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Unmanned Aerial Vehicle (UAV) deployment has risen rapidly in recent years. They are now used in a wide range of applications, from critical safety-of-life scenarios like nuclear power plant surveillance to entertainment and hobby applications. While the popularity of drones has grown lately, the associated intentional and unintentional security threats require adequate consideration. Thus, there is an urgent need for real-time accurate detection and classification of drones. This article provides an overview of drone detection approaches, highlighting their benefits and limitations. We analyze detection techniques that employ radars, acoustic and optical sensors, and emitted radio frequency (RF) signals. We compare their performance, accuracy, and cost under different operating conditions. We conclude that multi-sensor detection systems offer more compelling results, but further research is required.

Background: The air borne SARS-CoV-2 poses a high threat to the elderly and people with underlying diseases. COVID-19 spread quickly in South German nursing homes and for this reason called for preventive measures by the German government. The aim of this paper is to showcase the testing strategies implemented by the Public Health Department Reutlingen to control the spread of COVID-19 in local nursing homes and to report the results thereof., Methods: This study reports COVID-19 outbreaks in nursing homes in Reutlingen County and how they were dealt with through extensive testing, contact tracing, isolation and hygiene inspections. The testing strategy consisted of three phases: In phase 1 only suspected cases, in phase 2 all staff and residents, and in phase 3 all suspected cases and their contacts were tested., Results: Nearly all residents (98%) and staff members (92%) of all nursing homes in Reutlingen County were tested for SARS-COV-2. 25 of 37 nursing homes had COVID-19 cases, 5 had 30-81 cases/home. 62% of the 395 nursing homes cases were residents, but less than half of them exhibited symptoms (41%). The cases uncovered in nursing homes represented 26% of all 1529 cases in Reutlingen County during the time of this study., Conclusions: Many COVID-19 cases were discovered through extensive testing, allowing for early interventions. The results shed light on the COVID-19 situation in nursing homes and allowed for individually designed preventive measures. The results also lead to a change in the German legislation. The outbreak management methods of the Public Health Department Reutlingen may also be applicable in other countries., (© 2022. The Author(s).)

Background: Children with Robin sequence (RS) are at risk of growth failure, mainly due to their increased work of breathing and feeding difficulties. Various conservative and surgical treatment approaches exist, but their impact on weight gain has not yet been adequately addressed. A functional treatment concept, used in our center for > 20 years, includes a pre-epiglottic baton plate (Tuebingen palatal plate) and intensive feeding training., Objective: To investigate the effect of the Tuebingen treatment protocol on growth and weight trajectories during infancy., Methods: This retrospective study analyzed longitudinal data from infants with isolated RS admitted to Tuebingen University Children's Hospital, Germany between 1998 and 2019. Through our electronic patient database, we evaluated anthropometric parameters until reaching 1-year follow-up. Results are shown as median (IQR)., Results: In 307 infants analyzed, median Z-score for weight decreased from - 0.28 at birth to - 1.12 upon admission to our center at a median age of 22 days. Z-score then remained largely unchanged until discharge (Z-score difference, - 0.08), while the proportion of infants receiving tube feedings decreased from 55.1 to 13.7%. Z-score subsequently increased from - 1.17 at discharge to - 0.44 at the 1-year follow-up (p < 0.001)., Conclusion: Based on a comparatively large cohort, this functional treatment was associated with better weight gain and improved feeding. As RS infants often show postnatal growth failure, weight monitoring may be a valuable parameter for monitoring treatment effectiveness. Clinical Trial Registration Not necessary due to the retrospective design., (© 2021. The Author(s).)

The ability to forecast sea ice (both extent and thickness) and weather conditions are the major factors when it comes to safe marine transportation in the Arctic Ocean. This paper presents findings focusing on sea ice and weather prediction in the Arctic Ocean for navigation purposes, in particular along the Northeast Passage. Based on comparison with the observed sea ice concentrations for validation, the best performing Earth system models from the Intergovernmental Panel on Climate Change (IPCC) program (CMIP5-Coupled Model Intercomparison Project phase 5) were selected to provide ranges of potential future sea ice conditions. Our results showed that, despite a general tendency toward less sea ice cover in summer, internal variability will still be large and shipping along the Northeast Passage might still be hampered by sea ice blocking narrow passages. This will make sea ice forecasts on shorter time and space scales and Arctic weather prediction even more important.

A significant share of the world's undiscovered oil and natural gas resources are assumed to lie under the seabed of the Arctic Ocean. Up until now, the exploitation of the resources especially under the European Arctic has largely been prevented by the challenges posed by sea ice coverage, harsh weather conditions, darkness, remoteness of the fields, and lack of infrastructure. Gradual warming has, however, improved the accessibility of the Arctic Ocean. We show for the most resource-abundant European Arctic Seas whether and how a climate induced reduction in sea ice might impact future accessibility of offshore natural gas and crude oil resources. Based on this analysis we show for a number of illustrative but representative locations which technology options exist based on a cost-minimization assessment. We find that under current hydrocarbon prices, oil and gas from the European offshore Arctic is not competitive on world markets.

Pacific Water (PW) enters the Arctic Ocean through Bering Strait and brings in heat, fresh water, and nutrients from the northern Bering Sea. The circulation of PW in the central Arctic Ocean is only partially understood due to the lack of observations. In this paper, pathways of PW are investigated using simulations with six state-of-the art regional and global Ocean General Circulation Models (OGCMs). In the simulations, PW is tracked by a passive tracer, released in Bering Strait. Simulated PW spreads from the Bering Strait region in three major branches. One of them starts in the Barrow Canyon, bringing PW along the continental slope of Alaska into the Canadian Straits and then into Baffin Bay. The second begins in the vicinity of the Herald Canyon and transports PW along the continental slope of the East Siberian Sea into the Transpolar Drift, and then through Fram Strait and the Greenland Sea. The third branch begins near the Herald Shoal and the central Chukchi shelf and brings PW into the Beaufort Gyre. In the models, the wind, acting via Ekman pumping, drives the seasonal and interannual variability of PW in the Canadian Basin of the Arctic Ocean. The wind affects the simulated PW pathways by changing the vertical shear of the relative vorticity of the ocean flow in the Canada Basin.

