Management of chronic wasting disease in ranched elk: conclusions from a longitudinal three-year study.

Chronic wasting disease is a fatal, horizontally transmissible prion disease of cervid species that has been reported in free-ranging and farmed animals in North America, Scandinavia, and Korea. Like other prion diseases, CWD susceptibility is partly dependent on the sequence of the prion protein encoded by the host's PRNP gene; it is unknown if variations in PRNP have any meaningful effects on other aspects of health. Conventional diagnosis of CWD relies on ELISA or IHC testing of samples collected post-mortem, with recent efforts focused on antemortem testing approaches. We report on the conclusions of a study evaluating the role of antemortem testing of rectal biopsies collected from over 570 elk in a privately managed herd, and the results of both an amplification assay (RT-QuIC) and conventional IHC among animals with a several PRNP genotypes. Links between PRNP genotype and potential markers of evolutionary fitness, including pregnancy rates, body condition, and annual return rates were also examined. We found that the RT-QuIC assay identified significantly more CWD positive animals than conventional IHC across the course of the study, and was less affected by factors known to influence IHC sensitivity - including follicle count and PRNP genotype. We also found that several evolutionary markers of fitness were not adversely correlated with specific PRNP genotypes. While the financial burden of the disease in this herd was ultimately unsustainable for the herd owners, our scientific findings and the hurdles encountered will assist future CWD management strategies in both wild and farmed elk and deer.

Vimentin regulates lung cancer cell adhesion through a VAV2-Rac1 pathway to control focal adhesion kinase activity.

Vimentin is an intermediate filament protein whose expression correlates with increased metastatic disease, reduced patient survival and poor prognosis across multiple tumor types. Despite these well-characterized correlations, the molecular role of vimentin in cancer cell motility remains undefined. To approach this, we used an unbiased phosphoproteomics screen in lung cancer cell lines to discover cell motility proteins that show significant changes in phosphorylation upon vimentin depletion. We identified the guanine nucleotide exchange factor (GEF), VAV2, as having the greatest loss of phosphorylation owing to vimentin depletion. Since VAV2 serves as a GEF for the small Rho GTPase Rac1, a key player in cell motility and adhesion, we explored the vimentin-VAV2 pathway as a potential novel regulator of lung cancer cell motility. We show that VAV2 localizes to vimentin-positive focal adhesions (FAs) in lung cancer cells and complexes with vimentin and FA kinase (FAK). Vimentin loss impairs both pY142-VAV2 and downstream pY397-FAK activity showing that vimentin is critical for maintaining VAV2 and FAK activity. Importantly, vimentin depletion reduces the activity of the VAV2 target, Rac1, and a constitutively active Rac1 rescues defects in FAK and cell adhesion when vimentin or VAV2 is compromised. Based upon this data, we propose a model whereby vimentin promotes FAK stabilization through VAV2-mediated Rac1 activation. This model may explain why vimentin expressing metastatic lung cancer cells are more motile and invasive.

Chronic treatment with a GPR30 antagonist impairs acquisition of a spatial learning task in young female rats.

We hypothesize that the beneficial effects of estradiol on cognitive performance may be mediated through GPR30, a putative membrane target of estrogens. Recently we showed that administration of a selective GPR30 agonist (G-1) to ovariectomized rats enhanced acquisition of a delayed matching-to-position (DMP) T-maze task and increased potassium-stimulated acetylcholine release in the hippocampus, similar to estradiol (E2) (Hammond et al., 2009). The present study tested whether treating with a selective GPR30 antagonist (G-15) would impair spatial learning in gonadally intact rats and in ovariectomized (OVX) rats treated with E2. As predicted, G-15 dose-dependently impaired DMP acquisition both in gonadally intact rats and in OVX rats treated with E2. G-15 specifically reduced the rate of acquisition, and this effect was associated with an increased predisposition to adopt a persistent turn. In contrast, G-15 alone at the highest dose had no significant effect on DMP acquisition in OVX controls. The effects were task dependent, as similar effects of G-15 were not observed in gonadally intact rats tested on an operant discrimination/reversal learning task motivated by the same food reward. This suggests that the effects on DMP acquisition were not due to effects on motivation for food. Effects of G-15 on DMP acquisition were similar to previously published work showing significant impairment produced by selective cholinergic denervation of the hippocampus. These data suggest that GPR30 can play an important role in mediating the effects of estradiol on spatial learning, possibly by mediating estradiol effects on basal forebrain cholinergic function.

