A Cyanobacteria Enriched Layer of Shark Bay Stromatolites Reveals a New Acaryochloris Strain Living in Near Infrared Light.

The genus Acaryochloris is unique among phototrophic organisms due to the dominance of chlorophyll d in its photosynthetic reaction centres and light-harvesting proteins. This allows Acaryochloris to capture light energy for photosynthesis over an extended spectrum of up to ~760 nm in the near infra-red (NIR) spectrum. Acaryochloris sp. has been reported in a variety of ecological niches, ranging from polar to tropical shallow aquatic sites. Here, we report a new Acarychloris strain isolated from an NIR-enriched stratified microbial layer 4-6 mm under the surface of stromatolite mats located in the Hamelin Pool of Shark Bay, Western Australia. Pigment analysis by spectrometry/fluorometry, flow cytometry and spectral confocal microscopy identifies unique patterns in pigment content that likely reflect niche adaption. For example, unlike the original A. marina species (type strain MBIC11017), this new strain, Acarychloris LARK001, shows little change in the chlorophyll d/a ratio in response to changes in light wavelength, displays a different Fv/Fm response and lacks detectable levels of phycocyanin. Indeed, 16S rRNA analysis supports the identity of the A. marina LARK001 strain as close to but distinct from from the A. marina HICR111A strain first isolated from Heron Island and previously found on the Great Barrier Reef under coral rubble on the reef flat. Taken together, A. marina LARK001 is a new cyanobacterial strain adapted to the stromatolite mats in Shark Bay.

Analysis insights for three FRET pairs of chemically unlinked two-molecule FRET cytometry.

Förster resonance energy transfer (FRET) is the direct energy exchange between two-component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein-protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor-1 (TNFR1) molecules expressed as recombinant C-terminal fusion proteins of cyan, yellow, or red fluorescent protein (-CFP, -YFP, or -RFP) to evaluate two-molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET-1), YFP→RFP (FRET-2) and CFP→RFP (FRET-3), by comparing TNFR1+TNFR1 with non-interacting TNFR1+CD27 proteins, on both LSR-II and Fortessa X-20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of "spiked" FRET donor cells, together with TNFR1+TNFR1 co-transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single-cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single-cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1-based system the mean CFP→YFP FRET-1 efficiency is 0.43 (LSR-II) and 0.41 (Fortessa X-20), the mean YFP→RFP FRET-2 efficiency is 0.30 (LSR-II) and 0.29 (Fortessa X-20), and the mean CFP→RFP FRET-3 efficiency is 0.56 (LSR-II) and 0.54 (Fortessa X-20). This study also embraces multi-dimensional clustering using t-SNE, Fit-SNE, UMAP, Tri-Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.

ß-Methylamino-L-alanine-induced protein aggregation in vitro and protection by L-serine.

The cyanobacterial non-protein amino acid α-amino-ß-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system xc_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.

A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose.

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected by liposuction surgery, was digested with collagenase to separate adipocytes from other tissue cells, comprising blood and lymphatic endothelial cells, fibroblasts, and all vessel- and tissue-resident leukocytes - the stromal vascular fraction (SVF). SVF cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, adding Brefeldin-A to prevent cytokine secretion during the final 4 hours. Cells were incubated with CD11b-FITC and CD40-APC (M1 MØ)' or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then incubated with either (1) anti-TNF-PE, (2) anti- IL-1ß-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGFß-PE or (6) isotype-IgG-PE (control), and stained with Hoechst 33342, preserved in permanent mounting media and examined by confocal microscopy. The FITC, PE and APC fluorescence channels were set to achieve minimal cross-channel emission using single-colour controls and voltages set for optimal detection by thresholding on isotype-IgG stained activated cells. Finally, transmission and z-stack images were captured. Cells were analysed as regions of interest (ROI) based on Hoechst-33342 then enumerated as FITC+, FITC+APC+ or FITC+APC+PE+ using an ImageJ script and exported into Excel. This permitted the examination of >9000 SVF cells individually, per LE sample. This method allows for the analysis of a high number of heterogeneous cells defined into any subtype or combination by the investigators' choice of surface and intracellular expression profiles. Fibroblasts, or other cytokine producing cells, can also be analysed by using other antibodies, and the cell count data can be correlated with any clinical or laboratory data.

Vibrio cholerae residing in food vacuoles expelled by protozoa are more infectious in vivo.

