GONe: software for estimating effective population size in species with generational overlap.

GONe is a user-friendly, Windows-based program for estimating effective size (N(e) ) in populations with overlapping generations. It uses the Jorde-Ryman modification to the temporal method to account for age structure in populations. This method requires estimates of age-specific survival and birth rate and allele frequencies measured in two or more consecutive cohorts. Allele frequencies are acquired by reading in genotypic data from files formatted for either GENEPOP or TEMPOFS. For each interval between consecutive cohorts, N(e) is estimated at each locus and over all loci. Furthermore, N(e) estimates are output for three different genetic drift estimators (F(s) , F(c) and F(k) ). Confidence intervals are derived from a chi-square distribution with degrees of freedom equal to the number of independent alleles. GONe has been validated over a wide range of N(e) values, and for scenarios where survival and birth rates differ between sexes, sex ratios are unequal and reproductive variances differ. GONe is freely available for download at https://bcrc.bio.umass.edu/pedigreesoftware/.

Improved pain, physical functioning and health status in patients with rheumatoid arthritis treated with CP-690,550, an orally active Janus kinase (JAK) inhibitor: results from a randomised, double-blind, placebo-controlled trial.

OBJECTIVES: To determine the efficacy of CP-690,550 in improving pain, function and health status in patients with moderate to severe active rheumatoid arthritis (RA) and an inadequate response to methotrexate or a tumour necrosis factor alpha inhibitor. METHODS: Patients were randomised equally to placebo, CP-690,550 5, 15 or 30 mg twice daily for 6 weeks, with 6 weeks' follow-up. The patient's assessment of arthritis pain (pain), patient's assessment of disease activity, Health Assessment Questionnaire-Disability Index (HAQ-DI) and Short Form-36 (SF-36) were recorded. RESULTS: At week 6, significantly more patients in the CP-690,550 5, 15 and 30 mg twice-daily groups experienced a 50% improvement in pain compared with placebo (44%, 66%, 78% and 14%, respectively), clinically meaningful reductions in HAQ-DI (> or =0.3 units) (57%, 75%, 76% and 36%, respectively) and clinically meaningful improvements in SF-36 domains and physical and mental components. CONCLUSIONS: CP-690,550 was efficacious in improving the pain, function and health status of patients with RA, from week 1 to week 6.

create: a software to create input files from diploid genotypic data for 52 genetic software programs.

create is a Windows program for the creation of new and conversion of existing data input files for 52 genetic data analysis software programs. Programs are grouped into areas of sibship reconstruction, parentage assignment, genetic data analysis, and specialized applications. create is able to read in data from text, Microsoft Excel and Access sources and allows the user to specify columns containing individual and population identifiers, birth and death data, sex data, relationship information, and spatial location data. create's only constraints on source data are that one individual is contained in one row, and the genotypic data is contiguous. create is available for download at http://www.lsc.usgs.gov/CAFL/Ecology/Software.html.

Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.

Macrophage infiltration of the kidney is a prominent feature associated with the severity of renal injury and progressive renal failure. To determine the influence of macrophages in renal disease models in the absence of endogenous T and B cells, we performed adoptive transfer of macrophages into severe combined immunodeficient (SCID) mice. In this study, macrophages were isolated from the spleens of BALB/c mice and stimulated with lipopolysaccharide to induce classically activated M1 macrophages or with interleukin-4 (IL-4) and IL-13 to induce alternatively activated M2 macrophages. These macrophages were then infused into SCID mice with adriamycin nephropathy; an in vivo model of chronic inflammatory renal disease analogous to human focal segmental glomerulosclerosis. Mice infused with M1 macrophages had a more severe histological and functional injury, whereas M2 macrophage-induced transfused mice had reduced histological and functional injury. Both M1 and M2 macrophages localized preferentially to the area of injury and maintained their phenotypes even after 4 weeks. The protective effect of M2 macrophages was associated with reduced accumulation and possibly downregulated chemokine and inflammatory cytokine expression of the host infiltrating macrophages. Our findings demonstrate that macrophages not only act as effectors of immune injury but can be induced to provide protection against immune injury.

