Genome skimming provides new insight into the relationships in Ludwigia section Macrocarpon, a polyploid complex.

PREMISE OF THE STUDY: Interpreting relationships within groups containing polyploids, which are frequent in angiosperms, can be greatly assisted by genomic techniques. In this study, we used a genome-skimming approach to investigate the evolutionary relationships and origins of polyploids in the monophyletic group, Ludwigia section Macrocarpon (Onagraceae), which includes diploid, tetraploid, and hexaploid taxa. METHODS: We sampled all known taxa and ploidy levels in the section and conducted shotgun sequencing. We assembled plastomes, mitochondrial sequences, and completed nuclear ribosomal regions, reconstructed phylogenies, and conducted comparative genomic analyses for plastomes to gain insights into the relationships among studied taxa. KEY RESULTS: Within the section, results showed that the South American diploid taxa L. bonariensis and L. lagunae were closely related. We reported the first chromosome count (2n = 4× = 32) for L. neograndiflora, which is closely related to the two South American diploid taxa, although its exact origin remains unclear. The samples of the widespread, polyploid taxon L. octovalvis do not form a monophyletic group. Both tetraploid and hexaploid L. octovalvis lineages have originated more than once. At least one tetraploid in the L. octovalvis lineage may have been involved in the origins of hexaploids. One or more extinct/unsampled intermediate tetraploids in the L. octovalvis lineages had also likely been involved in the origins of hexaploids. CONCLUSIONS: Genome skimming provided important insights into the complex evolutionary relationships within sect. Macrocarpon, but additional sampling and data from single-copy nuclear regions are necessary to further elucidate the origins of the polyploids in this section.

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.

D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.

Molecular phylogeny of tribe Stachydeae (Lamiaceae subfamily Lamioideae).

Although tribe Stachydeae (Lamiaceae) is considered monophyletic, relationships within the tribe are still poorly understood. The complexity of Stachydeae includes paraphyletic genera, considerable morphological plasticity, a range of ploidy levels, and presumably frequent natural hybridization. We performed parsimony and Bayesian phylogenetic analyses of nuclear (ribosomal ITS) and plastid (trnL intron, trnL-trnF spacer, rps16 intron) DNA sequence data from a taxonomically and geographically broad sampling of the tribe to identify major evolutionary lineages and to test taxonomic hypotheses within this largest of all lamioid tribes. We included 143 accessions corresponding to 121 species, representing both Old and New World species, and all 12 recognized genera of tribe Stachydeae. Both nuclear and plastid data corroborate monophyly of the tribe, with Melittis as sister to all remaining Stachydeae. For the latter well-supported clade, we suggest the phylogenetic name Eurystachys. Within Eurystachys, although monophyly is supported by both nuclear and plastid data for several named and unnamed groups, the majority of recognized taxa appear to be para- or polyphyletic. The taxon compositions of most subclades are congruent between the plastid and nuclear tree topologies, whereas their relative phylogenetic placements are often not. This level of plastid-nuclear incongruence suggests considerable impact of hybridization in the evolution of Stachydeae.

The effect of perceived health status on patient satisfaction.

OBJECTIVE: To examine the effect of perceived health status on three components of patient satisfaction. METHODS: The Household Component of the 1999 Medical Expenditure Panel Survey for people 35-64 years of age was used to examine the effect of perceived health status on patient satisfaction measured in terms of access to care, provider quality and quality of care. Descriptive statistics and multivariate regression were used to describe the subjects and to examine the relationship between patient satisfaction and perceived health status controlling for patient demographic factors, health factors and provider characteristics. All analyses used STATA 8.0 which is designed to analyze weighted data. RESULTS: A total of 4,417 patients (71% women) met the inclusion criteria for the study. Patients who rated their health excellent or good scored higher on the three dimensions of patient satisfaction. Higher scores on one or more components of patient satisfaction were associated with being older, married, better educated and having higher income, health insurance and good mental health. Seeing the health-care provider for an old problem resulted in lower levels of patient satisfaction. Provider characteristics significantly related to patient satisfaction were listening to the patient, being a specialist, seeing patients in an office setting and being located in the South. CONCLUSIONS: This study has shown that patient satisfaction is influenced by a person's self-perceived health status and other personal characteristics that are external to the delivery of health care. These findings suggest that patient satisfaction data should be used judiciously because a significant portion of the variation may be attributed to factors endogenous to the patient and therefore are not amenable to provider intervention.