Vibrio cholerae interacts with many organisms in the environment, including heterotrophic protists (protozoa). Several species of protozoa have been reported to release undigested bacteria in expelled food vacuoles (EFVs) when feeding on some pathogens. While the production of EFVs has been reported, their biological role as a vector for the transmission of pathogens remains unknown. Here we report that ciliated protozoa release EFVs containing V. cholerae. The EFVs are stable, the cells inside them are protected from multiple stresses, and large numbers of cells escape when incubated at 37 °C or in the presence of nutrients. We show that OmpU, a major outer membrane protein positively regulated by ToxR, has a role in the production of EFVs. Notably, cells released from EFVs have growth and colonization advantages over planktonic cells both in vitro and in vivo. Our results suggest that EFVs facilitate V. cholerae survival in the environment, enhancing their infectious potential and may contribute to the dissemination of epidemic V. cholerae strains. These results improve our understanding of the mechanisms of persistence and the modes of transmission of V. cholerae and may further apply to other opportunistic pathogens that have been shown to be released by protists in EFVs.

Microparticles shed from multidrug resistant breast cancer cells provide a parallel survival pathway through immune evasion.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer in women. Resident macrophages at distant sites provide a highly responsive and immunologically dynamic innate immune response against foreign infiltrates. Despite extensive characterization of the role of macrophages and other immune cells in malignant tissues, there is very little known about the mechanisms which facilitate metastatic breast cancer spread to distant sites of immunological integrity. The mechanisms by which a key healthy defense mechanism fails to protect distant sites from infiltration by metastatic cells in cancer patients remain undefined. Breast tumors, typical of many tumor types, shed membrane vesicles called microparticles (MPs), ranging in size from 0.1-1 µm in diameter. MPs serve as vectors in the intercellular transfer of functional proteins and nucleic acids and in drug sequestration. In addition, MPs are also emerging to be important players in the evasion of cancer cell immune surveillance. METHODS: A comparative analysis of effects of MPs isolated from human breast cancer cells and non-malignant human brain endothelial cells were examined on THP-1 derived macrophages in vitro. MP-mediated effects on cell phenotype and functionality was assessed by cytokine analysis, cell chemotaxis and phagocytosis, immunolabelling, flow cytometry and confocal imaging. Student's t-test or a one-way analysis of variance (ANOVA) was used for comparison and statistical analysis. RESULTS: In this paper we report on the discovery of a new cellular basis for immune evasion, which is mediated by breast cancer derived MPs. MPs shed from multidrug resistant (MDR) cells were shown to selectively polarize macrophage cells to a functionally incapacitated state and facilitate their engulfment by foreign cells. CONCLUSIONS: We propose this mechanism may serve to physically disrupt the inherent immune response prior to cancer cell colonization whilst releasing mediators required for the recruitment of distant immune cells. These findings introduce a new paradigm in cancer cell biology with significant implications in understanding breast cancer colonization at distant sites. Most importantly, this is also the first demonstration that MPs serve as conduits in a parallel pathway supporting the cellular survival of MDR cancer cells through immune evasion.

Phylogenomics provides new insight into evolutionary relationships and genealogical discordance in the reef-building coral genus Acropora.

Understanding the genetic basis of reproductive isolation is a long-standing goal of speciation research. In recently diverged populations, genealogical discordance may reveal genes and genomic regions that contribute to the speciation process. Previous work has shown that conspecific colonies of Acropora that spawn in different seasons (spring and autumn) are associated with highly diverged lineages of the phylogenetic marker PaxC Here, we used 10 034 single-nucleotide polymorphisms to generate a genome-wide phylogeny and compared it with gene genealogies from the PaxC intron and the mtDNA Control Region in 20 species of Acropora, including three species with spring- and autumn-spawning cohorts. The PaxC phylogeny separated conspecific autumn and spring spawners into different genetic clusters in all three species; however, this pattern was not supported in two of the three species at the genome level, suggesting a selective connection between PaxC and reproductive timing in Acropora corals. This genome-wide phylogeny provides an improved foundation for resolving phylogenetic relationships in Acropora and, combined with PaxC, provides a fascinating platform for future research into regions of the genome that influence reproductive isolation and speciation in corals.

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype.

BACKGROUND: Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. METHODS: Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization. RESULTS: EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes. CONCLUSIONS: This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.

Biogeographic discordance of molecular phylogenetic and phenotypic variation in a continental archipelago radiation of land snails.