Morphological properties of mouse retinal ganglion cells.

The mouse retina offers an increasingly valuable model for vision research given the possibilities for genetic manipulation. Here we assess how the structural properties of mouse retinal ganglion cells relate to the stratification pattern of the dendrites of these neurons within the inner plexiform layer. For this purpose, we used 14 morphological measures to classify mouse retinal ganglion cells parametrically into different clusters. Retinal ganglion cells were labeled in one of three ways: Lucifer Yellow injection, 'DiOlistics' or transgenic expression of yellow fluorescent protein. The resulting analysis of 182 cells revealed 10 clusters of monostratified cells, with dendrites confined to either On or Off sublaminae of the inner plexiform layer, and four clusters of bistratified cells, dendrites spanning the On and Off sublaminae. We also sought to establish how these parametrically identified retinal ganglion cell clusters relate to cell types identified previously on the basis of immunocytochemical staining and the expression of yellow fluorescent protein. Cells labeled with an antibody against melanopsin were found to be located within a single cluster, while those labeled with the SMI-32 antibody were in four different clusters. Yellow fluorescent protein expressing cells were distributed within 13 of the 14 clusters identified here, which demonstrates that yellow fluorescent protein expression is a useful method for labeling virtually the entire population of mouse retinal ganglion cells. Collectively, these findings provide a valuable baseline for future studies dealing with the effects of genetic mutations on the morphological development of these neurons.

New findings on evolution of metal homeostasis genes: evidence from comparative genome analysis of bacteria and archaea.

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P(IB)-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P(IB)-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P(IB)-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.

Field and storage testing Bt potatoes for resistance to potato tuberworm (Lepidoptera: Gelichiidae).

Potato tuberworm, Phthorimaea operculella (Zeller), is the most serious insect pest of potatoes worldwide. The introduction of the Bacillus thuringiensis (Bt) toxin gene through genetic engineering offers host plant resistance for the management of potato tuberworm. We report on the field and storage studies to evaluate Bt-cry5 potato lines for resistance to potato tuberworm in Egypt under natural infestations and their agronomic performance in both Egypt and Michigan. From 1997 to 2001, field experiments were conducted at the International Potato Center (CIP) Research Station, Kafr El-Zyat, Egypt, and/or Agricultural Genetic Engineering Institute (AGERI), Giza, Egypt, to evaluate resistance to tuberworm. A total of 27 Bt-transgenic potato lines from six different Bt constructs were evaluated over a 5-yr period. After harvest and evaluation of the agronomic trials, storage evaluation of potato tuberworm damage was done at the CIP Research Station. The 1997 field trial was the first field test of genetically engineered crops in Egypt. Field tests to assess potato tuberworm resistance in Egypt were able to differentiate between the Bt-transgenic lines and the nontransgenic lines/cultivars in 1999, 2000, and 2001. The Bt-cry5-Spunta lines (Spunta-G2, Spunta-G3, and Spunta-6a3) were the most resistant lines in field with 99-100% of tubers free of damage. In the 2001 storage study, these lines were also over 90% free of tuberworm damage after 3 mo. NYL235-4.13, which combines glandular trichomes with the Bt-cry5/gus fusion construct, also had a high percentage of clean tubers in the field studies. In agronomic field trials in Michigan from 1997 to 2001, the Bt-transgenic lines in most instances performed similar to the nontransgenic line in the agronomic trials; however, in Egypt (1998-1999), the yields were less than one-half of those in Michigan. Expression of the Bt-cry5 gene in the potato tuber and foliage will provide the seed producer and grower a tool in which to reduce potato tuberworm damage to the tuber crop in the field and storage.

Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface.

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.

Neutral locus heterozygosity, inbreeding, and survival in Darwin's ground finches (Geospiza fortis and G. scandens).

