Initial descriptions of the HIV engagement continuum are limited by short-term follow-up and incomplete data. We evaluated engagement in a newly HIV-diagnosed cohort. Our goals were to assess long-term engagement-in-care, evaluate the effects of out-of-state migration on engagement estimates, and determine whether engagement has improved in more recently diagnosed individuals. This is a retrospective cohort study of individuals newly HIV-diagnosed at two large HIV care centers in the Denver metropolitan area from 2005 to 2009. Clinical data were obtained from three public HIV providers and two clinical trial groups. For statewide evaluation, we used mandated laboratory reporting databases for CD4 lymphocyte counts and HIV-1 RNA levels. From 2005 to 2009, 615 individuals were diagnosed with HIV. By 18 months after HIV diagnosis, 84% of the cohort had linked to care, 73% were retained in care, 49% were prescribed antiretroviral therapy, and 36% had viral suppression. By 5 years after HIV diagnosis, 55% of the cohort were retained in care, 37% had viral suppression, 15% had moved out of state, and 3% were deceased. When censoring for outmigration and death, 66% of the cohort were retained in care and 45% of the cohort had viral suppression 5 years after HIV diagnosis. Engagement-in-care 18 months after diagnosis was better in individuals diagnosed more recently. Retention in care declined while viral suppression increased over time after HIV diagnosis. Accounting for outmigration and death significantly increased estimates of engagement-in-care. Performance in the engagement continuum 18 months after diagnosis improved significantly in individuals more recently diagnosed with HIV.
Randomized controlled trials are considered the “gold standard” for scientific rigor in the assessment of benefits and harms of interventions in healthcare. They may not always be feasible, however, when evaluating quality improvement interventions in real-world healthcare settings. Non-randomized controlled trials (NCTs) are designed to answer questions of effectiveness of interventions in routine clinical practice to inform a decision or process. The on-off NCT design is a relatively new design where participant allocation is by alternation. In alternation, eligible patients are allocated to the intervention “on” or control “off ” groups in time series dependent sequential clusters.
We used two quality improvement studies undertaken in a Canadian primary care setting to illustrate the features of the on-off design. We also explored the perceptions and experiences of healthcare providers tasked with implementing the on-off study design.
Results and discussion
The on-off design successfully allocated patients to intervention and control groups. Imbalances between baseline variables were attributed to chance, with no detectable biases. However, healthcare providers’ perspectives and experiences with the design in practice reveal some conflict. Specifically, providers described the process of allocating patients to the off group as unethical and immoral, feeling it was in direct conflict with their professional principle of providing care for all. The degree of dissatisfaction seemed exacerbated by: 1) the patient population involved (e.g., patient population viewed as high-risk (e.g., depressed or suicidal)), 2) conducting assessments without taking action (e.g., administering the PHQ-9 and not acting on the results), and 3) the (non-blinded) allocation process.
Alternation, as in the on-off design, is a credible form of allocation. The conflict reported by healthcare providers in implementing the design, while not unique to the on-off design, may be alleviated by greater emphasis on the purpose of the research and having research assistants allocate patients and collect data instead of the healthcare providers implementing the trial. In addition, consultation with front-line staff implementing the trials with an on-off design on appropriateness to the setting (e.g., alignment with professional values and the patient population served) may be beneficial.
Health Eating and Active Living with Diabetes: ClinicalTrials.gov identifier: NCT00991380 Date registered: 7 October 2009.
Controlled trial of a collaborative primary care team model for patients with diabetes and depression: Clintrials.gov Identifier: NCT01328639 Date registered: 30 March 2011.
Alternation; Controlled trials; Quality improvement; Primary healthcare
CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.
•First structure of consecutive scavenger receptor cysteine rich domains in CD6•Structure of the two N-terminal domains of CD166 which is the ligand for CD6•Mapping binding sites on CD6 and CD166•Insight into how CD6 and its interactions are perturbed by polymorphisms and mAbs
Chappell et al. present structures of the T-cell surface receptor, CD6, the first of consecutive scavenger receptor cysteine rich domains and its ligand, CD166. The structures give insight into how CD6 and its interactions are perturbed by competition between homophilic and heterophilic interactions, SNPs, and mAbs.