Hospice effect on government expenditures among nursing home residents.

OBJECTIVE: To examine the effect of the Medicare hospice benefit on Medicare and Medicaid expenditures by dual-eligible Medicare-Medicaid nursing home (NH) residents. DATA SOURCES/STUDY SETTING: Secondary data for NH residents for 1998-1999. STUDY DESIGN: Retrospective cohort study of NH residents in the state of Florida who died between July and December 1999 (N=5,774). Medicare claims identified hospice enrollment, and Medicare and Medicaid claims identified expenditures by categories of care. Nursing home resident assessments were used to control for case-mix differences. Geocoding of nursing homes, hospice providers and hospitals was used to identify and characterize local health care markets. DATA COLLECTION/EXTRACTION METHODS: A file was constructed linking Medicare and Medicaid claims to Minimum Data Set assessments of NH residents, and NH provider (Online Survey and Certification Automated Record) and hospice provider files. PRINCIPAL FINDINGS: Hospice enrollment results in substantial savings in government expenditures (22 percent) among all short-stay (< or =90 days) dying NH residents. For long-stay (>90 days) dying NH residents, hospice provides some savings (8 percent) among cancer residents while it is cost-neutral among dementia residents and adds some cost (10 percent) for residents with a diagnosis other than cancer or dementia. There is evidence of selection bias, particularly among residents with cancer (19 percent savings unadjusted versus 8 percent adjusted). Among short-stay NH residents, hospice greatly reduces Medicare expenditures but increases Medicaid expenditures. CONCLUSIONS: Hospice enrollment results in lower combined Medicare/Medicaid expenditures in the last month of life, particularly among short-stay NH residents. This effect, however, varies by diagnosis and NH length of stay. In addition, for short-stay NH residents, current payment policy creates a Medicare incentive and Medicaid disincentive for promoting residents' referral to hospice.

Geography shapes the phylogeny of frailejones (Espeletiinae Cuatrec., Asteraceae): a remarkable example of recent rapid radiation in sky islands.

BACKGROUND: The páramo ecosystem, located above the timberline in the tropical Andes, has been the setting for some of the most dramatic plant radiations, and it is one of the world's fastest evolving and most diverse high-altitude ecosystems. Today 144+ species of frailejones (subtribe Espeletiinae Cuatrec., Asteraceae) dominate the páramo. Frailejones have intrigued naturalists and botanists, not just for their appealing beauty and impressive morphological diversity, but also for their remarkable adaptations to the extremely harsh environmental conditions of the páramo. Previous attempts to reconstruct the evolutionary history of this group failed to resolve relationships among genera and species, and there is no agreement regarding the classification of the group. Thus, our goal was to reconstruct the phylogeny of the frailejones and to test the influence of the geography on it as a first step to understanding the patterns of radiation of these plants. METHODS: Field expeditions in 70 páramos of Colombia and Venezuela resulted in 555 collected samples from 110 species. Additional material was obtained from herbarium specimens. Sequence data included nrDNA (ITS and ETS) and cpDNA (rpl16), for an aligned total of 2,954 bp. Fragment analysis was performed with AFLP data using 28 primer combinations and yielding 1,665 fragments. Phylogenies based on sequence data were reconstructed under maximum parsimony, maximum likelihood and Bayesian inference. The AFLP dataset employed minimum evolution analyses. A Monte Carlo permutation test was used to infer the influence of the geography on the phylogeny. RESULTS: Phylogenies reconstructed suggest that most genera are paraphyletic, but the phylogenetic signal may be misled by hybridization and incomplete lineage sorting. A tree with all the available molecular data shows two large clades: one of primarily Venezuelan species that includes a few neighboring Colombian species; and a second clade of only Colombian species. Results from the Monte Carlo permutation test suggests a very strong influence of the geography on the phylogenetic relationships. Venezuelan páramos tend to hold taxa that are more distantly-related to each other than Colombian páramos, where taxa are more closely-related to each other. CONCLUSIONS: Our data suggest the presence of two independent radiations: one in Venezuela and the other in Colombia. In addition, the current generic classification will need to be deeply revised. Analyses show a strong geographic structure in the phylogeny, with large clades grouped in hotspots of diversity at a regional scale, and in páramo localities at a local scale. Differences in the degrees of relatedness between sympatric species of Venezuelan and Colombian páramos may be explained because of the younger age of the latter páramos, and the lesser time for speciation of Espeletiinae in them.