Comprehensive long-term studies of isolated populations provide valuable comparative data that may be used to evaluate different methods for quantifying the relationship between genetic diversity and fitness. Here, we report on data collected from large and well-characterized cohorts of the two numerically dominant species of Darwin's finches on Isla Daphne Major, Galápagos, Ecuador - Geospiza fortis and G. scandens. Multilocus microsatellite (SSR) genetic diversity estimates (heterozygosity and d2) and pedigree-based estimates of the inbreeding coefficient (f) were compared to each other and to two fitness components: lifespan and recruitment. In the larger sample of G. fortis, heterozygosity (H) was correlated with both fitness components, but no relationship was detected in the smaller sample of G. scandens. Analyses of the inbreeding coefficient detected highly significant relationships between f and recruitment, but no relationship between f and overall lifespan. The d2 statistic showed no relationship to either fitness component. When the two SSR-based estimators were compared to f, d2 was correlated with f in G. fortis in the predicted direction, while in G. scandens the relationship was positive. Multilocus heterozygosity was correlated with f in G. fortis but not in the G. scandens sample. A pedigree simulation demonstrated that the variation in true autozygosity can be large among individuals with the same level of inbreeding. This observation may supplement the interpretation of patterns relevant to the local (locus-specific) and general (genome-wide) effects hypotheses, which have been proposed to explain the mechanism responsible for associations between genetic diversity and fitness.

Pigment identification in artwork using graphite furnace atomic absorption spectrometry.

The use of a sampling technique is described for the identification of metals from inorganic pigments in paint. The sampling technique involves gently contacting a cotton swab with the painted surface to physically remove a minute quantity ( approximately 1-2mug) of pigment. The amount of material removed from the painted surface is invisible to the unaided eye and does not cause any visible effect to the painted surface. The cotton swab was then placed in a 1.5ml polystyrene beaker containing HNO(3) to extract pigment metals prior to analysis using graphite furnace atomic absorption spectrometry (GFAAS). GFAAS is well suited for identifying pigment metals since it requires small samples and many pigments consist of main group elements (e.g. Al) as well as transition metals (e.g. Zn, Fe and Cd). Using Cd (cadmium red) as the test element, the reproducibility of sampling a paint surface with the cotton swab was approximately 13% in either a water or oil medium. To test the feasibility of cotton sampling for pigment identification, samples were obtained from paintings (watercolour and oil) of a local collection. Raman spectra provided complementary information to the GFAAS, which together are essential for positive identification of some pigments. For example, GFAAS indicated the presence of Cu, but the Raman spectra positively identified the modern copper pigment phthalocyanine green (Cu(C(32)Cl(16)N(8)). Both Raman spectroscopy and GFAAS were useful for identifying ZnO as a white pigment.

Metal resistance among aerobic chemoheterotrophic bacteria from the deep terrestrial subsurface.

The metal resistance of 350 subsurface bacterial strains from two U.S. Department of Energy facilities, the Savannah River Site (SRS), South Carolina, and the Hanford site, Washington, was determined to assess the effect of metal toxicity on microorganisms in the deep terrestrial subsurface. Resistance was measured by growth inhibition around discs containing optimized amounts of Hg(II), Pb(II), and Cr(VI). A broad range of resistance levels was observed, with some strains of Arthrobacter spp. demonstrating exceptional tolerance. A higher level of resistance to Hg(II) and Pb(II) (P < 0.05) and a higher occurrence of multiple resistances suggested that metals more effectively influenced microbial evolution in subsurface sediments of the SRS than in those of the Hanford site. Common resistance to heavy metals suggests that toxic metals are unlikely to inhibit bioremediation in deep subsurface environments that are contaminated with mixed wastes.

Responsiveness of the Acne-Specific Quality of Life Questionnaire (Acne-QoL) to treatment for acne vulgaris in placebo-controlled clinical trials.