Molecular crowding plays a significant role in regulating molecular conformation in cellular environments. It is also likely to be important wherever high molecular densities are required, for example in surface-phase studies, in which molecular densities generally far exceed those observed in solution. Using on-surface circular dichroism (CD) spectroscopy, we have investigated the structure of a synthetic peptide assembled into a highly packed monolayer. The immobilized peptide undergoes a structural transition between α-helical and random coil conformation upon changes in pH and ionic concentration, but critically the threshold for conformational change is altered dramatically by molecular crowding within the peptide monolayer. This study highlights the often overlooked role molecular crowding plays in regulating molecular structure and function in surface-phase studies of biological molecules.
coiled coil; molecular crowding; circular dichroism; peptide conformation; surface analysis
Fishing and pollution are chronic stressors that can prolong recovery of coral reefs and contribute to ecosystem decline. While this premise is generally accepted, management interventions are complicated because the contributions from individual stressors are difficult to distinguish. The present study examined the extent to which fishing pressure and pollution predicted progress towards the Micronesia Challenge, an international conservation strategy initiated by the political leaders of 6 nations to conserve at least 30% of marine resources by 2020. The analyses were rooted in a defined measure of coral-reef-ecosystem condition, comprised of biological metrics that described functional processes on coral reefs. We report that only 42% of the major reef habitats exceeded the ecosystem-condition threshold established by the Micronesia Challenge. Fishing pressure acting alone on outer reefs, or in combination with pollution in some lagoons, best predicted both the decline and variance in ecosystem condition. High variances among ecosystem-condition scores reflected the large gaps between the best and worst reefs, and suggested that the current scores were unlikely to remain stable through time because of low redundancy. Accounting for the presence of marine protected area (MPA) networks in statistical models did little to improve the models’ predictive capabilities, suggesting limited efficacy of MPAs when grouped together across the region. Yet, localized benefits of MPAs existed and are expected to increase over time. Sensitivity analyses suggested that (i) grazing by large herbivores, (ii) high functional diversity of herbivores, and (iii) high predator biomass were most sensitive to fishing pressure, and were required for high ecosystem-condition scores. Linking comprehensive fisheries management policies with these sensitive metrics, and targeting the management of pollution, will strengthen the Micronesia Challenge and preserve ecosystem services that coral reefs provide to societies in the face of climate change.
Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.
Most animal cells contain structures known as centrioles. Typically, a cell that is not dividing contains a pair of centrioles. But when a cell prepares to divide, the centrioles are duplicated. The two pairs of centrioles then organize the scaffolding that shares the genetic material equally between the newly formed cells at cell division.
Centriole assembly is tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Centrioles likely contain several hundred proteins, but only a few of these are strictly needed for centriole assembly. New centrioles usually assemble from a cartwheel-like arrangement of proteins, which includes a protein called SAS-6. Previous work has suggested that in the fruit fly Drosophila melanogaster, Sas-6 can only form this cartwheel when another protein called Ana2 is also present, but the details of this process are unclear.
Now, Cottee, Muschalik et al. have investigated potential features in the Ana2 protein that might be important for centriole assembly. These experiments revealed that a region in the Ana2 protein, called the ‘central coiled-coil domain’, is required to target Ana2 to centrioles. Furthermore, purified coiled-coil domains were found to bind together in groups of four (called tetramers). Cottee, Muschalik et al. then used a technique called X-ray crystallography to work out the three-dimensional structure of one of these tetramers and part of the Sas-6 protein with a high level of detail. These structures confirmed that Sas-6 proteins also associate with each other.
When fruit flies were engineered to produce either Ana2 or Sas-6 proteins that cannot self-associate, the flies' cells were unable to efficiently make centrioles. Furthermore, an independent study by Rogala et al. found similar results for a protein that is related to Ana2: a protein called SAS-5 from the microscopic worm Caenorhabditis elegans.
Further work is needed to understand how Sas-6 and Ana2 work with each other to form the cartwheel-like arrangement at the core of centrioles.
centriole; centrosome; ultra-high resolution structure; SAS-6; Ana2; STIL; D. melanogaster; human
Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ.
This paper describes new instrumentation developments at the LCLS for materials studies using THz laser excitation and resonant soft X-ray scattering.
This paper describes the development of new instrumentation at the Linac Coherent Light Source for conducting THz excitation experiments in an ultra high vacuum environment probed by soft X-ray diffraction. This consists of a cantilevered, fully motorized mirror system which can provide 600 kV cm−1 electric field strengths across the sample and an X-ray detector that can span the full Ewald sphere with in-vacuum motion. The scientific applications motivated by this development, the details of the instrument, and spectra demonstrating the field strengths achieved using this newly developed system are discussed.
