Investigating genetic discrimination in Australia: opportunities and challenges in the early stages

Genetic discrimination, defined as the differential treatment of individuals or their relatives on the basis of actual or presumed genetic differences, is an emerging issue of interest in academic, clinical, social and legal contexts. While its potential significance has been discussed widely, verified empirical data are scarce. Genetic discrimination is a complex phenomenon to describe and investigate, as evidenced by the recent Australian Law Reform Commission inquiry in Australia. The authors research project, which commenced in 2002, aims to document the multiple perspectives and experiences regarding genetic discrimination in Australia and inform future policy development and law reform. Data are being collected from consumers, employers, insurers and the legal system. Attempted verification of alleged accounts of genetic discrimination will be a novel feature of the research. This paper overviews the early stages of the research, including conceptual challenges and their methodological implications.     

Effects of flooding on recruitment and abundance of Golden Perch (Macquaria ambigua ambigua) in the lower River Murray

Flooding is often considered a stimulus for production of fish in floodplain rivers. In the southern Murray–Darling Basin (MDB), Australia, however, few native fish species have been shown to use the floodplain for spawning, and recruitment has been positively and negatively associated with flooding. In 2010/11, extensive flooding in the lower River Murray provided an opportunity to investigate the recruitment response of Golden Perch (Macquaria ambigua ambigua) following 10 years of drought and floodplain isolation. Annual variation in Golden Perch abundance and recruitment were investigated in anabranch and main channel habitats at Chowilla in the floodplain geomorphic region of the lower River Murray over a 7‐year period incorporating the flood and 6 years of in‐channel flow. Spatial variation in recruitment in the lower River Murray was also investigated by comparing the age structure of Golden Perch in the swamplands/lakes, gorge and floodplain geomorphic regions. Golden Perch abundance in the Chowilla region increased significantly postflooding compared with drought years. Age structures indicated that increased abundance was due predominantly to fish spawned during the flood (2010/11) and the previous year (2009/10), which was characterised by in‐channel flows. Age structure was similar in the nearby Katarapko Anabranch system indicating a uniform postflood recruitment response in the floodplain geomorphic region. Juvenile Golden Perch from the 2010/11 and 2009/10 cohorts were less apparent in the gorge and swamplands/lakes regions. Golden Perch have flexible life histories and will spawn and recruit in association with in‐channel rises in flow and overbank flows, but significant increases in abundance in the lower River Murray may result from overbank flooding. Contemporary approaches to flow restoration in the MDB emphasise overbank flows and floodplain processes. We suggest, however, that environmental flow management that incorporates floodplain and in‐channel processes, at appropriate spatio‐temporal scales, will result in more robust populations of Golden Perch.     

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD

Background

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.

Methods

Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.

Results

There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10^-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).

Conclusions

Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Assessing the Role of Cell-Surface Molecules in Central Synaptogenesis in the Drosophila Visual System

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.     

Connectivity Mapping for Candidate Therapeutics Identification Using Next Generation Sequencing RNA-Seq Data

The advent of next generation sequencing technologies (NGS) has expanded the area of genomic research, offering high coverage and increased sensitivity over older microarray platforms. Although the current cost of next generation sequencing is still exceeding that of microarray approaches, the rapid advances in NGS will likely make it the platform of choice for future research in differential gene expression. Connectivity mapping is a procedure for examining the connections among diseases, genes and drugs by differential gene expression initially based on microarray technology, with which a large collection of compound-induced reference gene expression profiles have been accumulated. In this work, we aim to test the feasibility of incorporating NGS RNA-Seq data into the current connectivity mapping framework by utilizing the microarray based reference profiles and the construction of a differentially expressed gene signature from a NGS dataset. This would allow for the establishment of connections between the NGS gene signature and those microarray reference profiles, alleviating the associated incurring cost of re-creating drug profiles with NGS technology. We examined the connectivity mapping approach on a publicly available NGS dataset with androgen stimulation of LNCaP cells in order to extract candidate compounds that could inhibit the proliferative phenotype of LNCaP cells and to elucidate their potential in a laboratory setting. In addition, we also analyzed an independent microarray dataset of similar experimental settings. We found a high level of concordance between the top compounds identified using the gene signatures from the two datasets. The nicotine derivative cotinine was returned as the top candidate among the overlapping compounds with potential to suppress this proliferative phenotype. Subsequent lab experiments validated this connectivity mapping hit, showing that cotinine inhibits cell proliferation in an androgen dependent manner. Thus the results in this study suggest a promising prospect of integrating NGS data with connectivity mapping.     

Proteomic Profiling of Burkholderia cenocepacia Clonal Isolates with Different Virulence Potential Retrieved from a Cystic Fibrosis Patient during Chronic Lung Infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient’s death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.     

A Subset of Mouse Colonic Goblet Cells Expresses the Bitter Taste Receptor Tas2r131

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics.