whereas in a separate collection, none of the eggs that passed the saltwater test ( N = 85) were found by a later candling to have been incubated prior to collection. Immersion of eggs in saltwater did not affect embryo survival. Although use of the saltwater method is likely limited to colonial species and requires calibrating a saltwater solution, it is a faster and more accurate method of identifying unincubated eggs than the traditional method of floating eggs in freshwater.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.     

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

Weighted Interaction SNP Hub (WISH) network method for building genetic networks for complex diseases and traits using whole genome genotype data

Background

High-throughput genotype (HTG) data has been used primarily in genome-wide association (GWA) studies; however, GWA results explain only a limited part of the complete genetic variation of traits. In systems genetics, network approaches have been shown to be able to identify pathways and their underlying causal genes to unravel the biological and genetic background of complex diseases and traits, e.g., the Weighted Gene Co-expression Network Analysis (WGCNA) method based on microarray gene expression data. The main objective of this study was to develop a scale-free weighted genetic interaction network method using whole genome HTG data in order to detect biologically relevant pathways and potential genetic biomarkers for complex diseases and traits.

Results

We developed the Weighted Interaction SNP Hub (WISH) network method that uses HTG data to detect genome-wide interactions between single nucleotide polymorphism (SNPs) and its relationship with complex traits. Data dimensionality reduction was achieved by selecting SNPs based on its: 1) degree of genome-wide significance and 2) degree of genetic variation in a population. Network construction was based on pairwise Pearson's correlation between SNP genotypes or the epistatic interaction effect between SNP pairs. To identify modules the Topological Overlap Measure (TOM) was calculated, reflecting the degree of overlap in shared neighbours between SNP pairs. Modules, clusters of highly interconnected SNPs, were defined using a tree-cutting algorithm on the SNP dendrogram created from the dissimilarity TOM (1-TOM). Modules were selected for functional annotation based on their association with the trait of interest, defined by the Genome-wide Module Association Test (GMAT). We successfully tested the established WISH network method using simulated and real SNP interaction data and GWA study results for carcass weight in a pig resource population; this resulted in detecting modules and key functional and biological pathways related to carcass weight.

Conclusions

We developed the WISH network method which is a novel 'systems genetics' approach to study genetic networks underlying complex trait variation. The WISH network method reduces data dimensionality and statistical complexity in associating genotypes with phenotypes in GWA studies and enables researchers to identify biologically relevant pathways and potential genetic biomarkers for any complex trait of interest.

     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Research into the (Cost-) effectiveness of the ketogenic diet among children and adolescents with intractable epilepsy: design of a randomized controlled trial

Background  

Epilepsy is a neurological disorder, characterized by recurrent unprovoked seizures which have a high impact on the individual as well as on society as a whole. In addition to the economic burden, epilepsy imposes a substantial burden on the patients and their surroundings. Patients with uncontrolled epilepsy depend heavily on informal care and on health care professionals. About 30% of patients suffer from drug-resistant epilepsy. The ketogenic diet can be a treatment of last resort, especially for children. The beneficial effect of the ketogenic diet has been proven, but information is lacking about its cost-effectiveness. In the current study we will evaluate the (cost-) effectiveness of the ketogenic diet in children and adolescents with intractable epilepsy.     

Glycogen storage and degradation during in vitro growth and differentiation of Giardia intestinalis

Giardia intestinalis is the causative agent of human giardiasis, a common diarrheal illness worldwide. Despite its global distribution and prevalence, many questions regarding its basic biology and metabolism remain unanswered. In this study, we examine the accumulation and degradation of glycogen, an important source of stored carbon and energy, during the in vitro growth and differentiation of G. intestinalis . We report that, as G. intestinalis progresses through its growth cycle, cultures of trophozoites accumulate glycogen during the lag and early logarithmic phases of growth and then utilize this compound during their remaining logarithmic growth. As cultures enter the stationary phase of growth, they re-accumulate glycogen stores. The activity of glycogen phosphorylase, an enzyme involved in glycogen metabolism, also varied throughout in vitro trophozoite growth. During the in vitro induction of trophozoite differentiation into water-resistant cyst forms, the cultures initially accumulated stores of glycogen which diminished throughout transition to the cyst form. This observation is suggestive of a role for glycogen in the differentiation process. These studies represent the first thorough analysis of changes in glycogen content and glycogen phosphorylase activity during G. intestinalis growth and differentiation.