FEL; X-ray diffraction; THz; instrumentation
Health care data included in clinical data repositories (CDRs) are increasingly used for quality reporting, business analytics and research; however, extended clinical data from interprofessional practice are seldom included. With the increasing emphasis on care coordination across settings, CDRs need to include data from all clinicians and be harmonized to understand the impact of their collaborative efforts on patient safety, effectiveness and efficiency. This study characterizes the extended clinical data derived from EHR flowsheet data that is available in the University of Minnesota’s CDR and describes a process for creating an ontology that organizes that data so that it is more useful and accessible to researchers. The process is illustrated using a pressure ulcer ontology and compares ease of finding concepts in i2b2 for different data organization approaches. The challenges of the manual process and difficulties combining similar concepts are discussed.
Malaria remains a major health concern for a large percentage of the world’s population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitos. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block P. falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.
Plasmodium falciparum; PfCDPK4; PfCDPK1; Pyrazolopyrimidine; Exflagellation
This clinical trial identifies a significantly earlier time to virologic failure in women randomized to atazanavir/ritonavir compared to women randomized to efavirenz.
Background. We aimed to evaluate treatment responses to atazanavir plus ritonavir (ATV/r) or efavirenz (EFV) in initial antiretroviral regimens among women and men, and determine if treatment outcomes differ by sex.
Methods. We performed a randomized trial of open-label ATV/r or EFV combined with abacavir/lamivudine (ABC/3TC) or tenofovir/emtricitabine (TDF/FTC) in 1857 human immunodeficiency virus type 1–infected, treatment-naive persons enrolled between September 2005 and November 2007 at 59 sites in the United States and Puerto Rico. Associations of sex with 3 primary study endpoints of time to virologic failure, safety, and tolerability events were analyzed using Cox proportional hazards models. Model-based population pharmacokinetic analysis was performed using nonlinear mixed effects modeling (NONMEM version VII).
Results. Of 1857 participants, 322 were women. Women assigned to ATV/r had a higher risk of virologic failure with either nucleoside reverse transcriptase inhibitor backbone than women assigned to EFV, or men assigned to ATV/r. The effects of ATV/r and EFV upon safety and tolerability risk did not differ significantly by sex. With ABC/3TC, women had a significantly higher (32%) safety risk compared to men; with TDF/FTC, the safety risk was 20% larger for women compared to men, but not statistically significant. Women had slower ATV clearance and higher predose levels of ATV compared to men. Self-reported adherence did not differ significantly by sex.
Conclusions. This is the first randomized clinical trial to identify a significantly earlier time to virologic failure in women randomized to ATV/r compared to women randomized to EFV. This finding has important clinical implications given that boosted protease inhibitors are often favored over EFV in women of childbearing potential.
Clinical Trials Registration NCT00118898.
sex; atazanavir; efavirenz; abacavir; tenofovir
Background: The hemoprotein TsdA catalyzes the oxidation of two thiosulfate molecules to form tetrathionate.
Results: The mechanism of TsdA has been probed using biochemical and structural methods.
Conclusion: The TsdA reaction proceeds via a cysteine S-thiosulfonate intermediate formed on a cysteine ligand to the active site heme.
Significance: TsdA provides a catalytic model for other members of the SoxA enzyme family.
Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.
Bacterial Metabolism; Cytochrome; Electron Transport; Heme; Metalloenzyme; Sox System; Sulfur Oxidizing Bacteria; Thiosulfate Dehydrogenase
Background and Aims
According to the Grant–Stebbins model of pollinator-driven divergence, plants that disperse beyond the range of their specialized pollinator may adapt to a new pollination system. Although this model provides a compelling explanation for pollination ecotype formation, few studies have directly tested its validity in nature. Here we investigate the distribution and pollination biology of several subspecies of the shrub Erica plukenetii from the Cape Floristic Region in South Africa. We analyse these data in a phylogenetic context and combine these results with information on pollinator ranges to test whether the evolution of pollination ecotypes is consistent with the Grant–Stebbins model.
