Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

BackgroundThe potential for an expanded HIV epidemic in Papua New Guinea (PNG) demands an effective, evidence-based and locally-appropriate national response. As sexually transmitted infections (STIs) may be important co-factors in HIV transmission nationally, it is timely to conduct a systematic review of STI prevalences to inform national policy on sexual health and HIV/STI prevention.Methodology/Principal FindingsWe undertook a systematic review and meta-analysis of HIV and STI prevalences in PNG, reported in peer-reviewed and non-peer-reviewed publications for the period 1950–2010. Prevalence estimates were stratified by study site (community or clinic-based), geographic area and socio-demographic characteristics. The search strategy identified 105 reports, of which 25 studies (10 community-based; 10 clinic-based; and 5 among self-identified female sex workers) reported STI prevalences and were included in the systematic review. High prevalences of chlamydia, gonorrhoea, syphilis and trichomonas were reported in all settings, particularly among female sex workers, where pooled estimates of 26.1%, 33.6%, 33.1% and 39.3% respectively were observed. Pooled HIV prevalence in community-based studies was 1.8% (95% CI:1.2–2.4) in men; 2.6% (95% CI:1.7–3.5) in women; and 11.8% (95% CI:5.8–17.7) among female sex workers.Conclusions/SignificanceThe epidemiology of STIs and HIV in PNG shows considerable heterogeneity by geographical setting and sexual risk group. Prevalences from community-based studies in PNG were higher than in many other countries in the Asia-Pacific. A renewed focus on national STI/HIV surveillance priorities and systems for routine and periodic data collection will be essential to building effective culturally-relevant behavioural and biomedical STI/HIV prevention programs in PNG.

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.

Male circumcision (MC) significantly reduces the risk of HIV acquisition in men. The geographical, linguistic and cultural diversity of Papua New Guinea (PNG) makes issues of acceptability and implementation complex, and culturally appropriate HIV and Sexually Transmissible Infection (STI) prevention strategies are crucial in this setting. A modified Delphi approach was conducted with sexual health specialists to document and classify variants of penile cutting as part of a programme of research being carried out to investigate the acceptability and potential epidemiological impact of MC for HIV prevention in PNG, and options for future roll-out. Three broad categories were identified: circumcision, longitudinal incisions (including dorsal slit procedures) and incisions that did not alter the profile of penis or foreskin. The typology provides a universal language for health practitioners and policy makers that will inform future sexual health deliberations. The popularity of dorsal slit procedures in PNG has significant implications due to its procedural simplicity and limited resource requirements, making it an attractive provider option compared to medical circumcision. Further research is urgently required to examine the effectiveness of dorsal slit procedures for HIV prevention in PNG, the prevalence of various forms of penile cutting and the extent to which health staff are currently engaged in dorsal slit procedures.

ObjectivePapua New Guinea (PNG) has among the highest estimated prevalences of genital Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) of any country in the Asia-Pacific region. Diagnosis and treatment of these infections have relied on the WHO-endorsed syndromic management strategy that uses clinical presentation without laboratory confirmation to make treatment decisions. We evaluated the performance of this strategy in clinical settings in PNG.DesignWomen attending antenatal (ANC), well woman (WWC) and sexual health (SHC) clinics in four provinces were invited to participate, completed a face-to-face interview and clinical examination, and provided genital specimens for laboratory testing. We estimated the performance characteristics of syndromic diagnoses against combined laboratory diagnoses.Results1764 women were enrolled (ANC=765; WWC=614; SHC=385). The prevalences of CT, NG and TV were highest among women attending ANC and SHC. Among antenatal women, syndromic diagnosis of sexually transmitted infection had low sensitivity (9%–21%) and positive predictive value (PPV) (7%–37%), but high specificity (76%–89%) and moderate negative predictive value (NPV) (55%–86%) for the combined endpoint of laboratory-confirmed CT, NG or TV. Among women attending WWC and SHC, ‘vaginal discharge syndrome’ had moderate to high sensitivity (72%–78%) and NPV (62%–94%), but low specificity (26%–33%) and PPV (8%–38%). ‘Lower abdominal pain syndrome’ had low sensitivity (26%–41%) and PPV (8%–23%) but moderate specificity (66%–68%) and high NPV (74%–93%) among women attending WWC, and moderate-high sensitivity (67%–79%) and NPV (62%–86%) but low specificity (26%–28%) and PPV (14%–33%) among SHC attendees.ConclusionThe performance of syndromic management for the detection and treatment of genital chlamydia, gonorrhoea and trichomonas was poor among women in different clinical settings in PNG. New diagnostic strategies are needed to control these infections and to prevent their adverse health outcomes in PNG and other high-burden countries.

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We have developed a system in which the fusion of pancreatic zymogen granules with plasma membranes can be studied in vitro. Here we show that this membrane fusion event is stimulated specifically by peptides of the effector domain of rab3, a small, monomeric GTP-binding protein.In addition, we demonstrate that the stimulatory effect of the peptides involves their binding to a target on the plasma membrane, and is both qualitatively and quantitatively different from the effect of GTPyS, which also enhances membrane fusion. We suggest that regulated exocytosis in the pancreatic acinar cell may be under the control of more than one type of GTP-binding protein.rab3; GTP-binding protein; Membrane fusion; Exocytosis