NMR measurement of 39K detectability and relaxation constants in rat tissue

Differences in the NMR detectability of 39K in various excised rat tissues (liver, brain, kidney, muscle, and testes) have been observed. The lowest NMR detectability occurs for liver (61 ± 3% of potassium as measured by flame photometry) and highest for erythrocytes (100 ± 7%). These differences in detectability correlate with differences in the measured 39K NMR relaxation constants in the same tissues. 39K detectabilities were also found to correlate inversely with the mitochondrial content of the tissues. Mitochondria prepared from liver showed greatly reduced 39K NMR detectability when compared with the tissue from which it was derived, 31.6 ± 9% of potassium measured by flame photometry compared to 61 ± 3%. The detectability of potassium in mitochondria was too low to enable the measurement of relaxation constants. This study indicates that differences in tissue structure, particularly mitochondrial content are important in determining 39K detectability and measured relaxation rates.

Are we there yet? Early years reform in Queensland : stakeholder perspectives on the introduction of funded preschool programs in long day care services

Australian educators are currently engaging with wide-ranging, national early childhood reform that is reshaping early childhood education and care. The Australian reform agenda reflects many of the early childhood policy directions championed by bodies such as the Organisation for Economic Cooperation and Development and the United Nations Children's Education Fund, and is based on the dual discourse of (i) starting strong and (ii) investing in the early years. However, despite its traction in policy rhetoric and policy there is little empirical evidence of how reform is being played out. This paper reports on research undertaken in collaboration with the Queensland Office for Early Childhood Education and Care to generate sector feedback on one element of the reform agenda, the implementation of universal preschool in Queensland. The study aimed to determine the efficacy of the new policy in supporting the provision of 'approved preschool programs' within long day care services. Drawing together the views and experiences of a range of stakeholders, including peak organisations, service providers, directors, preschool teachers and government policy officers, it provides a situated case study of the implementation of universal preschool, and offers empirical evidence of how this policy is being played out at the local level. The paper identifies the opportunities and challenges in implementing universal preschool in Queensland that may have bearing on early childhood reform in Australia as well as other countries. Discussion of key findings is set within an overview of the ECEC policy agenda in Australia, with a particular focus on the commitment to universal preschool. Les éducateurs australiens s’engagent présentement dans une vaste réforme nationale de la petite enfance qui remodèle l'éducation et l’accueil de la petite enfance. Le programme de la réforme australienne reflète plusieurs des orientations en politique de la petite enfance soutenues par des organismes comme l'Organisation de coopération et de développement économiques (OCDE) et le Fonds des Nations Unies pour les enfants (UNICEF). Il s’appuie sur le double discours de (i) un bon départ et de (ii) l’investissement dans les premières années. Cependant, en dépit de son attrait en rhétorique de politique et en politique il y a peu de données empiriques sur la façon dont la réforme se déroule. Cet article rend compte de la recherche entreprise en collaboration avec le bureau de l'éducation et l’accueil à la petite enfance du Queensland afin d’obtenir une rétroaction du secteur sur un élément de la réforme, la mise sur pied du préscolaire universel dans le Queensland. L'étude visait à déterminer l'efficacité de la nouvelle politique pour soutenir la disposition «programmes préscolaires approuvés» dans les services de garde à temps plein. En regroupant les perspectives et les expériences d'une gamme d’intervenants, y compris d’importantes organisations, des prestataires de service, des directeurs, des enseignants du préscolaire et des fonctionnaires de politique gouvernementale, elle constitue une étude de cas localisée de l'exécution la mise sur pied du préscolaire universel, et fournit des données empiriques sur la façon dont cette politique se met en place au niveau local. L’article identifie les opportunités et les défis liés à l’implantation du préscolaire universel au Queensland, qui pourraient avoir une portée sur la réforme de petite enfance en Australie ainsi que dans d'autres pays. La discussion des principaux résultats est faite en lien avec un aperçu global de la politique d'éducation et d’accueil de la petite enfance en Australie, avec un accent particulier sur l'engagement envers le préscolaire universel. Los educadores australianos actualmente están involucrados en una amplia reforma de la educación temprana nacional que está revolucionando la educación preescolar y los servicios de cuidado. El programa de reforma Australiana refleja muchas de las direcciones políticas relacionadas con la infancia temprana incitadas por organismos como la Organización de Cooperación y Desarrollo Económicos y el Fondo Educacional Infantil de las Naciones Unidas, y se basa en el doble discurso de (i) empezando fuertemente e (ii) invertir en los primeros años. Sin embargo, a pesar de su política de tracción en retórica y política, hay pocos datos empíricos de cómo la reforma se está llevando a cabo. Este documento informa sobre las investigaciones llevadas a cabo en colaboración con la Oficina de Queensland de Educación tempana y cuidados, para generar comentarios del sector, sobre uno de los elementos de la agenda de reforma, la aplicación del preescolar universal en Queensland. El estudio tiene como objetivo determinar la eficacia de la nueva política para apoyar la prestación de "programas preescolares aprovados" dentro se los servicios de guardería y cuidado. Reuniendo los puntos de vista y las experiencias de una serie de interesados, entre ellos algunas organizaciones cumbre, proveedores de servicios, los directores, los maestros preescolares y oficiales de política y gobierno, se logra un estudio simulado de la implementación del preescolar universal, y ofrece evidencia empírica de cómo esta política se está llevando a cabo en el plano local. El documento identifica las oportunidades y desafíos en la implementación del preescolar universal en Queensland, que puede repercutir en la reforma de la indancia temprana en Australia, así como en otros países. La discusión de los resultados claves se encuentra en el interior de una visión de la agenda política de ECEC en Australia, con un enfoque particular en el compromiso con el preescolar universal.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

Orchids : My Intersex Adventure

Orchids: My Intersex Adventure is a multi-award winning autobiographical documentary film. The film follows documentary filmmaker, Phoebe Hart, as she comes clean on her journey of self-discovery to embrace her future and reconcile the past shame and family secrecy surrounding her intersex condition. Despite her mother’s outright refusal to be in the film, Phoebe decides she must push on with her quest to resolve her life story and connect with other intersex people on camera. With the help of her sister Bonnie and support from her partner James, she hits the open road and reflects on her youth. Phoebe’s happy and carefree childhood came to an abrupt end at puberty when she was told she would never menstruate nor have children. But the reasons why were never discussed and the topic was taboo. At the age of 17, Phoebe’s mother felt she was old enough to understand the true nature of her body and the family secret was finally revealed. Phoebe then faced an orchidectomy, invasive surgery to remove her undescended testes, the emotional scars of which are still raw today. Phoebe’s road trip around Australia exposes her to the stories of other intersex people and holds a mirror to her own experience. She learns valuable lessons in resilience and healing but also sees the pervasive impact her condition has on all her relationships. At home, Phoebe and James want to start a family but dealing with infertility and the stress of the adoption process puts pressure on their marriage. Phoebe also starts to understand the difficult decisions her parents faced and is excited but apprehensive when they eventually agree to be interviewed. Will talking openly with her mother give Phoebe the answers she has been looking for? The film was produced and directed by Phoebe Hart and commissioned by the Australian Broadcasting Commission. The film premiered at the Brisbane International Film Festival in 2010 where it was voted the number one film of the festival by audiences. Orchids was broadcast on ABC1 in Australia in 2012, appeared in more than 50 film festivals internationally and has since been broadcast nationally in Switzerland, Sweden, Israel, Spain, France, Russia, Poland, Germany and the USA.

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.