Methods and Key Results
Pollinator observations showed that the most common form of E. plukenetii with intermediate corolla length is pollinated by short-billed Orange-breasted sunbirds. Populations at the northern fringe of the distribution are characterized by long corollas, and are mainly pollinated by long-billed Malachite sunbirds. A population with short corollas in the centre of the range was mainly pollinated by insects, particularly short-tongued noctuid moths. Bird exclusion in this population did not have an effect on fruit set, while insect exclusion reduced fruit set. An analysis of floral scent across the range, using coupled gas chromatography–mass spectrometry, showed that the scent bouquets of flowers from moth-pollinated populations are characterized by a larger number of scent compounds and higher emission rates than those in bird-pollinated populations. This was also reflected in clear separation of moth- and bird-pollinated populations in a two-dimensional phenotype space based on non-metric multidimensional scaling analysis of scent data. Phylogenetic analyses of chloroplast and nuclear DNA sequences strongly supported monophyly of E. plukenetii, but not of all the subspecies. Reconstruction of ancestral character states suggests two shifts from traits associated with short-billed Orange-breasted sunbird pollination: one towards traits associated with moth pollination, and one towards traits associated with pollination by long-billed Malachite sunbirds. The latter shift coincided with the colonization of Namaqualand in which Orange-breasted sunbirds are absent.
Conclusions Erica plukenetii
is characterized by three pollination ecotypes, but only the evolutionary transition from short- to long-billed sunbird pollination can be clearly explained by the Grant–Stebbins model. Corolla length is a key character for both ecotype transitions, while floral scent emission was important for the transition from bird to moth pollination.
Helicoverpa armigera; speciation; benzene propanoids; phylogeny; niche; floral scent; bird pollination; floral trait; ancestral character state; Erica plukenetii; pollination ecotype; Cape flora
Background and Aims
Floral diversification driven by shifts between pollinators has been one of the key explanations for the radiation of angiosperms. According to the Grant–Stebbins model of pollinator-driven speciation, these shifts result in morphologically distinct ‘ecotypes’ which may eventually become recognizable as species. The current circumscription of the food-deceptive southern African orchid Eulophia parviflora encompasses a highly variable monophyletic species complex. In this study, two forms were identified within this complex that differ in distribution, floral morphology, scent chemistry and phenology, and a test was made of whether these differences represent adaptations for different pollinators.
Methods and Results
Multivariate analysis of floral and vegetative traits revealed that there are at least two discrete morphological forms in the species complex. Field observations revealed that each form is pollinated by a different insect species, and thus represent distinct ecotypes. The early-flowering coastal form which has long spurs and floral scent dominated by sesquiterpene compounds is pollinated exclusively by the long-tongued bee Amegilla fallax (Apidae, Anthophorinae), while the late-flowering inland form with short spurs and floral scent dominated by benzenoid compounds is pollinated exclusively by the beetle Cyrtothyrea marginalis (Cetoniinae; Scarabaeidae). Choice experiments in a Y-maze olfactometer showed that beetles are preferentially attracted to the scent of the short-spurred form. A spur-shortening experiment showed that long spurs are required for effective pollination of the bee-pollinated form. Although it was initially thought likely that divergence occurred across a geographical pollinator gradient, plants of the long-spurred form were effectively pollinated when transplanted to an inland locality outside the natural coastal range of this form. Thus, the underlying geographical basis for the evolution of ecotypes in the E. parviflora complex remains uncertain, although early flowering in the long-spurred form to exploit the emergence of naïve bees may restrict this form to coastal areas where there is no frost that would damage flower buds. Later flowering of the short-spurred form coincides closely with the emergence of the pollinating beetles following winter frosts.
This study identifies a shift between bee and beetle pollination as the main driver of floral divergence in an orchid species complex. Floral scent and spur length appear to be key traits in mediating this evolutionary transition.
Grant–Stebbins model; pollinator-driven speciation; pollination ecotypes; scent; Eulophia; Orchidaceae; phenology; beetle pollination; Cetoniinae; bee pollination; Y-maze olfactometer; Amegilla
The hypothesis that pollinators have been important drivers of angiosperm diversity dates back to Darwin, and remains an important research topic today. Mounting evidence indicates that pollinators have the potential to drive diversification at several different stages of the evolutionary process. Microevolutionary studies have provided evidence for pollinator-mediated floral adaptation, while macroevolutionary evidence supports a general pattern of pollinator-driven diversification of angiosperms. However, the overarching issue of whether, and how, shifts in pollination system drive plant speciation represents a critical gap in knowledge. Bridging this gap is crucial to fully understand whether pollinator-driven microevolution accounts for the observed macroevolutionary patterns. Testable predictions about pollinator-driven speciation can be derived from the theory of ecological speciation, according to which adaptation (microevolution) and speciation (macroevolution) are directly linked. This theory is a particularly suitable framework for evaluating evidence for the processes underlying shifts in pollination systems and their potential consequences for the evolution of reproductive isolation and speciation.