Complete Plastome Sequence of Ludwigia octovalvis (Onagraceae), a Globally Distributed Wetland Plant.

Here, we present the first plastome of Ludwigia octovalvis (Onagraceae, Myrtales) as well as the first plastome in the subfamily Ludwigioideae. This genome is notable for its contracted inverted repeat regions and an expanded small single-copy region compared to other species in the orders Myrtales and Geraniales.

Perspectives of Stakeholders on Implementing a Farm-to-University Program at an HBCU.

OBJECTIVE: To explore the perspectives of various stakeholders on whether an HBCU has the resources to establish a farm-to-university program that can improve fruits and vegetables intake among African American students. Additionally, this study assessed students' satisfaction with fruits and vegetables served in University dining halls, and their desire for changes in policies to increase local fruits and vegetables access on campus. METHODS: This study employed a mixed method data collection strategy. Semi-structured interviews were used to explore the stakeholders' perspectives and self-administered questionnaires were used to assess students' satisfaction with fruits and vegetables and desire for policy changes. RESULTS: Barriers reported by both food service administrators and farmers were cost and variation in supply and demand. Students expressed lack of satisfaction with fresh produce served in campus dining halls and a desire for change in policies to increase local fruits and vegetables access on campus. CONCLUSION: While there is student desire for improved access to fresh produce on campus, there are perceived barriers to overcome. University partnerships are needed to address the desired nutritional improvements.

Government expenditures at the end of life for short- and long-stay nursing home residents: differences by hospice enrollment status.

OBJECTIVES: To examine end-of-life government expenditures for short- and long-stay Medicare- and Medicaid-eligible (dual-eligible) nursing home (NH) hospice and nonhospice residents. DESIGN: A retrospective cohort study. SETTING: Six hundred fifty-seven Florida NHs. PARTICIPANTS: Dual-eligible NH residents who died in Florida NHs between July and December 1999 (N=5,774). MEASUREMENTS: Nursing home stays of 90 days or less were considered short stays (n=1,739), and those over 90 days were long stays (n=4,035). Three diagnosis groups were studied: cancer without Alzheimer's disease or dementia, Alzheimer's disease or dementia, and other diagnoses. Eligibility and expenditure claims data for 1998 and 1999 were merged with vital statistics and NH resident assessment data to determine diagnoses, location of death, hospice enrollment, eligibility, and expenditures. RESULTS: Twenty percent of short-stay (n=350) and 26% of long-stay (n=958) NH decedents elected hospice; of these, 73% of short-stay and 58% of long-stay NH residents had hospice stays of 30 days or less. Overall, mean government expenditures in the last month of life were significantly less for hospice than nonhospice residents (7,365 dollars; 95% confidence interval (CI)=7,144-7586 dollars vs 8,134 dollars; 95% CI=7,896-8,372 dollars), but 1-month expenditures were only significantly lower for hospice residents with short NH stays, not for those with long NH stays. CONCLUSION: Overall, hospice care in NHs does not appear to increase government expenditures. Because significantly lower expenditures are observed for short-stay NH hospice residents, policy restricting access to Medicare hospice for Medicare skilled nursing facility residents may represent a missed opportunity for savings.

Origin and evolution of the endemic genera of Gonosperminae (Asteraceae: Anthemideae) from the Canary Islands: evidence from nucleotide sequences of the internal transcribed spacers of the nuclear ribosomal DNA.