The Acne-Specific Quality of Life Questionnaire (Acne-QoL) was developed to measure the impact of facial acne across four dimensions of patient quality of life. The main objective of the current study was to evaluate the responsiveness of this instrument. Secondarily, this study provided an opportunity to extend the developer's psychometric validation. The Acne-QoL was utilized in two randomized, double-blind, placebo-controlled studies of the efficacy of Estrostep (norethindrone acetate/ethinyl estradiol) in the treatment of facial acne; a total of 296 Estrostep and 295 placebo patients were evaluated. The Acne-QoL was completed at the beginning, middle (cycle 3), and end (cycle 6) of the 6-month treatment period. The responsiveness of the Acne-QoL was demonstrated through its ability to detect both small (baseline to mid-study) and moderate (baseline to study end) treatment advantages for Estrostep patients. Confirmatory factor analysis supported the subscale structure, and internal consistency estimates were excellent. Convergent and discriminant validity were supported by correlations between Acne-QoL scores and clinical measures that were both in the direction and relative magnitude hypothesized. Finally, item response theory analyses confirmed that each item is highly related to its subscale's latent construct and that each subscale is sensitive across a broad range of the underlying continuum. The results of this evaluation confirm that the Acne-QoL is responsive, internally consistent, and valid.

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.

Compliance from self-reported versus pharmacy claims data with metered-dose inhalers.

OBJECTIVE: To determine how well self-reported compliance with metered-dose inhalers (MDIs) agrees with compliance determined from pharmacy claims data. Additional analyses were conducted to establish phychmetric properties of the questionnaire. METHODS: A cross-sectional survey was performed of all adult enrollees with asthma of a managed care organization. The overall return rate was 63.4% (696/1098). Patents with persistent asthma and at least one prescription claim for a controller MDI during the four months preceding the survey were included. Pharmacy claims data were used to calculate a compliance ratio. Self-reported compliance was based on a four-item questionnaire. The primary end points were the percent-agreement and the overall correlation between the two measures. The questionnaire's internal reliability and construct validity were assessed using Cronbach's alpha and Pearson's correlations, respectively. Mean item response scores for compliant patients were compared with scores of noncompliant patients using the Student's t-test. RESULTS: One hundred questionnaire respondents met the study criteria. The mean +/- SD compliance ratio was 0.52+/-0.27, the mean +/- SD self-reported compliance score was 4.07+/-1.0. The overall percent agreement and correlation between self-reported compliance score and the compliance ratio were 75.5% and r = 0.348 (p = 0.01), respectively. The percent agreement was highest (85%) when noncompliant patients (by compliance ratio <0.8) reported being noncompliant (questionnaire score <5). Cronbach's alpha was 0.86. CONCLUSIONS: This study demonstrated a moderate correlation between self-reported asthma compliance and the compliance ratio determined using claims data. The questionnaire may be useful as a screening tool to detect noncompliant behavior in populations or individual patients with asthma.

From concept to culture: the WWAMI program at the University of Washington School of Medicine.

Shortages of primary care physicians have historically affected rural areas more severely than urban and suburban areas. In 1970, the University of Washington School of Medicine (UWSOM) administrators and faculty initiated a four-state, community-based program to increase the number of generalist physicians throughout a predominantly rural and underserved region in the U.S. Northwest. The program developed regional medical education for three neighboring states that lacked their own medical schools, and encouraged physicians in training to practice in the region. Now serving five Northwest states (Washington, Wyoming, Alaska, Montana, and Idaho), the WWAMI program has solidified and expanded throughout its 30-year history. Factors important to success include widespread participation in and ownership of the program by the participating physicians, faculty, institutions, legislatures, and associations; partnership among constituents; educational equivalency among training sites; and development of an educational continuum with recruitment and/or training at multiple levels, including K--12, undergraduate, graduate training, residency, and practice. The program's positive influences on the UWSOM have included historically early attention to primary care and community-based clinical training and development of an ethic of closely monitored innovation. The use of new information technologies promises to further expand the ability to organize and offer medical education in the WWAMI region.

A process for reducing workload and enhancing residents' education at an academic medical center.