This Viewpoint paper focuses on evidence for the four components of ecological speciation in the context of plant-pollinator interactions, namely (1) the role of pollinators as selective agents, (2) floral trait divergence, including the evolution of ‘pollination ecotypes‘, (3) the geographical context of selection on floral traits, and (4) the role of pollinators in the evolution of reproductive isolation. This Viewpoint also serves as the introduction to a Special Issue on Pollinator-Driven Speciation in Plants. The 13 papers in this Special Issue range from microevolutionary studies of ecotypes to macroevolutionary studies of historical ecological shifts, and span a wide range of geographical areas and plant families. These studies further illustrate innovative experimental approaches, and they employ modern tools in genetics and floral trait quantification. Future advances to the field require better quantification of selection through male fitness and pollinator isolation, for instance by exploiting next-generation sequencing technologies. By combining these new tools with strategically chosen study systems, and smart experimental design, we predict that examples of pollinator-driven speciation will be among the most widespread and compelling of all cases of ecological speciation.
Geography; floral odour; flower colour; flower shape; specialization; reproductive isolation; next-generation sequencing; pollination ecotypes; pollination; nectar tube; adaptation; natural selection; geographical mosaic of selection; Grant–Stebbins model
Background. Childhood arterial ischemic stroke (AIS) is rare and may be difficult to diagnose. Management of acute stroke in any age group is time sensitive, so awareness of the manifestations and appropriate diagnostic procedures for pediatric AIS is vital to establishing care. We present a pediatric case of arterial ischemic stroke that presented to the emergency department (ED) after two seizures. Case Report. A five-year-old female with an existing seizure disorder presented to a pediatric ED after having two seizures. Postictal upon arrival, she underwent a computed tomography (CT) scan of her head. Family reported that she had complained of a severe headache and vomited; her seizures were described as different from those she had experienced in the past. Loss of grey white matter differentiation on the CT warranted magnetic resonance imaging (MRI), which demonstrated a right-sided stroke. After a complicated course in the hospital, the patient was discharged to a rehabilitation hospital. Why Should an Emergency Physician Be Aware of This? It is important that emergency physicians recognize that a seizure may be the initial symptom of a pediatric stroke regardless of an established seizure history. Pediatric seizures are relatively common; however consideration of the diagnosis of pediatric stroke may prevent unnecessary delays in treatment.
We present an efficient solid phase synthesis methodology that provides easy access to a range of functionalised long-chain alkanethiol–oligoethyleneglycols that form well-defined self-assembled monolayers on gold and are compatible with pre- or post-assembly conjugation of (bio)molecules. We demonstrate the versatility of our synthetic route by synthesising LCAT–OEGs with a range of functional moieties, including peptides, electro-active redox groups, chemical handles for post-assembly conjugation of (bio)molecules, and demonstrate the application of our LCAT–OEG monolayers in immunosensing, where they show good biocompatibility with minimal biofouling.
Background and Aims
The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production.
The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species.
Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification.
Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.
Disa; Disinae; Orchidaceae; orchid; deceit pollination; modified stoma; nectar; nectary; reward; rewardless; evolution; functional convergence
An outstanding feature of the orchid family is that approximately 30–40% of the species have non-rewarding flowers and deploy various modes of deception to attract pollinators, whereas the remaining species engage in pollination mutualisms based on provision of floral rewards. Here, we explore the direction, frequency and reversibility of transitions between deceptive and rewarding pollination systems in the radiation of the large African genus Disa, and test whether these transitions had consequences for diversification. By optimizing nectar production data for 111 species on a well-resolved phylogeny, we confirmed that floral deception was the ancestral condition and that nectar production evolved at least nine times and was lost at least once. Transitions to nectar production first occurred ca 17 million years ago but did not significantly affect either speciation or extinction rates. Nectar evolved independently of a spur, which was lost and gained multiple times. These results show that nectar production can be a highly labile trait and highlight the need for further studies of the genetic architecture of nectar production and the selective factors underlying transitions between deception and mutualism.
diversification rates; floral deception; mimicry; nectary; phylogenetic signal; pollination
Background: PhoD family enzymes liberate phosphate from organic compounds.