Degradation of aromatic hydrocarbons by Sphingomonas paucimobilis strain EPA505.

To determine the substrate range capability of Sphingomonas paucimobilis strain EPA505, a number of aromatic compounds were tested as potential growth substrates. Strain EPA505 grew on phenanthrene, naphthalene, fluoranthene, toluene, benzoic acid, 2,3- and 3,4-dihydroxybenzoic acids, 1-chloro-2,4-dinitrobenzene, anthracene, 2-hydroxy-3-naphthoic acid and 1-hydroxy- 2-naphthoic acid, salicylic acid, and catechol. Strain EPA505 was unable to grow on coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, pyrene, benzo[b]fluoranthene, and fluorene. Catabolic products were not detected or identified when the bacterium was incubated with coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, or benzo[b]fluoranthene. Dihydroxypyrene, the ortho ring fission product of pyrene, and 10-hydroxy-1- phenanthroic acid were detected when the bacterium was incubated with pyrene. The open rings of benzo[b]fluoranthene, hydroxyacephenanthroic acid, hydroxyacephenanthrene, and phenanthrene anhydride, catabolites of benzo[b]fluoranthene degradation, were detected with Tn5 mutants of EPA505. With strain EPA505, both 9-fluorenone and an open ring fission product accumulated during incubation with fluorene. Other catabolites beyond the open ring of fluorene were detected, specifically dihydroxyfluorene, hydroxy-9-fluorenone, dihydroxy-9-fluorenone, hydroxyindane, and a putative glutathione-conjugated benzylanhydride. Benzylanhydride appeared to be a final end product of fluorene degradation by strain EPA505.

Transcription and replication of a natural hepatitis B virus nucleocapsid promoter variant is regulated in vivo by peroxisome proliferators.

A hepatitis B virus (HBV) transgenic mouse containing a naturally occurring mutation in the nucleocapsid promoter (A1764T plus G1766A) that inhibits the retinoid X receptor alpha (RXRalpha) plus peroxisome proliferator-activated receptor alpha (PPARalpha) heterodimer from binding to the proximal nuclear hormone receptor recognition sequence has been generated. Viral transcription and replication occur in the liver and kidney. The nucleocapsid promoter mutation does not prevent peroxisome proliferators from increasing viral transcription and replication in the liver of these variant HBV transgenic mice. This suggests that peroxisome proliferators may enhance viral transcription directly in a PPARalpha-dependent manner through the nuclear hormone receptor recognition site in the enhancer 1 region of the HBV genome. Hepatocyte nuclear factor 4 (HNF4) binding to the proximal nuclear hormone receptor recognition sequence in the nucleocapsid promoter appears to limit RNA synthesis from the precore transcription initiation site. Consequently, the variant HBV transgenic mice transcribe very little precore RNA and secrete extremely low levels of hepatitis B e antigen (HBeAg) compared with the wild-type HBV transgenic mice. This is consistent with the suggestion that viruses expressing HBeAg are preferentially eliminated in infected individuals when they seroconvert from HBeAg positive to anti-HBe antibody-positive status, leaving escape HBV variants that have reduced HBeAg expression.

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505.

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.

Colonization of mine tailings by marine invertebrates.

An experiment was conducted to determine if a tailings substrate would inhibit recolonization of benthic macrofauna upon closure of a submarine tailings disposal (STD) operation. Trays of defaunated marine sediment, serving as a reference, and trays of tailings from a proposed gold mine were placed at 21 m depth on the ocean floor to allow colonization via settlement from the water column. Trays of reference sediment and tailings, and cores of ambient sediment were collected 9, 17, and 22 months after tray placement. Probable tray effects, which were desirable given the objectives of this study, precluded direct comparison of ecological succession in the tray sediments to the ambient assemblage. The ambient macrofauna assemblage was distinguishable from the reference sediment and tailings assemblages throughout the experiment and displayed more pronounced seasonality. Differences between the reference sediment and tailings assemblages were generally insignificant, including total abundance, total biomass, number of taxa, average size of individuals, numerically dominant taxa, abundance by ecological guilds, and overall community composition. Upon cessation of STD, recolonization of a benthic macrofauna community should not be inhibited by the presence of these tailings as a benthic substrate.

