Effects of soil nitrogen on diploid advantage in fireweed,Chamerion angustifolium (Onagraceae)

In many ecosystems, plant growth and reproduction are nitrogen limited. Current and predicted increases of global reactive nitrogen could alter the ecological and evolutionary trajectories of plant populations. Nitrogen is a major component of nucleic acids and cell structures, and it has been predicted that organisms with larger genomes should require more nitrogen for growth and reproduction and be more negatively affected by nitrogen scarcities than organisms with smaller genomes. In a greenhouse experiment, we tested this hypothesis by examining whether the amount of soil nitrogen supplied differentially influenced the performance (fitness, growth, and resource allocation strategies) of diploid and autotetraploid fireweed (Chamerion angustifolium). We found that soil nitrogen levels differentially impacted cytotype performance, and in general, diploids were favored under low nitrogen conditions, but this diploid advantage disappeared under nitrogen enrichment. Specifically, when nitrogen was scarce, diploids produced more seeds and allocated more biomass toward seed production relative to investment in plant biomass or total plant nitrogen than did tetraploids. As nitrogen supplied increased, such discrepancies between cytotypes disappeared. We also found that cytotype resource allocation strategies were differentially dependent on soil nitrogen, and that whereas diploids adopted resource allocation strategies that favored current season reproduction when nitrogen was limiting and future reproduction when nitrogen was more plentiful, tetraploids adopted resource allocation strategies that favored current season reproduction under nitrogen enrichment. Together these results suggest nitrogen enrichment could differentially affect cytotype performance, which could have implications for cytotypes’ ecological and evolutionary dynamics under a globally changing climate.     

Public Opinions about Overdiagnosis: A National Community Survey

BackgroundDespite evidence about the "modern epidemic" of overdiagnosis, and expanding disease definitions that medicalize more people, data are lacking on public views about these issues. Our objective was to measure public perceptions about overdiagnosis and views about financial ties of panels setting disease definitions.MethodsWe conducted a 15 minute Computer Assisted Telephone Interview with a randomly selected community sample of 500 Australians in January 2014. We iteratively developed and piloted a questionnaire, with a convenience sample (n=20), then with participants recruited by a research company (n=20). Questions included whether respondents had been informed about overdiagnosis; opinions on informing people; and views about financial ties among panels writing disease definitions.FindingsOur sample was generally representative, but included a higher proportion of females and seniors, typical of similar surveys. American Association for Public Opinion Research response rate was 20% and cooperation rate was 44%. Only 10% (95% CI 8%–13%) of people reported ever being told about overdiagnosis by a doctor. 18% (95% CI 11%–28%) of men who reported having prostate cancer screening, and 10% (95% CI 6%–15%) of women who reported having mammography said they were told about overdiagnosis. 93% (95% CI 90%–95%) agreed along with screening benefits, people should be informed about overdiagnosis. On panels setting disease definitions, 78% (95% CI 74%–82%) felt ties to pharmaceutical companies inappropriate, and 91% (95% CI 82%–100%) believed panels should have a minority or no members with ties. Limitations included questionnaire novelty and complexity.ConclusionsA small minority of Australians surveyed, including those reporting being screened for prostate or breast cancer, reported being informed of overdiagnosis; most believed people should be informed; and a majority felt it inappropriate that doctors with ties to pharmaceutical companies write disease definitions. Results suggest strategies to better inform people about overdiagnosis, and review disease definition processes, have significant public sympathy.     

Redox‐dependent lipoylation of mitochondrial proteins in Plasmodium falciparum

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H‐protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α‐ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H‐protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H‐protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H‐protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.     

A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.

Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.     

A Higher Mutational Burden in Females Supports a “Female Protective Model” in Neurodevelopmental Disorders

Increased male prevalence has been repeatedly reported in several neurodevelopmental disorders (NDs), leading to the concept of a “female protective model.” We investigated the molecular basis of this sex-based difference in liability and demonstrated an excess of deleterious autosomal copy-number variants (CNVs) in females compared to males (odds ratio [OR] = 1.46, p = 8 × 10⁻¹⁰) in a cohort of 15,585 probands ascertained for NDs. In an independent autism spectrum disorder (ASD) cohort of 762 families, we found a 3-fold increase in deleterious autosomal CNVs (p = 7 × 10⁻⁴) and an excess of private deleterious single-nucleotide variants (SNVs) in female compared to male probands (OR = 1.34, p = 0.03). We also showed that the deleteriousness of autosomal SNVs was significantly higher in female probands (p = 0.0006). A similar bias was observed in parents of probands ascertained for NDs. Deleterious CNVs (>400 kb) were maternally inherited more often (up to 64%, p = 10⁻¹⁵) than small CNVs < 400 kb (OR = 1.45, p = 0.0003). In the ASD cohort, increased maternal transmission was also observed for deleterious CNVs and SNVs. Although ASD females showed higher mutational burden and lower cognition, the excess mutational burden remained, even after adjustment for those cognitive differences. These results strongly suggest that females have an increased etiological burden unlinked to rare deleterious variants on the X chromosome. Carefully phenotyped and genotyped cohorts will be required for identifying the symptoms, which show gender-specific liability to mutational burden.     

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.     

Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death. These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.