INVESTIGATING PROCESSES OF NEOTROPICAL RAIN FOREST TREE DIVERSIFICATION BY EXAMINING THE EVOLUTION AND HISTORICAL BIOGEOGRAPHY OF THE PROTIEAE (BURSERACEAE)

Andean uplift and the collision of North and South America are thought to have major implications for the diversification of the Neotropical biota. However, few studies have investigated how these geological events may have influenced diversification. We present a multilocus phylogeny of 102 Protieae taxa (73% of published species), sampled pantropically, to test hypotheses about the relative importance of dispersal, vicariance, habitat specialization, and biotic factors in the diversification of this ecologically dominant tribe of Neotropical trees. Bayesian fossil‐calibrated analyses date the Protieae stem at 55 Mya. Biogeographic analyses reconstruct an initial late Oligocene/early Miocene radiation in Amazonia for Neotropical Protieae, with several subsequent late Miocene dispersal events to Central America, the Caribbean, Brazil's Atlantic Forest, and the Chocó. Regional phylogenetic structure results indicate frequent dispersal among regions throughout the Miocene and many instances of more recent regional in situ speciation. Habitat specialization to white sand or flooded soils was common, especially in Amazonia. There was one significant increase in diversification rate coincident with colonization of the Neotropics, followed by a gradual decrease consistent with models of diversity‐dependent cladogenesis. Dispersal, biotic interactions, and habitat specialization are thus hypothesized to be the most important processes underlying the diversification of the Protieae.     

Genome-wide association study of circulating estradiol, testosterone, and sex hormone-binding globulin in postmenopausal women

Genome-wide association studies (GWAS) have successfully identified common genetic variants that contribute to breast cancer risk. Discovering additional variants has become difficult, as power to detect variants of weaker effect with present sample sizes is limited. An alternative approach is to look for variants associated with quantitative traits that in turn affect disease risk. As exposure to high circulating estradiol and testosterone, and low sex hormone-binding globulin (SHBG) levels is implicated in breast cancer etiology, we conducted GWAS analyses of plasma estradiol, testosterone, and SHBG to identify new susceptibility alleles. Cancer Genetic Markers of Susceptibility (CGEMS) data from the Nurses' Health Study (NHS), and Sisters in Breast Cancer Screening data were used to carry out primary meta-analyses among ~1600 postmenopausal women who were not taking postmenopausal hormones at blood draw. We observed a genome-wide significant association between SHBG levels and rs727428 (joint β = -0.126; joint P = 2.09 × 10(-16)), downstream of the SHBG gene. No genome-wide significant associations were observed with estradiol or testosterone levels. Among variants that were suggestively associated with estradiol (P<10(-5)), several were located at the CYP19A1 gene locus. Overall results were similar in secondary meta-analyses that included ~900 NHS current postmenopausal hormone users. No variant associated with estradiol, testosterone, or SHBG at P<10(-5) was associated with postmenopausal breast cancer risk among CGEMS participants. Our results suggest that the small magnitude of difference in hormone levels associated with common genetic variants is likely insufficient to detectably contribute to breast cancer risk.     

Evidence for the functions of surface‐active behaviors in humpback whales (Megaptera novaeangliae)

As part of their social sound repertoire, migrating humpback whales (Megaptera novaeangliae) perform a large variety of surface‐active behaviors, such as breaching and repetitive slapping of the pectoral fins and tail flukes; however, little is known about what factors influence these behaviors and what their functions might be. We investigated the potential functions of surface‐active behaviors in humpback whale groups by examining the social and environmental contexts in which they occurred. Focal observations on 94 different groups of whales were collected in conjunction with continuous acoustic monitoring, and data on the social and environmental context of each group. We propose that breaching may play a role in communication between distant groups as the probability of observing this behavior decreased significantly when the nearest whale group was within 4,000 m compared to beyond 4,000 m. Involvement in group interactions, such as the splitting of a group or a group joining with other whales, was an important factor in predicting the occurrence of pectoral, fluke, and peduncle slapping, and we suggest that they play a role in close‐range or within‐group communication. This study highlights the potentially important and diverse roles of surface‐active behaviors in the communication of migrating humpback whales.     