Nuclear covalently closed circular viral genomic DNA in the liver of hepatocyte nuclear factor 1 alpha-null hepatitis B virus transgenic mice.

The role of hepatocyte nuclear factor 1alpha (HNF1 alpha) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1 alpha-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1 alpha transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1 alpha protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1 alpha-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1 alpha-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.

Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences.

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method.

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species.

Competitive PCR-DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy.

Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments from environmental samples by denaturing gradients of chemicals or heat [denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have addressed this problem by the construction and evaluation of a quantitative standard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the psyllid Anomoneura mori, a gamma-proteobacterium. This sequence is the most AT-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatured under exceptionally low stringency denaturing conditions. The native sequence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molecules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost.

Identification of four structural genes and two putative promoters necessary for utilization of naphthalene, phenanthrene, fluoranthene by Sphingomonas paucimobilis var. EPA505.

Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tc(p-)) or luminescence (luxAB(p-)). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap(-)phe(-)fla(-)), classII (nap(-)phe(-)), and classIII (fla(-)). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.

Frequency, predictors, and appropriateness of blood transfusion after percutaneous coronary interventions.

Increased awareness of the risks of blood-borne infections has recently led to profound changes in the practice of transfusion medicine. These changes include, among others, the development of guidelines by the American College of Physicians (ACP) for transfusion. Although the incidence and predictors of vascular complications of percutaneous interventions have been well defined, there are currently no data on frequency, risk factors, and appropriateness of blood transfusions. We performed a retrospective analysis of 628 consecutive percutaneous coronary revascularization procedures. Predictors of blood transfusion were identified using multivariate logistic regression analysis. Appropriateness of transfusions was determined using modified ACP guidelines. Transfusions were administered after 8.9% of interventions (56 of 628). Multivariate analysis identified age >70 years, female gender, procedure duration, coronary stenting, acute myocardial infarction, postprocedural use of heparin and intra-aortic balloon pump placement as independent predictors of blood transfusions (all p <0.05). According to the ACP guidelines, 36 of 56 patients (64%) received transfusions inappropriately. Transfusion reactions (fever) occurred in 10% of patients who received tranfusions appropriately and in 5% of patients who received tranfusions inappropriately. The estimated additional costs per procedure related to transfusions were $551 and $419, respectively. In conclusion, unnecessary transfusions were performed frequently after percutaneous coronary interventions. Application of available guidelines could reduce the number of unnecessary transfusions, thus avoiding exposure of patients to additional risks and reducing procedural costs.

Design and construction of model stream ecosystems.

A series of 12 outdoor model stream mesocosms was designed to evaluate the effects of chemicals and mixtures on the biota in stream ecosystems. An integrated design plan incorporated physical, chemical, and biological factors as well as the specific experimental objectives and effects parameters to be evaluated. Analysis of biological assemblages such as macroinvertebrates and periphyton in the model stream mesocosms demonstrated the presence of diverse and sensitive taxa. These model stream mesocosms also included the ability to evaluate responses of sentinel fish species such as Pimephales promelas and Lepomis macrochirus as well as macrophytes. The design and construction of the stream are discussed in detail and a brief description of a typical experimental protocol is provided. Experiments, to date, have spanned 14-30 days pretreatment "colonization" with 30 days of treatment and 15 days of posttreatment observation. Precision and accuracy of test chemical delivery to the stream have been excellent and the overall experimental design has been useful for delivering threshold toxicity concentrations and evaluating ecosystem potency for the chemicals tested.

Health care network communications infrastructure: an engineering design for the Military Health Service System.

