Characterization of a Multiple-Scan-Rate Voltammetric Waveform for Real-Time Detection of Met-Enkephalin.

Opioid peptides are critically involved in a variety of physiological functions necessary for adaptation and survival, and as such, understanding the precise actions of endogenous opioid peptides will aid in identification of potential therapeutic strategies to treat a variety of disorders. However, few analytical tools are currently available that offer both the sensitivity and spatial resolution required to monitor peptidergic concentration fluctuations in situ on a time scale commensurate with that of neuronal communication. Our group has developed a multiple-scan-rate waveform to enable real-time voltammetric detection of tyrosine containing neuropeptides. Herein, we have evaluated the waveform parameters to increase sensitivity to methionine-enkephalin (M-ENK), an endogenous opioid neuropeptide implicated in pain, stress, and reward circuits. M-ENK dynamics were monitored in adrenal gland tissue, as well as in the dorsal striatum of anesthetized and freely behaving animals. The data reveal cofluctuations of catecholamine and M-ENK in both locations and provide measurements of M-ENK dynamics in the brain with subsecond temporal resolution. Importantly, this work also demonstrates how voltammetric waveforms can be customized to enhance detection of specific target analytes, broadly speaking.

Assessing the cumulative impacts of geographically isolated wetlands on watershed hydrology using the SWAT model coupled with improved wetland modules.

Despite recognizing the importance of wetlands in the Coastal Plain of the Chesapeake Bay Watershed (CBW) in terms of ecosystem services, our understanding of wetland functions has mostly been limited to individual wetlands and overall catchment-scale wetland functions have rarely been investigated. This study is aimed at assessing the cumulative impacts of wetlands on watershed hydrology for an agricultural watershed within the Coastal Plain of the CBW using the Soil and Water Assessment Tool (SWAT). We employed two improved wetland modules for enhanced representation of physical processes and spatial distribution of riparian wetlands (RWs) and geographically isolated wetlands (GIWs). This study focused on GIWs as their hydrological impacts on watershed hydrology are poorly understood and GIWs are poorly protected. Multiple wetland scenarios were prepared by removing all or portions of the baseline GIW condition indicated by the U.S. Fish and Wildlife Service National Wetlands Inventory geospatial dataset. We further compared the impacts of GIWs and RWs on downstream flow (i.e., streamflow at the watershed outlet). Our simulation results showed that GIWs strongly influenced downstream flow by altering water transport mechanisms in upstream areas. Loss of all GIWs reduced both water routed to GIWs and water infiltrated into the soil through the bottom of GIWs, leading to an increase in surface runoff of 9% and a decrease in groundwater flow of 7% in upstream areas. These changes resulted in increased variability of downstream flow in response to extreme flow conditions. GIW loss also induced an increase in month to month variability of downstream flow and a decrease in the baseflow contribution to streamflow. Loss of all GIWs was shown to cause a greater fluctuation of downstream flow than loss of all RWs for this study site, due to a greater total water storage capacity of GIWs. Our findings indicate that GIWs play a significant role in controlling hydrological processes in upstream areas and downstream flow and, therefore, protecting GIWs is important for enhanced hydrological resilience to extreme flow conditions in this region.

Carbon in Natural, Cultivated, and Restored Depressional Wetlands in the Mid-Atlantic Coastal Plain.

Aerial extent of wetland ecosystems has decreased dramatically since precolonial times due to the conversion of these areas for human use. Wetlands provide various ecosystem services, and conservation efforts are being made to restore wetlands and their functions, including soil carbon storage. This Mid-Atlantic Regional USDA Wetland Conservation Effects Assessment Project study was conducted to evaluate the effects and effectiveness of wetland conservation practices along the Mid-Atlantic Coastal Plain. This study examined 48 wetland sites in Delaware, Maryland, Virginia, and North Carolina under natural, prior converted cropland, and 5- to 10-yr post wetland restoration states. The North Carolina sites mainly contained soils dominated by organic soil materials and therefore were analyzed separately from the rest of the sites, which primarily contained mineral soils. Soil samples were collected using the bulk density core method by horizon to a depth of 1 m and were analyzed for percent carbon. The natural wetlands were found to have significantly greater carbon stocks (21.5 ± 5.2 kg C m) than prior converted croplands (7.95 ± 1.93 kg C m; < 0.01) and restored wetlands (4.82 ± 1.13 kg C m; < 0.001). The restored and prior converted sites did not differ significantly, possibly the result of the methods used to restore the wetlands, and the relatively young age of the restored sites. Wetlands were either restored by plugging drainage structures, with minimal surface disturbance, or by scraping the surface (i.e., excavation) to increase hydroperiod. Sites restored with the scraping technique had significantly lower carbon stocks (2.70 ± 0.38 kg C m) than those restored by passive techniques (6.06 ± 1.50 kg C m; = 0.09). Therefore, techniques that involve excavation and scraping to restore hydrology appear to negatively affect C storage.

RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma.

RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.

Soil physicochemical conditions, denitrification rates, and abundance in north Carolina coastal plain restored wetlands.

Over the last century, North Carolina has seen a severe reduction in the percentage of wetlands and a rise in negative environmental impacts related to this loss. To counter these effects, efforts have been enacted to mitigate wetland loss and create new wetland areas. The objective of this study was to assess the impact of hydrological restoration at several sites in the North Carolina coastal plain. Nine sites were selected for study. Hydrologically restored wetlands were compared with natural wetlands and prior converted (PC) croplands (i.e., historic wetlands under agricultural production). Each site was analyzed along a relative wetness gradient, and physicochemical properties, denitrification enzyme activity, and NO reductase gene () abundances using real-time PCR were measured. Physicochemically, restoration resulted in significantly increased levels of total C as compared with PC cropland sites. Restored wetland sites also saw pH, soil moisture, P, and NO+NO approximate levels similar to those of natural wetlands. Denitrification enzyme activity rates varied based on relative wetness within individual sites, generally increasing with increasing soil moisture. However, denitrification tended to be lower in restored wetland sites relative to natural wetlands. Gene abundances of saw statistically significant decreases in restored wetland soils. In conclusion, although analysis of restored wetlands reveals clear changes in several physicochemical characteristics and significant decreases in gene abundances, restoration efforts appear to have not significantly affected the denitrification component of the N cycle.

Response of coliform populations in streambed sediment and water column to changes in nutrient concentrations in water.

As sediments increasingly become recognized as reservoirs of indicator and pathogen microorganisms, an understanding of the persistence of indicator organisms becomes important for assessment and predictions of microbial water quality. The objective of this work was to observe the response of water column and sediment coliform populations to the change in nutrient concentrations in the water column. Survival experiments were conducted in flow-through chambers containing sandy sediments. Bovine feces were collected fresh and introduced into sediment. Sixteen days later, the same fecal material was autoclaved and diluted to provide three levels - 1×, 0.5×, and 0.1× of nutrient concentrations - spike in water column. Total coliforms, Escherichia coli, and total aerobic heterotrophic bacterial concentrations were monitored in water and sediment. Bacteria responded to the nutrient spike with initial growth both in the water column and in sediment. The response of bacterial concentrations in water column was nonlinear, with no significant changes at 0.1 and .5× spikes, but a substantial change at 1× spike. Bacteria in sediment responded to the spikes at all added nutrient levels. Coliform inactivation rates both in sediment and in water after the initial growth occurred, were not significantly different from the inactivation rates before spike. These results indicate that introduction of nutrients into the water column results in nonlinear response of E. coli concentrations both in water and in sediments, followed by the inactivation with the same rate as before introduction of nutrients.

The impact of agricultural soil erosion on the global carbon cycle.

Agricultural soil erosion is thought to perturb the global carbon cycle, but estimates of its effect range from a source of 1 petagram per year(-1) to a sink of the same magnitude. By using caesium-137 and carbon inventory measurements from a large-scale survey, we found consistent evidence for an erosion-induced sink of atmospheric carbon equivalent to approximately 26% of the carbon transported by erosion. Based on this relationship, we estimated a global carbon sink of 0.12 (range 0.06 to 0.27) petagrams of carbon per year(-1) resulting from erosion in the world's agricultural landscapes. Our analysis directly challenges the view that agricultural erosion represents an important source or sink for atmospheric CO2.

Isoforms of Wilms' tumor suppressor gene (WT1) have distinct effects on mammary epithelial cells.

