A genetic assessment of a successful seagrass meadow (Posidonia australis) restoration trial

Seagrass meadows are in decline globally. Although numerous experimental methods have been implemented to restore meadows, few have been successful in the long term. Poor decisions on the sourcing of transplants from donor sites, including poor genetic integration and/or low genetic diversity, may impact on restoration success. However, despite evidence to suggest a positive association between genetic diversity and ecological resilience, there is usually little or no input from genetic data to inform on the genetic management of ecological restoration. Cockburn Sound has seen a 77% decline in seagrass cover since 1967. A transplant trial was conducted between 2004 and 2008 with sprigs of Posidonia australis being planted into a bare sand area. Survival was monitored annually, and in 2012, we compared genetic diversity in this transplant area with the original donor site. Genetic diversity in the restored meadow was very high and comparable to the donor site, with no genetic differentiation detected. The high level of genetic diversity and choice of site may have played an important role in the success of this restoration trial. The observed natural recruits around the site after establishment of transplants suggest that local restoration efforts may improve seafloor habitat and facilitate natural expansion of the meadow.     

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.     

Contrasting relatedness patterns in bottlenose dolphins (Tursiops sp.) with different alliance strategies

Male bottlenose dolphins (Tursiops sp.) in Shark Bay have one of the most complex male societies outside humans. Two broad mating strategies have been identified in males. In the first strategy, there are two types of alliances: stable 'first-order' pairs and trios that herd individual females in reproductive condition, and 'second-order' teams of two first-order alliances (five or six individuals) that join forces against rivals in contests for females. In the alternative strategy, a 'super-alliance' of ca. 14 individuals, males form pairs or trios to herd females, but in contrast to the stable alliances, these pairs and trios are highly labile. Here, we show that males in stable first-order alliances and the derived second-order alliances are often strongly related, so that they may gain inclusive fitness benefits from alliance membership. By contrast, members of the super-alliance are no more closely related than expected by chance. Further, the strength of the association of alliance partners within the super-alliance, as measured by an index of joint participation in consorting a female, was not correlated with their genetic relatedness. Thus, within one population and one sex, it appears that there may be simultaneous operation of more than one mode of group formation.     

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map,validated SNP assay resource,and high-throughput instrumentation system for large-scale genetic studies

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.     

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).     

Phylogeny of the core Malvales: evidence from ndhF sequence data

The monophyly of the group comprising the core malvalean families, Bombacaceae, Malvaceae, Sterculiaceae, and Tiliaceae, was recently confirmed by molecular studies, but the internal structure of this clade is poorly understood. In this study, we examined sequences of the chloroplast ndhF gene (aligned length 2226 bp) from 70 exemplars representing 35 of the 39 putative tribes of core Malvales. The monophyly of one traditional family, the Malvaceae, was supported in the trees resulting from these data, but the other three families, as traditionally circumscribed, are nonmonophyletic. In addition, the following relationships were well supported: (1) a clade, /Malvatheca, consisting of traditional Malvaceae and Bombacaceae (except some members of tribe Durioneae), plus Fremontodendron and Chiranthodendron, which are usually treated as Sterculiaceae; (2) a clade, /Malvadendrina, supported by a unique 21-bp (base pair) deletion and consisting of /Malvatheca, plus five additional subclades, including representatives of Sterculiaceae and Tiliaceae, and Durionieae; (3) a clade, /Byttneriina, with genera traditionally assigned to several tribes of Tiliaceae, plus exemplars of tribes Byttnerieae, Hermannieae, and Lasiopetaleae of Sterculiaceae. The most striking departures from traditional classifications are the following: Durio and relatives appear to be more closely related to Helicteres and Reevesia (Sterculiaceae) than to Bombacaceae; several genera traditionally considered as Bombacaceae (Camptostemon, Matisia, Phragmotheca, and Quararibea) or Sterculiaceae (Chiranthodendron and Fremontodendron) appear as sister lineages to the traditional Malvaceae; the traditional tribe Helictereae (Sterculiaceae) is polyphyletic; and Sterculiaceae and Tiliaceae, as traditionally circumscribed, represent polyphyletic groups that cannot sensibly be maintained with their traditional limits for purposes of classification. We discuss morphological characters and conclude that there has been extensive homoplasy in characters previously used to delineate major taxonomic groups in core Malvales. The topologies here also suggest that /Malvatheca do not have as a synapormophy monothecate anthers, as has been previously supposed but, instead, may be united by dithecate, transversely septate (polysporangiate) anthers, as found in basal members of both /Bombacoideae and /Malvoideae. Thus, “monothecate” anthers may have been derived at least twice, independently, within the /Bombacoideae (core Bombacaceae) and /Malvoideae (traditional Malvaceae).     

Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed "unlinkase," that recognizes and cleaves the unique 5' tyrosyl-RNA phosphodiester bond found at the 5' end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.     

The Role of Hormones in the Differences in the Incidence of Breast Cancer between Mongolia and the United Kingdom

Background

There are striking differences in breast cancer incidence between Asian and western women. Rates vary substantially within Asia also, with Mongolia''s even lower than China''s. These profound differences have been speculated to be due in part to diet, mediated by circulating hormone concentrations.

Methods

Sex steroid hormone concentrations were measured in women living in Ulaanbaatar, Mongolia and the United Kingdom (U.K.). Diet was obtained by interview and national survey data. Mean hormone differences were compared by country, and systematic variation by number of days since last menstrual period was modeled and adjusted for age and parity; difference in overall area under the curves was assessed.

Findings

The diet in Mongolia was higher in meat and dairy than in the U.K. Mean testosterone concentrations were 18.5% lower (p<0.0001) while estradiol concentrations were 19.1% higher (p = 0.02) in Mongolian than British women, adjusted for age and parity. Progesterone was almost 50% higher in Mongolian women (p = 0.04), particularly during the follicular phase and early luteal surge. Hormone concentrations generally were similar in Mongolian women born in Ulaanbaatar compared with those born in rural areas, although there was a decreasing progesterone trend by degree of westernization (rural Mongolia; urban Mongolia; U.K.). Mean hormone differences were similar when restricted to parous women, and with further adjustment for body mass index, height, and smoking status.

Interpretation

These data augment accumulating evidence that circulating estrogens are unlikely to explain reduced breast cancer rates in Asia compared with the west, and suggest casting a wider net with respect to biomarkers. Lower testosterone and higher progesterone in Mongolian women raise the possibility that these hormones may be important to consider. In addition, the almost exclusive dietary reliance of Mongolians on meat and dairy argues against beneficial effects of a low-fat diet on circulating hormones explaining international breast cancer differences.