Golden bananas in the field: elevated fruit pro‐vitamin A from the expression of a single banana transgene

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro‐vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA‐biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β‐carotene equivalent (β‐CE) in the fruit. Expression of a Fe'i banana‐derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β‐CE . Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1‐aminocyclopropane‐1‐carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild‐type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate‐limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.     

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.     

Co-circulating microorganisms in questing Ixodes scapularis nymphs in Maryland

Ixodes scapularis can be infected with Borrelia burgdorferi, Anaplasma phagocytophilum, Bartonella spp., Babesia microti, and Rickettsia spp., including spotted-fever group Rickettsia. As all of these microorganisms have been reported in Maryland, the potential for these ticks to have concurrent infections exists in this region. To assess the frequency of these complex infections, 348 I. scapularis nymphs collected in 2003 were screened for these microorganisms by PCR with positives being confirmed by DNA sequencing. Borrelia burgdorferi was detected in 14.7% of nymphs. Anaplasma phagocytophilum (0.3%), Rickettsia spp. (19.5%), and an uncategorized agent (0.9%) was also detected. Dual infections were detected with B. burgdorferi and Rickettsia spp. as well as a triple infection with B. burgdorferi, Rickettsia spp., and an uncategorized agent. Infections with B. burgdorferi and Rickettsia spp. were statistically independent of one another. However, infection with B. burgdorferi and any one of these other microorganisms appears to occur more frequently than by chance alone, probably as a result of shared enzootic cycles. This study confirms that multiple microorganisms co-circulate with B. burgdorferi in I. scapularis in Maryland and demonstrates that Rickettsia spp. and B. burgdorferi circulate independently and at nearly equal frequencies, while A. phagocytophilum and other unrecognized organisms are less common.     

Interactions between the environmental pathogen Listeria monocytogenes and a free-living protozoan (Acanthamoeba castellanii)

Listeria monocytogenes can cause severe disease in animal hosts, but it has no recognized animal host reservoir. We tested the hypothesis that L. monocytogenes retains virulence traits to survive predation by amoebae and that listeriolysin O plays a crucial role in this process. Co-culturing of L. monocytogenes and Acanthamoeba castellanii demonstrated that L. monocytogenes does not actively kill amoebae, but in the presence of amoebae, high bacterial population densities can be maintained over a period of at least 96 h. A gentamicin protection assay demonstrated that there is no significant difference in the ability to survive predation between serovars (4b versus 1/2a and 1/2c; P  = 0.08) and between five species of Listeria ( P  = 0.14). Three of these species do not harbour the hly gene responsible for listeriolysin O production. A hly knockout strain had poorer survival compared with the parental strain ( P  = 0.04 at 24 h; P  = 0.04 at 48 h; P  = 0.02 at 72 h) and electron microscopy was consistent with a wild-type strain being able to escape the phagosome whereas the hly knockout strain did not appear to have this ability. Thus, while there is weak evidence that listeriolysin O can contribute to improved survival after ingestion by amoebae, listeriolysin O does not appear to provide a significant selective advantage under the conditions of this study.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

The origin and diversification of angiosperms

The angiosperms, one of five groups of extant seed plants, are the largest group of land plants. Despite their relatively recent origin, this clade is extremely diverse morphologically and ecologically. However, angiosperms are clearly united by several synapomorphies. During the past 10 years, higher-level relationships of the angiosperms have been resolved. For example, most analyses are consistent in identifying Amborella, Nymphaeaceae, and Austrobaileyales as the basalmost branches of the angiosperm tree. Other basal lineages include Chloranthaceae, magnoliids, and monocots. Approximately three quarters of all angiosperm species belong to the eudicot clade, which is strongly supported by molecular data but united morphologically by a single synapomorphy-triaperturate pollen. Major clades of eudicots include Ranunculales, which are sister to all other eudicots, and a clade of core eudicots, the largest members of which are Saxifragales, Caryophyllales, rosids, and asterids. Despite rapid progress in resolving angiosperm relationships, several significant problems remain: (1) relationships among the monocots, Chloranthaceae, magnoliids, and eudicots, (2) branching order among basal eudicots, (3) relationships among the major clades of core eudicots, (4) relationships within rosids, (5) relationships of the many lineages of parasitic plants, and (6) integration of fossils with extant taxa into a comprehensive tree of angiosperm phylogeny.