The role of WT1 (Wilm's tumor suppressor gene) in breast cancer is controversial, with evidence for both tumor-promoting and tumor-suppressing activities. In order to address this question, we expressed different WT1 isoforms in the mammary epithelial cell line H16N-2, which does not express endogenous WT1. Cells were stably transfected with either WT1 (-Ex5/-KTS) or WT1 (+Ex5/+KTS) under the control of the inducible metallothionein promoter. Induction of WT1 (-Ex5/-KTS) upregulated p21, causing a slowing of proliferation and a G2-phase cell cycle arrest. In artificial basement membrane, the WT1 (-Ex5/-KTS) isoform promoted the appearance of highly organized acinar cellular aggregates. In contrast, WT1 (+Ex5/+KTS) had no effect on p21 or proliferation, but rather caused an epithelial-mesenchymal transition and a redistribution of E-cadherin from the cell membrane to the cytoplasm. This isoform also causes the cellular aggregates growing in artificial basement membrane to appear significantly less organized than control cells. Thus, different WT1 isoforms have distinct effects in this cell line, suggesting that depending on the ratio of WT1 isoform expression in mammary epithelial cells, WT1 could function to either promote or suppress a transformed phenotype.

Fate of dietary perchlorate in lactating dairy cows: Relevance to animal health and levels in the milk supply.

Perchlorate is a goitrogenic anion that competitively inhibits the sodium iodide transporter and has been detected in forages and in commercial milk throughout the U.S. The fate of perchlorate and its effect on animal health were studied in lactating cows, ruminally infused with perchlorate for 5 weeks. Milk perchlorate levels were highly correlated with perchlorate intake, but milk iodine was unaffected, and there were no demonstrable health effects. We provide evidence that up to 80% of dietary perchlorate was metabolized, most likely in the rumen, which would provide cattle with a degree of refractoriness to perchlorate. Data presented are important for assessing the environmental impact on perchlorate concentrations in milk and potential for relevance to human health.

Cardiac disease in the antiphospholipid syndrome: recommendations for treatment. Committee consensus report.

The Committee reviewed cardiac involvement in the antiphospholipid antibody syndrome. The Committee's recommendations are: Valve abnormalities: anticoagulation is recommended for symptomatic patients with valvulopathy. Prophylactic antiplatelet therapy may be appropriate for asymptomatic patients (recommended by 13/17 experts in an independent review). Committee members disagreed whether corticosteroid therapy is helpful, but agree that distinguishing among presumptive valvulitis (valve thickening on echocardiogram), valve deformity and vegetations is important, as treatment implications may differ. Occlusive arterial disease (angina, myocardial infarction): the Committee recommends aggressive treatment of all risk factors for atherosclerosis (hypertension, hypercholesterolaemia, smoking) and liberal use of folic acid, B vitamins and cholesterol-lowering drugs (preferably statins). Hydroxychloroquine for cardiac protection in APS patients may be considered. The Committee also recommends warfarin anticoagulation for those who have suffered thrombosis in the absence of atherosclerosis, but recognizes that developing data may support the use of antiplatelet agents instead. Intracardiac thrombi: the Committee recommends intensive warfarin anticoagulation, and consultation with cardiac surgeons when appropriate. Ventricular dysfunction: the Committee has no recommendations on this aspect of cardiac disease. Pulmonary hypertension: the Committee recommends intensive anticoagulation with warfarin and clinical trials of bosentan, epoprostenol and other new agents.

Response of soil microbiological activities to cadmium, lead, and zinc salt amendments.

Heavy metal pollution of soil has been recognized as a major factor impeding soil microbial processes. From this perspective, we studied responses of the soil biological activities to metal stress simulated by soil amendment with Zn, Pb, and Cd chlorides. The amounts of heavy metal salts added to five metal-polluted soils and four nonpolluted soils were selected to match the total metal concentrations typically found in polluted soils of the Silesia region of Poland. From the perspective of soil quality, metal mobility in amended soils could not be described by simple functions of pH or organic matter. Reaction of Pb with the soil caused strong immobilization with less than 1% of the Pb amendment recovered by 0.01 M CaCl2 extractions. Immobilization of Cd was also significant, whereas immobilization of the Zn amendment was much weaker than that of Cd or Pb. The Zn amendment had substantial inhibitory effect on soil dehydrogenase, acid and alkaline phosphatase, arylsulfatase, urease, and nitrification potential. Generally, Cd and Pb had limited or stimulatory effect on most of these biological activities, with an exception of Pb strongly inhibiting soil urease. The effect of the metal amendments on biological activities could not be satisfactorily accounted for by metal toxicity because no strong relationship was observed between extractable metal content and the degree of inhibition. The Zn amendment had a significant effect on soil pH, resulting in confounding effects of pH and Zn toxicity on activities. Metal amendment experiments seem to be of limited utility for meaningful assessment of metal contamination effects on soil quality.