BACKGROUND: In island archipelagos, where islands have experienced repeated periods of fragmentation and connection through cyclic changes in sea level, complex among-island distributions might reflect historical distributional changes or local evolution. We test the relative importance of these mechanisms in an endemic radiation of Rhagada land snails in the Dampier Archipelago, a continental archipelago off the coast of Western Australia, where ten morphospecies have complex, overlapping distributions. RESULTS: We obtained partial mtDNA sequence (COI) for 1015 snails collected from 213 locations across 30 Islands, and used Bayesian phylogenetic analysis and Analysis of Molecular Variance (AMOVA) to determine whether geography or the morphological taxonomy best explains the pattern of molecular evolution. Rather than forming distinct monophyletic groups, as would be expected if they had single, independent origins, all of the widely distributed morphospecies were polyphyletic, distributed among several well-supported clades, each of which included several morphospecies. Each mitochondrial clade had a clear, cohesive geographic distribution, together forming a series of parapatric replacements separated by narrow contact zones. AMOVA revealed further incongruence between mtDNA diversity and morphological variation within clades, as the taxonomic hypothesis always explained a low or non-significant proportion of the molecular variation. In contrast, the pattern of mtDNA evolution closely reflected contemporary and historical marine barriers. CONCLUSIONS: Despite opportunities for distributional changes during periods when the islands were connected, there is no evidence that dispersal has contributed to the geographic variation of shell form at the broad scale. Based on an estimate of dispersal made previously for Rhagada, we conclude that the periods of connection have been too short in duration to allow for extensive overland dispersal or deep mitochondrial introgression. The result is a sharp and resilient phylogeographic pattern. The distribution of morphotypes among clades and distant islands is explained most simply by their parallel evolution.

Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens.

Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant 2-fold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a 2-fold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin.

Long-term genetic monitoring reveals contrasting changes in the genetic composition of newly established populations of the intertidal snail Bembicium vittatum.

Newly established populations are susceptible to founder events that reduce genetic variation. This may be counterbalanced by gene flow after populations become established or founders coming from genetically different populations. However, initial gains in genetic diversity may be short-lived if there is limited mixing between lineages and subsequent inbreeding, or if one lineage sweeps to fixation through selection or genetic drift. Here, we report on the genetic changes taking place within two newly established populations of intertidal snail over a 15-year period (∼ 10 generations). Each translocation was set up using multiple, genetically distinct source populations. Our data show that higher levels of variation in the translocated populations compared to the source populations were maintained over time for both nuclear (microsatellite) and mitochondrial genes. Small changes in allele and haplotype frequencies were observed in the source populations and in one of the translocated populations, but marked changes were evident in the other, where there was a dramatic shift towards the genetic make-up of one of the source populations. These genetic changes occurred despite relatively large numbers of founders (200-374 adults) and no evidence of the population experiencing a severe reduction in effective population size. Our study shows that the genetic composition of newly established populations can vary greatly over time and that genetic outcomes can be highly variable, and significantly different from initial expectations, even when they are established using high numbers of individuals and involve source populations from the same geographic regions.

Biogeography of the stygobitic isopod Pygolabis (Malacostraca: Tainisopidae) in the Pilbara, Western Australia: evidence for multiple colonisations of the groundwater.

COI sequence variation in groundwater amphipods of Western Australia showed multiple highly divergent lineages associated with tributaries. Estimates of the timing of this diversification are consistent with the hypothesis that aridification of the region during the Tertiary forced freshwater fauna into subterranean refugia. The groundwater isopod Pygolabis provides an opportunity to compare phylogenetic signals in co-distributed taxa that might share similar histories. A 549 bp fragment of COI was sequenced for 135 individuals of Pygolabis from 12 sites. Phylogenetic analyses revealed 12 highly divergent lineages associated with tributary boundaries. Lineages were separated by 11.8-27.8% sequence divergence. This level of divergence is consistent with diversification in the Pliocene, supporting the aridification hypothesis. While lineages were associated with distinct tributaries in both amphipods and Pygolabis, the two groups showed different phylogenetic patterns, suggesting that the mode of colonisation of the groundwater differed between the amphipods and Pygolabis.

Development and validation of a medication intensity scale derived from computerized pharmacy data that predicts emergency hospital utilization for persistent asthma.