The Military Health Service System (MHSS) provides health care for the Department of Defense (DOD). This system operates on an annual budget of $15 Billion, supports 127 medical treatment facilities (MTFs) and 500 clinics, and provides support to 8.7 million beneficiaries worldwide. To support these facilities and their patients, the MHSS uses more than 125 different networked automated medical systems. These systems rely on a heterogeneous telecommunications infrastructure for data communications. With the support of the Defense Medical Information Management (DMIM) Program Office, our goal was to identify the network requirements for DMIM migration and target systems and design a communications infrastructure to support all systems with an integrated network. This work used tools from Business Process Reengineering (BPR) and applied it to communications infrastructure design for the first time. The methodology and results are applicable to any health care enterprise, military or civilian.

An analysis of the cause of early mortality after administration of thrombolytic therapy. The Thrombolysis Angioplasty in Myocardial Infarction Study Group.

BACKGROUND: The use of thrombolytic therapy in myocardial infarction has been associated with a considerable improvement in survival rate; however, almost 40% of the deaths during hospitalization occur during the first 24 h. Clinical and angiographic characteristics identified through careful comparison of those patients who die early with those who survive may serve as important targets for the development of new strategies for the management of myocardial infarction. METHODS: Medical records and autopsy reports of 810 patients enrolled into four sequential studies evaluating thrombolytic therapy and angioplasty in acute myocardial infarction were reviewed. All patients were enrolled into four similar protocols with administration of thrombolytic therapy (intravenous tissue plasminogen activator in 561 patients, urokinase in 102, and a combination of tissue plasminogen activator and urokinase in 147) and acute cardiac catheterization performed 90 min after starting therapy. RESULTS: The overall in-hospital mortality rate was 6.8% (55 out of 810), with 21 of these deaths (38%) occurring within the first day. The median (25th, 75th percentile) time to death was 3 (0, 12) days. Infarct location was more frequently anterior in patients who died within the first day. Patients who died 24 h after admission to hospital had the lowest patency rate (45%) compared with patients who died within 24 h (59%) and those who survived (71%, P = 0.003). The deaths within the first day were more likely to be a result of cardiogenic shock (48%), ventricular arrhythmias (14%), or cardiac rupture (9%), whereas late deaths were more likely to be a result of recurrent ischemia or reinfarction (32%) and non-cardiac causes (18%). Two patients had an intracranial hemorrhage within the first 24 h which caused immediate death in one and death on the third day of hospitalization in the other. CONCLUSION: Mortality within the first 24 h of thrombolytic therapy administration can be defined by inadequate myocardial reperfusion in patients with cardiac failure, possibly associated reperfusion injury leading to cardiac rupture, and an increased risk of intracranial hemorrhage. These factors may serve as targets for the development of new treatment strategies in acute myocardial infarction that may alter prognosis.

Homeless women. The context of an urban shelter.

The role of the nurse in a shelter has just begun to emerge. The prevalence of homeless women is escalating, and nurses are playing primary roles in teaching, health care, social support, and case finding for women in shelters. This article describes some of the health, social, and psychologic issues facing women in a large urban shelter from the perspective of a senior nurse psychologist at Boston's Long Island Shelter.

Effects of proteolytic and thermal action on the activity of lactate dehydrogenase isozymes LD-1 and LD-5.

L-Lactate dehydrogenase (LD) catalyzes the interconversion of pyruvate and lactate. Using a spectrophotometric assay to determine LD activity, incubation of rabbit, porcine, and bovine LD-1 and LD-5 isozymes with the protease subtilisin (Carlsberg) gave first-order degradation kinetics. Degradation half-lives were significantly lower for the LD-5 isozymes from the three species when incubated with subtilisin at temperatures from 4 degrees C to 25 degrees C. The energy involved in the degradation process, however, was not different. The activation energy for the conversion of pyruvate to lactate by LD-1 at pH 7.4 was significantly higher than that for LD-5 for all three species examined (P less than 0.005). Thermocalorimetry showed that the LD-1 isozymes have both a higher mean temperature of denaturation and a higher heat uptake during the denaturation process than corresponding LD-5 forms. The results suggest that the LD-5 isozymes in the species studied are more metabolically efficient, whereas the LD-1 forms have greater structural stability.