The potential of diffuse reflectance spectroscopy for the determination of carbon inventories in soils.

Investigations have shown that near- and mid-infrared reflectance spectroscopy can accurately determine organic-C in soil. Efforts have also demonstrated that both can differentiate between organic and inorganic-C in soils, but the mid-infrared produces more accurate calibrations. Nevertheless, the greatest benefit would come with in situ determinations where factors such as particle size, sample heterogeneity and moisture can be important. While the variations in large (> 20 mesh) particle size can adversely effect calibration accuracy, efforts have demonstrated that the scanning of larger amounts of sample can overcome this, but the effects of moisture have not been fully explored. While under in situ conditions C distribution and sample heterogeneity are a problem for any analytical method, the rapid analysis possible with spectroscopic techniques will allow many more samples to be analyzed. In conclusion, near- and mid-infrared spectroscopy have great potential for providing the C values needed for C sequestration studies.

Impact of soil movement on carbon sequestration in agricultural ecosystems.

Recent modeling studies indicate that soil erosion and terrestrial sedimentation may establish ecosystem disequilibria that promote carbon (C) sequestration within the biosphere. Movement of upland eroded soil into wetland systems with high net primary productivity may represent the greatest increase in storage capacity potential for C sequestration. The capacity of wetland systems to capture sediments and build up areas of deposition has been documented as well as the ability of these ecosystems to store substantial amounts of C. The purpose of our work was to assess rates of sediment deposition and C storage in a wetland site adjacent to a small first-order stream that drains an agricultural area. The soils of the wetland site consist of a histosol buried by sediments from the agricultural area. Samples of deposited sediments in the riparian zone were collected in 5 cm increments and the concentration of 137Cs was used to determine the 1964 and 1954 deposition layers. Agricultural activity in the watershed has caused increased sediment deposition to the wetland. The recent upland sediment is highly enriched in organic matter indicating that large amounts of organic C have been sequestered within this zone of sediment deposition. Rates of sequestration are much higher than rates that have occurred over the pre-modern history of the wetland. These data indicate the increased sedimentation rates in the wetland ecosystem are associated with increased C sequestration rates.

Indirect institutional revenue generated from an academic primary care clinical network.

BACKGROUND AND OBJECTIVES: As the financial performances of US academic health centers have faltered under managed care and the Balanced Budget Act of 1997, increasing attention has been paid to the costs and benefits of operating primary care networks. This study examines the indirect revenues to a university hospital and faculty group practice that result from such a primary care network using a method of abstracting billing data. METHODS: A primary care patient cohort was identified by selecting all patients who generated at least one charge in any of the 10 primary care clinics in the network over a 15-month period. All charges from the hospital and the faculty practice group for this cohort were then examined during a 6-month period, and the total charges generated in the primary care setting were compared with charges generated elsewhere in the health system. RESULTS: The primary care patient cohort included 56,459 patients and generated a total of $7,243,312 in charges for primary care services, $43,559,741 of charges in the hospital billing system for non-primary care services, and $8,825,611 of charges for services from specialty faculty. This cohort accounted for 18.5% of the gross charges for hospital care and 17.6% of charges generated by the specialty physicians. CONCLUSIONS: Using a simple and replicable methodology, this study estimates a substantial financial benefit to the hospital and specialty practices from a primary care network.

Mid-infrared diffuse reflectance spectroscopy for the quantitative analysis of agricultural soils.