Academic medical centers are under increasing pressure to find alternatives to residents for the provision of patient care and to expand and improve the educational opportunities for residents. To address these concerns, the authors performed a study of the medical wards at Harborview Medical Center, a county-owned medical center managed by the University of Washington School of Medicine. Admitting diagnoses, provider names, and billings were obtained from professional practice plan billing records. Based on the distribution of admitting diagnoses, a subset of patients was identified that could be removed from routine care by residents and could instead be cared for by non-physician providers (i.e., physician assistants and nurse practitioners) using clinical pathways. The cohort was large enough to reduce the number of patients per resident to within national accreditation guidelines, and to provide faculty with more time available for teaching. The authors summarize the approach used to identify the new model for care delivery indicated above and the plans made to implement that model and to analyze its impact on the quality of patient care, hospital costs, residents' education, and the process of implementing change. The authors conclude that solutions to the problems of workload and education that they confronted will vary by department and hospital setting. Yet a systematic approach to discovering solutions, such as they present, can be adapted to any setting.

Surveying graduates of one school to determine regional workforce demand.

PURPOSE: To study the demand for physician graduates from one school in one region of the country. The use of demand as a measure of potential regional variation should be of interest to medical educators and policymakers. METHOD: All residency graduates of the University of Washington School of Medicine between 1975 and 1995 (n = 3,824) were surveyed about their ability to gain employment in a timely manner and whether they were recruiting physicians for their practices. RESULTS: The response rate was 50.29%. A non-responder survey was done using a subsample (n = 200), with a 28% return. Over 95% of the graduates had found employment in their desired specialties and locations within two years of finishing their residencies. This was the same for graduates over all years. Approximately 30% of all practices of respondents within the Northwest region were recruiting for new physicians (26% of specialty practices and 34% of generalist practices were recruiting). There was no difference between recruitment in the urban and rural practices or between respondents to the initial survey and those responding to the follow up. CONCLUSIONS: Despite a significant oversupply of specialist physicians and at least a sufficient supply of generalist physicians nationally, there appears to be a strong demand for both specialists and generalists in the Northwest region of the country. This raises questions concerning the use of national averages to inform the education policies in specific regions of the country. More validated measures of demand are needed for future studies.

Postoperative inflammation in a patient with multiple sclerosis.

We report a 51-year old white woman with a history of relapsing episodes of multiple sclerosis (MS) who developed acute anterior uveitis 11 days after having uneventful phacoemulsification with posterior chamber intraocular lens implantation. Topical corticosteroids relieved the pain and inflammation within hours. A week after the episode of anterior uveitis, the patient had a severe systemic relapse of MS. Acute inflammation in MS patients during the postoperative period may be noninfectious and could be a prodrome for a relapse of MS.

Effect of Host Plant Resistance and Reduced Rates and Frequencies of Fungicide Application to Control Potato Late Blight.

Field experiments were conducted during 1998 to 2000 to determine the response of commercial potato cultivars and advanced breeding lines (ABL) differing in susceptibility to foliar late blight (caused by Phytophthora infestans) to reduced rates and frequencies of residual, contact fungicide applications. When environmental conditions were most favorable for the development of late blight, the lowest application rate of the fungicides chlorothalonil or fluazinam (33% of the manufacturers' recommended application rate [MRAR]) gave unsatisfactory control of potato late blight. Under conditions moderately conducive for late blight development, effective control was achieved with 33 to 66% MRAR with either fungicide. The Michigan State University advanced selection, MSG274-3, was the least susceptible ABL tested and, during 1998 to 2000, late blight was effectively managed using reduced rates of fungicides. Application rates of chlorothalonil (33 to 100% MRAR) significantly reduced late blight in the cultivar Snowden (5-day application interval) compared with the nontreated control; whereas, late blight was not effectively controlled in Snowden even at 100% MRAR of chlorothalonil at either 10- or 15-day application intervals in 1999 or 2000. The ABL MSG274-3 was the least susceptible of all cultivars and ABL used in this study, and required minimal chemical protection against late blight. The study demonstrates that ABL with reduced susceptibility to late blight can be managed with reduced fungicide rates and longer application intervals, thus offering more economical control of this disease.