Genetic and functional dissection of ciliary genes

Ciliopathy disorders are associated with either abnormal formation or function of cilia. Mutations have been described in over 60 ciliary genes to date. With the identification of over 1,000 ciliary polypeptides, other disorders exhibiting ciliopathy features could result from mutations in other ciliary genes. A new ciliopathy disease gene, CENPF, has been identified in a kindred exhibiting midgestation lethality with congenital malformations suggestive of a novel ciliopathy phenotype. Where conventional approaches such as genome-wide linkage analysis and homozygosity mapping had failed, whole exome capture coupled with massive parallel deep sequencing was successful in elucidating the genetic cause in a single affected case. Identification of compound heterozygous mutations in the causative gene was facilitated through analysis of an unfiltered approach for depth of coverage. Utilising a combinatorial approach of comparative genomics and proteomics, a novel ciliogenic role for the causative gene was identified and proposed by modelling cenpf loss of function in D. rerio and supported by interactions found with I ft 88 and Kif3b, key regulators of ciliogenesis. These data support emerging vidence for the existence of acytoplasmic dynein 1-dependent multiprotein complex which has dual roles in mitosis and ciliogenesis

A core outcome set for clinical trials in oropharyngeal cancer

The impact of randomized controlled trials is frequently diminished by disparate outcome reporting, precluding the comparison of results between trials or synthesis of data in meta-analyses. This is particularly problematic in lower incidence conditions such as oropharyngeal squamous cell carcinoma (OPSCC), where the need to synthesise data from competing trials is greater. Minimum outcome reporting standards, known as Core Outcome Sets (COS) have been shown to increase the consistency of outcome reporting between trials of comparable interventions, thus facilitating the comparison or synthesis of trial data. The objective of the work in this thesis was to identify outcomes of importance to patients, carers and healthcare professionals and define a COS for OPSCC. The methods used comprised a systematic review of OPSCC RCTs to identify the outcomes reported and establish whether there was outcomes heterogeneity as suggested by other studies; semi-structured qualitative interviews with patients and carers to establish their outcomes of importance; a Delphi Study of patients, carers and healthcare professionals, to reach consensus on the outcomes that should be included in a COS for OPSCC. The systematic review described in chapter two identified significant heterogeneity in outcome reporting; 58 outcomes were reported in 43 RCTs, only three outcomes were measured in more than 50% of studies, and only 41% of outcomes were measured in more than one study. The qualitative study identified 136 outcomes. Survival and late adverse effects of treatment are of greatest priority to patients and carers. The Delphi study successfully reached consensus on eight outcomes for inclusion in the COS. There is substantial heterogeneity in the outcomes measured in contemporary RCTs in OPSCC. Yet, there is strong consensus between stakeholder groups in the outcomes of importance. Implementation of the COS will increase the consistency of outcome reporting thus facilitating the comparison of data from competing trials and synthesis of data in meta-analyses. Further consideration must be given to ways in which the uptake of COS can be maximised to have the highest impact. The COS is applicable to trials of interventions used in current clinical practice, however the advent of new treatment strategies may require that this is reviewed and adapted

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

BACKGROUND: X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue. RESULTS: We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog. CONCLUSIONS: In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center.This project was funded by grants to JAMG and PDW from the Australian Research Council

The 69-mu m forsterite band as a dust temperature indicator

A band of pure crystalline forsterite (100 per cent Mg2SiO4) occurs at 69.67 μm at room temperature (295 K); for olivines with ≳10 per cent Fe the corresponding feature is at ≳73 μm. The Mg-rich forsterite feature is observed in a variety of ISO LWS spectra, but the corresponding Fe-rich olivine feature is not. For the 10 astronomical sources in our sample, the forsterite band peaks in the 68.9–69.3 μm range and narrows with decreasing peak wavelength. This is consistent with the shortwards shifting of the peak observed when laboratory samples are cooled to 77 K (69.07 μm) and 3.5 K (68.84 μm). The shifted peak is produced by lattice contraction and the sharpening is due to a decrease in phonon density at lower temperatures. However, the astronomical bands are narrower than those of the laboratory samples. By comparing the laboratory and astronomical peak wavelengths, we deduce characteristic forsterite 69-μm band temperatures that are in the 27–84 K range for the eight post-main-sequence objects in our sample. These values are shown to be consistent with the local continuum temperatures derived using a β=1.5 dust emissivity index, similar to derived interstellar values of the opacity index. For the pre-main sequence-objects HD 100546 and MWC 922, the characteristic 69-μm forsterite band temperatures (127±18 and 139±10 K, respectively) are significantly higher than those of the post-main-sequence objects and are more than twice as high as their local continuum temperatures deduced using β=1.5. The assumption of large grains (β=0) can produce agreement between the derived 69-μm and continuum temperatures for one of these objects but not for the other — a spatial separation between the forsterite and continuum-emitting grains may therefore be implied for it. We conclude that observations of the peak wavelength and FWHM of the 69-μm forsterite band show great promise as a new diagnostic of characteristic grain temperatures

Activation of podocyte Notch mediates early Wt1 glomerulopathy

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis

CD86 Is a Selective CD28 Ligand Supporting FoxP3+ Regulatory T Cell Homeostasis in the Presence of High Levels of CTLA-4

CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4

Insufficient protection by Neisseria meningitidis vaccination alone during eculizumab therapy

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D-cycloserine-augmented one-session treatment of specific phobias in children and adolescents.

BACKGROUND: D-Cycloserine has potential to enhance exposure therapy outcomes. The current study presents a preliminary randomized, placebo-controlled double-blind pilot trial of DCS-augmented one-session treatment (OST) for youth (7-14 years) with specific phobia. A secondary aim of this pilot study was to explore the effects of youth age and within-session fear reduction as potential moderators of DCS outcomes in order to generate hypotheses for a larger trial. It was hypothesized that DCS would be associated with greater improvements than placebo, that children (7-10 years) would have greater benefits than adolescents (11-14 years), and that DCS effects would be stronger for participants with the greater within-session fear reduction during the OST. METHODS: Thirty-five children and adolescents were randomized to either OST combined with DCS (n = 17), or OST combined with placebo (PBO; n = 18) and assessed at 1 week, 1 month, and 3 month following treatment. RESULTS: There were no significant pre- to post-treatment or follow-up benefits of DCS relative to placebo. Secondary analyses of age indicated that relative to PBO, DCS was associated with greater improvements for children (but not adolescents) on measures of severity at 1-month follow-up. Children in the DCS condition also showed significantly greater improvement to 1 month on global functioning relative to other groups. Conversely, adolescents had significant post-treatment benefits in the PBO condition on symptom severity measures relative to DCS, and adolescents in the DCS condition had significantly poorer functioning at 3 months relative to all other groups. Finally, there was a trend for within-session fear reduction to be associated with moderating effects of DCS, whereby greater reduction in fear was associated with greater functioning at one-month follow-up for children who received DCS, relative to PBO. LIMITATIONS: The study sample was small and therefore conclusions are tentative and require replication. CONCLUSIONS: Age and within-session fear reduction may be important moderators of DCS-augmented one-session exposure therapy, which requires testing in a fully powered randomized controlled trial