Results: The structure of PhoD reveals an active site with one Fe3+ and two Ca2+ ions.
Conclusion: PhoD represents a new class of phosphatase related to purple acid phosphatases.
Significance: The requirement of PhoD for iron ions may limit bacterial phosphate acquisition in low iron environments.
The PhoD family of extra-cytoplasmic phosphodiesterases are among the most commonly occurring bacterial phosphatases. The exemplars for this family are the PhoD protein of Bacillus subtilis and the phospholipase D of Streptomyces chromofuscus. We present the crystal structure of B. subtilis PhoD. PhoD is most closely related to purple acid phosphatases (PAPs) with both types of enzyme containing a tyrosinate-ligated Fe3+ ion. However, the PhoD active site diverges from that found in PAPs and uses two Ca2+ ions instead of the single extra Fe2+, Mn2+, or Zn2+ ion present in PAPs. The PhoD crystals contain a phosphate molecule that coordinates all three active site metal ions and that is proposed to represent a product complex. A C-terminal helix lies over the active site and controls access to the catalytic center. The structure of PhoD defines a new phosphatase active site architecture based on Fe3+ and Ca2+ ions.
Bacterial Metabolism; Catalysis; Metalloenzyme; Phosphatase; Structural Biology
Background and Aims
Ontogenetic patterns of odour emissions and heating associated with plant reproductive structures may have profound effects on insect behaviour, and consequently on pollination. In some cycads, notably Macrozamia, temporal changes in emission of specific odour compounds and temperature have been interpreted as a ‘push–pull’ interaction in which pollinators are either attracted or repelled according to the concentration of the emitted volatiles. To establish which mechanisms occur in the large Encephalartos cycad clade, the temporal patterns of volatile emissions, heating and pollinator activity of cones of Encephalartos villosus in the Eastern Cape (EC) and KwaZulu Natal (KZN) of South Africa were investigated.
Methods and Key Results
Gas chromatography–mass spectrometry (GC-MS) analyses of Encephalartos villosus cone volatiles showed that emissions, dominated by eucalyptol and 2-isopropyl-3-methoxypyrazine in EC populations and (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene in the KZN populations, varied across developmental stages but did not vary significantly on a daily cycle. Heating in male cones was higher at dehiscence than during pre- and post-dehiscence, and reached a maximum at about 1830 h when temperatures were between 7·0 and 12·0 °C above ambient. Daily heating of female cones was less pronounced and reached a maximum at about 1345 h when it was on average between 0·9 and 3·0 °C above ambient. Insect abundance on male cones was higher at dehiscence than at the other stages and significantly higher in the afternoon than in the morning and evening.
There are pronounced developmental changes in volatile emissions and heating in E. villosus cones, as well as strong daily changes in thermogenesis. Daily patterns of volatile emissions and pollinator abundance in E. villosus are different from those observed in some Macrozamia cycads and not consistent with the push–pull pattern as periods of peak odour emission do not coincide with mass exodus of insects from male cones.
Encephalartos villosus; Zamiaceae; cycads; monoterpenes; developmental stages; nitrogen-containing compounds; unsaturated hydrocarbons; gas chromatography–mass spectrometry; push–pull; odour emission; floral volatiles; thermogensis; pollination
We identified ticks submitted by the public from 2008 through 2012 in Ontario, Canada, and tested blacklegged ticks Ixodes scapularis for Borrelia burgdorferi and Anaplasma phagocytophilum. Among the 18 species of ticks identified, I. scapularis, Dermacentor variabilis, Ixodes cookei and Amblyomma americanum represented 98.1% of the 14,369 ticks submitted. Rates of blacklegged tick submission per 100,000 population were highest in Ontario's Eastern region; D. variabilis in Central West and Eastern regions; I. cookei in Eastern and South West regions; and A. americanum had a scattered distribution. Rates of blacklegged tick submission per 100,000 population were highest from children (0–9 years old) and older adults (55–74 years old). In two health units in the Eastern region (i.e., Leeds, Grenville & Lanark District and Kingston-Frontenac and Lennox & Addington), the rate of submission for engorged and B. burgdorferi-positive blacklegged ticks was 47× higher than the rest of Ontario. Rate of spread for blacklegged ticks was relatively faster and across a larger geographic area along the northern shore of Lake Ontario/St. Lawrence River, compared with slower spread from isolated populations along the northern shore of Lake Erie. The infection prevalence of B. burgdorferi in blacklegged ticks increased in Ontario over the study period from 8.4% in 2008 to 19.1% in 2012. The prevalence of B. burgdorferi-positive blacklegged ticks increased yearly during the surveillance period and, while increases were not uniform across all regions, increases were greatest in the Central West region, followed by Eastern and South West regions. The overall infection prevalence of A. phagocytophilum in blacklegged ticks was 0.3%. This study provides essential information on ticks of medical importance in Ontario, and identifies demographic and geographic areas for focused public education on the prevention of tick bites and tick-borne diseases.