OBJECTIVE: To validate a risk stratification scheme using computerized pharmacy data to predict emergency hospital utilization for persistent asthma. STUDY DESIGN: Retrospective cohort. METHODS: The development sample consisted of 1079 HMO members aged 18 to 56 years with persistent asthma. The scale used medication cut-points as predictors for next-year emergency hospital utilization in a stepwise logistic regression model. Prediction properties were evaluated in a validation sample of 24 370 patients aged 18 to 56 years in a separate persistent-asthma database. RESULTS: Increasing use of beta-agonists (odds ratio [OR] of 2.2 for 5-13 vs 0-4 canisters; OR of 2.4 for >13 vs 5-13 canisters) and oral corticosteroids (OR of 2.6 for >2 vs 0-2 dispensing events) in the first year independently predicted emergency hospital utilization in the second year. Assigning 1 point for exceeding each of the above 3 medication thresholds led to a 4-level medication intensity scale that was significantly (P <.0001) related to validated measures of asthma symptom severity, asthma control, and asthma quality of life in the development sample. In the validation sample, this scheme identified a high-risk group that was 6 times more likely than the low-risk group to require subsequent emergency hospital care, with overall sensitivity of 65% and specificity of 54%. This scale did not perform as well as a scale based on both baseline emergency hospital care and pharmacy data. CONCLUSION: This simple risk stratification scheme can be used for populations with persistent asthma for whom computerized pharmacy data, but not computerized prior utilization data, are available.

The controller-to-total asthma medication ratio is associated with patient-centered as well as utilization outcomes.

BACKGROUND: The ratio of controller medication to total asthma medications has been related to asthma utilization outcomes, but its relationship to patient-centered outcomes has not been explored. METHODS: Surveys that included validated asthma quality-of-life, control, and symptom severity tools were completed by a random sample of 2,250 health maintenance organization members aged 18 to 56 years who had persistent asthma. Linked computerized pharmacy data provided dispensing information on beta-agonist canisters and asthma controller medication. The ratio was calculated as the number of controller medications dispensed during the year of the survey divided by the total number medications (ie, inhaled beta-agonist plus controller medications) dispensed. The relationships of the optimal ratio cutoff to patient-centered outcomes and to subsequent acute asthma exacerbations were determined. RESULTS: Mean asthma quality-of-life, asthma control, and symptom severity scale scores were significantly (p < 0.0001) more favorable in patients with ratios of > or = 0.5. After adjusting for demographic characteristics, patients with ratios of > or = 0.5 were significantly less likely to have adverse results regarding asthma quality of life (odds ratio [OR], 0.65; 95% confidence interval [CI], 0.52 to 0.80), asthma control (OR, 0.62; 95% CI, 0.50 to 0.77), and symptom severity (OR, 0.53; 95% CI, 0.43 to 0.65), and were also less likely to experience subsequent asthma hospitalizations or emergency department visits (OR, 0.44; 95% CI, 0.26 to 0.74) than patients with lower ratios. CONCLUSION: A higher controller medication/total asthma medication ratio is associated with better patient-centered asthma outcomes as well as with reduced emergency hospital utilization. This adds further support to the use of the medication ratio as an asthma quality-of-care measure.

Validation of a beta-agonist long-term asthma control scale derived from computerized pharmacy data.

BACKGROUND: Asthma control has been defined clinically by using validated tools, but an asthma control scale using administrative data has not been reported. OBJECTIVE: We sought to validate a beta-agonist asthma control scale derived from administrative data. METHODS: Surveys that included validated asthma symptom and control tools were completed by a random sample of 2250 health maintenance organization members aged 18 to 56 years with persistent asthma. Linked computerized pharmacy data provided beta-agonist canister and oral corticosteroid dispensings. The proposed 4-level asthma control scale was based on the number of short-acting beta-agonist canisters dispensed in 12 months. Construct validity and predictive validity were assessed. RESULTS: For construct validity, factor analysis showed significant loading of the beta-agonist scale on the symptom control factor, and the beta-agonist scale was significantly related to the validated asthma control and symptom scales (r = 0.31, P < .0001). For predictive validity, each progressive level of the proposed beta-agonist control scale was associated with an increased risk of subsequent asthma hospitalizations or emergency department visits and oral corticosteroid use, independent of prior use. CONCLUSION: A scale based on the number of beta-agonists dispensed in a 1-year period and derived from administrative data reflects asthma symptom control over that period of time. This scale can help identify patients who are at risk for future acute asthma health care use. CLINICAL IMPLICATIONS: This information can be used in population management and by clinicians to assess long-term asthma control and identify patients who need intervention to prevent future morbidity.

Islands increase genetic subdivision and disrupt patterns of connectivity of intertidal snails in a complex archipelago.