The Gonosperminae (Asteraceae) are composed of three genera endemic to the Canary Islands (GONOSPERMUM: Less., and LUGOA: DC.) and southern Africa (INULANTHERA: Källersjö), and they are considered an example of a floristic link between these two regions. Phylogenetic analyses of ITS sequences reveal that the Canarian genera are not sister to INULANTHERA: and do not support the monophyly of the Gonosperminae. These results, coupled with previous phylogenetic studies of other groups, suggest that many of the putative biogeographic links between Macaronesia and southeast Africa need to be evaluated by rigorous phylogenetic analyses. INULANTHERA: forms part of the basal southern African radiation of the Anthemideae, and therefore it is closely related to other taxa from this region. Maximum likelihood and weighted parsimony analyses support a monophyletic group in the Canary Islands, that includes LUGOA:, Gonospermum, and three TANACETUM: species endemic to the island of Gran Canaria. Bootstrap support for the monophyly of this Canarian group is weak, and it collapses in the strict consensus tree based on unweighted parsimony. LUGOA: is nested within Gonospermum, and both interisland colonization among the western islands of La Gomera, El Hierro, La Palma and Tenerife, and radiation on the central island of Gran Canaria have been the major patterns of species diversification for these Canarian endemics.

Economic burden of dehydration among hospitalized elderly patients.

PURPOSE: The cost of unnecessary hospitalizations associated with dehydration in elderly patients was studied. METHODS: The study involved a retrospective examination of 1999 data on hospital discharges from the Healthcare Cost and Utilization Project Nationwide Inpatient Sample. The procedure code for volume depletion was used to extract hospitalization episodes for patients > or = 65 years of age who had a principal diagnosis of dehydration and were discharged alive. Hospitalizations with procedure codes unrelated to dehydration were omitted. RESULTS: The descriptive findings indicated that hospitalized older patients with a principal diagnosis of dehydration averaged 80.4 years of age, were primarily white (82.5%), and were more likely to live in the community than in a nursing home. Hospitalizations for dehydration were more common in the South and less common in the West. The average length of stay (LOS) was 4.6 days. The average total hospital charge was dollars 7,442, and the average per diem charge was dollars 1,628. Regression analysis explained nearly half of the variation in charges for dehydration (R2 = 0.457). Average LOS and number of diagnoses were directly related to hospital charges, and age was inversely related. Higher charges were associated with being nonwhite, entering the hospital via the emergency room, having private insurance, having no insurance, having comorbidities, and being hospitalized in the West or in teaching or urban hospitals. In 1999, the potential national saving from avoidable hospitalizations in these patients could have been as much as dollars 1.14 billion. CONCLUSION: The economic burden associated with avoidable hospitalizations due to dehydration in elderly patients was substantial.

Enhancement of cisplatin cytotoxicity by disulfiram involves activating transcription factor 3.

BACKGROUND: Activating transcription factor 3 (ATF3), a stress-inducible gene, is a regulator of cisplatin-induced cytotoxicity, and enhancement of the ATF3 expression potentiates this cytotoxicity. MATERIALS AND METHODS: ATF3 expression and its binding to the transcription target CHOP were evaluated by western blot and chromatin immunoprecipitation (ChIP), respectively, in a panel of five cell lines (WI38, MCF7, PC3, A549). MTT assays were employed to assess the effects of many drugs, including disulfiram, on cell viability. RESULTS: ATF3 protein expression was up-regulated after cytotoxic doses of cisplatin treatment and it directly bound to the CHOP gene promoter, increasing this pro-apoptotic protein's expression. In a library screen of 1200 compounds, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was synergistic and likely due to their ability to induce ATF3 independently. CONCLUSION: Understanding the role of ATF3 in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug's efficacy.

Timing and tempo of early and successive adaptive radiations in Macaronesia.