The individual contribution of natural disturbances, localized stressors, and environmental regimes upon longer-term reef dynamics remains poorly resolved for many locales despite its significance for management. This study examined coral reefs in the Commonwealth of the Northern Mariana Islands across a 12-year period that included elevated Crown-of-Thorns Starfish densities (COTS) and tropical storms that were drivers of spatially-inconsistent disturbance and recovery patterns. At the island scale, disturbance impacts were highest on Saipan with reduced fish sizes, grazing urchins, and water quality, despite having a more favorable geological foundation for coral growth compared with Rota. However, individual drivers of reef dynamics were better quantified through site-level investigations that built upon island generalizations. While COTS densities were the strongest predictors of coral decline as expected, interactive terms that included wave exposure and size of the overall fish assemblages improved models (R2 and AIC values). Both wave exposure and fish size diminished disturbance impacts and had negative associations with COTS. However, contrasting findings emerged when examining net ecological change across the 12-year period. Wave exposure had a ubiquitous, positive influence upon the net change in favorable benthic substrates (i.e. corals and other heavily calcifying substrates, R2 = 0.17 for all reeftypes grouped), yet including interactive terms for herbivore size and grazing urchin densities, as well as stratifying by major reeftypes, substantially improved models (R2 = 0.21 to 0.89, lower AIC scores). Net changes in coral assemblages (i.e., coral ordination scores) were more sensitive to herbivore size or the water quality proxy acting independently (R2 = 0.28 to 0.44). We conclude that COTS densities were the strongest drivers of coral decline, however, net ecological change was most influenced by localized stressors, especially herbivore sizes and grazing urchin densities. Interestingly, fish size, rather than biomass, was consistently a better predictor, supporting allometric, size-and-function relationships of fish assemblages. Management implications are discussed.
Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems.
Random digit dialing is often used in public health research initiatives to accrue and establish a study sample; however few studies have fully described the utility of this approach. The primary objective of this paper was to describe the implementation and utility of using random digit dialing and Computer Assisted Telephone Interviewing (CATI) for sampling, recruitment and data collection in a large population-based study of older adults [Alberta Older Adult Health Behavior (ALERT) study].
Using random digit dialing, older adults (> = 55 years) completed health behavior and outcome and demographic measures via CATI. After completing the CATI, participants were invited to receive a step pedometer and waist circumference tape measure via mail to gather objectively derived ambulatory activity and waist circumference assessments.
Overall, 36,000 telephone numbers were called of which 7,013 were deemed eligible for the study. Of those, 4,913 (70.1%) refused to participate in the study and 804 (11.4%) participants were not included due to a variety of call dispositions (e.g., difficult to reach, full quota for region). A total of 1,296 participants completed telephone interviews (18.5% of those eligible and 3.6% of all individuals approached). Overall, 22.8% of households did not have an age 55+ resident and 13.6% of individuals refused to participate, Average age was 66.5 years, and 43% were male. A total of 1,081 participants (83.4%) also submitted self-measured ambulatory activity (i.e., via step pedometer) and anthropometric data (i.e., waist circumference). With the exception of income (18.7%), the rate of missing data for demographics, health behaviors, and health measures was minimal (<1%).
Older adults are willing to participate in telephone-based health surveys when randomly contacted. Researchers can use this information to evaluate the feasibility and the logistics of planned studies using a similar population and study design.
Health behavior; Computer-assisted telephone interviews (CATI); Random digit dialing; Response rate; Older adults