The view that marine species with planktonic dispersal have highly connected, demographically open populations is giving way to recognition that populations may often be largely self-recruiting, or demographically closed. This raises the question of what local conditions might favor isolation of populations. To test the importance of islands for local isolation in species with planktonic larvae, we examined allozyme variation among 35 populations of the intertidal snail Austrocochlea constricta in the Houtman Abrolhos Islands, Western Australia, spanning 60 km. Heterogeneity of allozyme frequencies among populations was high, with average F(ST) of 0.237, indicating highly localized populations. Increased subdivision was associated with islands at different scales: between island groups, separated by deep water gaps, and between disconnected sets of islands within groups. At short distances, up to two km, subdivision increased fivefold between islands compared with that between populations on the same island. Along 11 km of continuous, sheltered shore, there was isolation by distance but among a linear series of islands over similar distance, there was greater subdivision at short distances but no association with distance. These patterns had been seen previously in the direct-developing snail Bembicium vittatum, but its finding in A. constricta confirms for a planktonic disperser the importance of this complex archipelago for both retention of locally produced larvae and disruption of patterns of connectivity. Taken together, these results indicate that islands can increase both the "open" and the "closed" components of recruitment and that applicable models of genetic connectivity depend substantially on local conditions.

Improved asthma outcomes from allergy specialist care: a population-based cross-sectional analysis.

BACKGROUND: Prior studies suggest that allergist care improves asthma outcomes, but many of these studies have methodological shortcomings. OBJECTIVE: We sought to compare patient-based and medical utilization outcomes in randomly selected asthmatic patients cared for by allergists versus primary care providers. METHODS: A random sample of 3568 patients enrolled in a staff model health maintenance organization who were given diagnoses of persistent asthma completed surveys. Of these participants, 1679 (47.1%) identified a primary care provider as their regular source of asthma care, 884 (24.8%) identified an allergist, 693 (19.4%) reported no regular source of asthma care, and 195 (5.5%) identified a pulmonologist. Validated quality of life, control, severity, patient satisfaction, and self-management knowledge tools and linked administrative data that captured medication use were compared between groups, adjusting for demographics and baseline hospital and corticosteroid use. RESULTS: Compared with those followed by primary care providers, patients of allergists reported significantly higher (P < .001) generic physical and asthma-specific quality of life, less asthma control problems, less severe symptoms, higher satisfaction with care, and greater self-management knowledge. Patients of allergists were less likely than patients of primary care providers to require an asthma hospitalization (odds ratio, 0.45) or unscheduled visit (odds ratio, 0.71) and to overuse beta-agonists (odds ratio, 0.47) and were more likely to receive inhaled steroids (odds ratio, 1.81) during their past year. CONCLUSIONS: Allergist care is associated with a wide range of improved outcomes in asthmatic patients compared with care provided by primary care providers.

Relationships among quality of life, severity, and control measures in asthma: an evaluation using factor analysis.

BACKGROUND: Validated psychometric tools measuring quality of life, asthma control, and asthma severity have been developed, but their relationships with each other and with other important patient-centered outcomes have not been rigorously assessed. OBJECTIVE: To use factor analysis to evaluate the relationships of these validated tools with each other and with other patient-centered outcomes. METHODS: Surveys were completed by a random sample of 2854 Health Maintenance Organization members age 18 to 56 years with persistent asthma. Surveys included demographic information; validated tools measuring generic (Short Form-12; SF-12) and asthma-specific (Juniper Mini Asthma Quality of Life Questionnaire; AQLQ) quality of life, asthma control (Asthma Therapy Assessment Questionnaire), and asthma symptom severity (Asthma Outcomes Monitoring System); self-described severity, control, and course over time; and history of acute exacerbations. RESULTS: Principal component analysis suggested a 5-factor model that accounted for approximately 59% of the variability. The most prominent rotated factor reflected asthma symptom frequency (19.4% of variability), was measured by the symptom subscale of the AQLQ, and was the only factor significantly related to the Asthma Therapy Assessment Questionnaire, Asthma Outcomes Monitoring System, or the self-reported assessments of severity, control, or course. Other factors included symptom bother (12.1% of variability), reflected by the environment and emotion AQLQ subscales; activity limitation (13.9% of variability), reflected by the activity AQLQ subscale and the SF-12 physical component scale; mental health (8.3% of variability), reflected by the SF-12 mental component scale; and acute exacerbations (5.0% of variability), not measured by any of the validated scales. CONCLUSION: Distinct components of patient-reported asthma health status can be identified by factor analysis. Distinct constructs of severity versus control cannot be identified by the use of these tools alone.