The flora of Macaronesia, which encompasses five Atlantic archipelagos (Azores, Canaries, Madeira, Cape Verde, and Salvage), is exceptionally rich and diverse. Spectacular radiation of numerous endemic plant groups has made the Macaronesian islands an outstanding area for studies of evolution and speciation. Despite intensive investigation in the last 15 years, absolute age and rate of diversification are poorly known for the flora of Macaronesia. Here we report molecular divergence estimates and rates of diversification for five representative, putative rapid radiations of monophyletic endemic plant lineages across the core eudicot clade of flowering plants. Three discrete windows of colonization during the Miocene and early Pliocene are suggested for these lineages, all of which are inferred to have had a single colonization event followed by rapid radiation. Subsequent inter-archipelago dispersal events into Madeira and the Cape Verdes took place very recently during the late Pliocene and Pleistocene after initial diversification on the Canary Islands. The tempo of adaptive radiations differs among the groups, but is relatively rapid compared to continental and other island radiations. Our results demonstrate that opportunity for island colonization and successful radiation may have been constrained to discrete time periods of profound climatic and geological changes in northern African and the Mediterranean.

Relationships of cucumbers and melons unraveled: molecular phylogenetics of Cucumis and related genera (Benincaseae, Cucurbitaceae).

Cucumis (Cucurbitaceae) comprises 33 species of annuals and perennials with a major native center of diversity in tropical and southern Africa. The genus includes some economically important and widely grown vegetables such as cucumbers and melons. Monophyly of the genus has been disputed in previous studies, but with only limited sampling. Relationships within Cucumis are thus poorly understood; moreover, the validity of the closely related genera has not been thoroughly tested. The present study was undertaken to test the monophyly of Cucumis and several closely related genera, to test sectional circumscriptions within Cucumis, and to understand the biogeographical history of the genus. We sequenced the nuclear ITS and plastid trnS-trnG regions for 40 ingroup and three outgroup taxa, representing all recognized subgenera and sections. Parsimony, maximum likelihood, and Bayesian analyses found Cucumella, Oreosyce, Mukia, Myrmecosicyos, and Dicaelospermum nested within Cucumis. The clades recovered within the Cucumis complex in some instances represent the first phylogenetically derived hypothesis of relationships, whereas others correspond to previous subgeneric and sectional classifications. At least four introductions from Africa to Asia, as well as one reintroduction to Africa, are suggested within the Cucumis complex. Cucumis sativus (cucumber) is strongly supported as sister to the eastern Asian C. hystrix, whereas C. melo (melon) is strongly supported as sister to C. sagittatus in southern Africa.

Pharmacologic management of integrative brain failure.

Integrative brain failure is often a comorbidity of critical illness, and although not uncommon, is perhaps the least understood of all the various organ failure phenomena. Factors that contribute to integrative brain failure include inadequate management of pain, stress, anxiety, and the several underlying mechanisms that create agitation in the intensive care unit patient. Delirium, a resultant organic mental syndrome, is reversible when promptly recognized and aggressively managed. The nurse who is astute in clinical assessments and skillful in the management of the environment of care can prevent and control states of anxiety, irritability, restlessness, and sleep disturbances that contribute to the development of delirium.

Origin of Macaronesian Sideritis L. (Lamioideae: Lamiaceae) inferred from nuclear and chloroplast sequence datasets.

Sideritis L. (Lamiaceae) comprises approximately 150 species of annuals and perennials distributed chiefly in the Mediterranean region. The majority of the species belong to the continental subgenus Sideritis which is divided into two perennial (Sideritis and Empedoclea) and two annual (Hesiodia and Burgsdorfia) sections. Twenty-three species are woody perennials endemic to the Macaronesian archipelagos of Madeira and the Canary Islands. In an effort to determine the continental origin of the insular group, we constructed independent phylogenies comprising sequence data from both chloroplast and nuclear markers. Sampling included 7 island taxa drawn from the Macaronesian subgenus Marrubiastrum and 25 continental taxa representing all four sections of subgenus Sideritis. Subgenus Marrubiastrum and the two continental perennial sections form well-supported monophyletic groups in both individual and combined analyses. The annual sections are not monophyletic in any analysis; further sampling of annual taxa is needed to resolve these relationships. All analyses identified Sideritis cossoniana, an annual species from Morocco, as the closest continental relative of the Macaronesian group. This contrasts with the hypothesis of earlier workers who suggested that the insular taxa were most closely related to eastern Mediterranean species of the genus. The phylogenies also demonstrate a distinct increase in woodiness among the Macaronesian species relative to their continental congeners, providing further support for the secondary nature of woodiness in island plants.