Soil samples were analyzed conventionally and by mid-infrared diffuse reflectance spectroscopy for total C, total N, pH, and measures of biological activity. Ground, non KBr diluted, samples (n = 180) from experimental plots (two locations, three replicate plots, under plow and no-till practices, three rates of N fertilizer, and from five depths) were scanned from 4000 to 400 cm(-1) (4-cm(-1) resolution, 64 co-added scans) on a DigiLab FTS-60 Fourier transform spectrometer using a custom-made linear sample transport (50 by 2 mm sample area scanned). Results using partial least-squares regression showed that accurate calibrations can be developed for the determination of a number of compositional parameters: total C, total N, pH, and many measures of biological activity. In general, the results achieved using mid-infrared spectra were at least as accurate as those found previously using near-infrared spectra and were sometimes significantly better, that is, pH.

An innovative approach for locating and evaluating subsurface pathways for nitrogen loss.

Fundamental watershed-scale processes governing chemical flux to neighboring ecosystems are so poorly understood that effective strategies for mitigating chemical contamination cannot be formulated. Characterization of evapotranspiration, surface runoff, plant uptake, subsurface preferential flow, behavior of the chemicals in neighboring ecosystems, and an understanding of how crop management practices influence these processes are needed. Adequate characterization of subsurface flow has been especially difficult because conventional sampling methods are ineffective for measuring preferential flow of water and solutes. A sampling strategy based on ground-penetrating radar (GPR) mapping of subsurface structures coupled with near real-time soil moisture data, surface topography, remotely sensed imagery, and a geographic information system (GIS) appears to offer a means of accurately identifying subsurface preferential flow pathways. Four small adjacent watersheds draining into a riparian wetland and first-order stream at the USDA-ARS Beltsville Agricultural Research Center, Beltsville, MD are being studied with this protocol. The spatial location of some preferential flow pathways for chemicals exiting these agricultural watersheds to the neighboring ecosystems have been identified. Confirmation of the pathways is via examination of patterns in yield monitor data and remote sensing imagery.

Surface and subsurface nitrate flow pathways on a watershed scale.

Determining the interaction and impact of surface runoff and subsurface flow processes on the environment has been hindered by our inability to characterize subsurface soil structures on a watershed scale. Ground penetrating radar (GPR) data were collected and evaluated in determining subsurface hydrology at four small watersheds in Beltsville, MD. The watersheds have similar textures, organic matter contents, and yield distributions. Although the surface slope was greater on one of the watersheds, slope alone could not explain why it also had a nitrate runoff flux that was 18 times greater than the other three watersheds. Only with knowledge of the subsurface hydrology could the surface runoff differences be explained. The subsurface hydrology was developed by combining GPR and surface topography in a geographic information system. Discrete subsurface flow pathways were identified and confirmed with color infrared imagery, real-time soil moisture monitoring, and yield monitoring. The discrete subsurface flow patterns were also useful in understanding observed nitrate levels entering the riparian wetland and first order stream. This study demonstrated the impact that subsurface stratigraphy can have on water and nitrate (NO3-N) fluxes exiting agricultural lands, even when soil properties, yield distributions, and climate are similar. Reliable protocols for measuring subsurface fluxes of water and chemicals need to be developed.

Impact of a first-order riparian zone on nitrogen removal and export from an agricultural ecosystem.

Riparian zones are reputed to be effective at preventing export of agricultural groundwater nitrogen (N) from local ecosystems. This is one impetus behind riparian zone regulations and initiatives. However, riparian zone function can vary under different conditions, with varying impacts on the regional (and ultimately global) environment. Rates of groundwater delivery to the surface appear to have significant effects on the N-removing capabilities of a riparian zone. Research conducted at a first-order agricultural watershed with a well-defined riparian zone in the Maryland coastal plain indicates that more than 2.5 kg/day of nitrate-N can be exported under moderate-to-high stream baseflow conditions. The total nitrate-N load that exits the system increases with increasing flow not simply because of the greater volume of water export. Stream water nitrate-N concentrations also increase by more than an order of magnitude as flow increases, at least during baseflow. This appears to be largely the result of changes in dominant groundwater delivery mechanisms. Higher rates of groundwater exfiltration lessen the contact time between nitrate-carrying groundwater and potentially reducing riparian soils. Subsurface preferential flow paths, in the wetland and adjacent field, also strongly influence N removal. Simple assumptions regarding riparian zone function may be inadequate because of complexities observed in response to changing hydrologic conditions.