The observed changes in physical properties of sea ice such as decreased thickness and increased melt pond cover severely impact the energy budget of Arctic sea ice. Increased light transmission leads to increased deposition of solar energy in the upper ocean and thus plays a crucial role for amount and timing of sea-ice-melt and under-ice primary production. Recent developments in underwater technology provide new opportunities to study light transmission below the largely inaccessible underside of sea ice. We measured spectral under-ice radiance and irradiance using the new Nereid Under-Ice (NUI) underwater robotic vehicle, during a cruise of the R/V Polarstern to 83°N 6°W in the Arctic Ocean in July 2014. NUI is a next generation hybrid remotely operated vehicle (H-ROV) designed for both remotely piloted and autonomous surveys underneath land-fast and moving sea ice. Here we present results from one of the first comprehensive scientific dives of NUI employing its interdisciplinary sensor suite. We combine under-ice optical measurements with three dimensional under-ice topography (multibeam sonar) and aerial images of the surface conditions. We investigate the influence of spatially varying ice-thickness and surface properties on the spatial variability of light transmittance during summer. Our results show that surface properties such as melt ponds dominate the spatial distribution of the under-ice light field on small scales (<1000 m 2 ), while sea ice-thickness is the most important predictor for light transmission on larger scales. In addition, we propose the use of an algorithm to obtain histograms of light transmission from distributions of sea ice thickness and surface albedo.

Aim: To examine cost and mortality differences in postmenopausal women with HR(+)/HER2(-) advanced breast cancer., Methods: Using claims data (2007-2013), women with newly diagnosed (de novo) stage IV, or early- or late-recurring metastatic breast cancer were identified., Results: Compared with de novo (n = 121) and late-recurrent (n = 106), early-recurrent (n = 172) patients had significantly higher costs in total and for anticancer systemic agents. Adjusted per patient per month costs for early-recurrent patients were US$13,404, versus US$9955 (de novo) and US$9721 (late-recurrent; p = 0.02). Early-recurrent patients' risk of death was twice that of de novo patients (p = 0.02)., Conclusion: Compared with new diagnosis or late recurrence, early recurrence of HR+/HER2- metastatic breast cancer was associated with higher mortality and healthcare costs.

In insects, the first extraction of motion and direction clues from local brightness modulations is thought to take place in the medulla. However, whether and how these computations are represented in the medulla stills remain widely unknown, because electrical recording of the neurons in the medulla is difficult. As an effort to overcome this difficulty, we employed local electroporation in vivo in the medulla of the blowfly (Calliphora vicina) to stain small ensembles of neurons with a calcium-sensitive dye. We studied the responses of these neuronal ensembles to spatial and temporal brightness modulations and found selectivity for grating orientation. In contrast, the responses to the two opposite directions of motion of a grating with the same orientation were similar in magnitude, indicating that strong directional selectivity is either not present in the types of neurons covered by our data set, or that direction-selective signals are too closely spaced to be distinguished by our calcium imaging. The calcium responses also showed a bell-shaped dependency on the temporal frequency of drifting gratings, with an optimum higher than that observed in one of the subsequent processing stages, i.e., the lobula plate. Medulla responses were elicited by on- as well as off-stimuli with some spatial heterogeneity in the sensitivity for "on" and "off", and in the polarity of the responses. Medulla neurons thus show similarities to some established principles of motion and edge detection in the vertebrate visual system.

A robust molecule that resists degradation via nucleophilic, electrophilic and radical attacks is described. Coordinated O(2) is reduced catalytically, producing efficiently thyil radicals in spite of the extreme electronic deficiency of the catalyst.

The first single-crystal X-ray structures of substituted vanadyl phthalocyanine materials reveal the high-valence vanadium ions (denoted as V(IV)), whose coordination by a highly electron-deficient ligand is facilitated by an axial oxo group. The metal center of the hydrophilic V═O core, encapsulated in F-rich hydrophobic pockets, reaches a coordination number of 6 by binding an additional H(2)O that, in turn, hydrogen-bonds with ketones, resulting in solvent-induced variable solid-state architectures. Fluoroalkyl (R(f)) ligand substituents hinder π-π stacking interactions and favor ordered long-range packing, as well as the facile formation of film materials that exhibit high thermal stability and oxidation resistance. Reversible redox chemistry and spectroscopic studies in both solution and the solid-state indicate single-site isolation in both phases and an R(f)-induced propensity for electron uptake and inhibition of electron loss. Repeated redox cycles reorganize the thin films to accommodate Li(+) ions and facilitate their migration. The facile reduction, combined with high stability and ease of sublimation imparted by the R(f) scaffold that suppresses oxidations, recommends the new materials for sensors, color displays, electronic materials, and redox catalysts, as well as other applications., (© 2011 American Chemical Society)

A series of Toxicity Identification Evaluations (TIEs) to identify the cause(s) of observed toxicity to Ceriodaphnia dubia have been conducted on a hard rock mining effluent. Characteristic of hard rock mining discharges, the effluent has elevated (∼3000 mg l(-1)) total dissolved solids (TDS) composed primarily of Ca(2+) and SO(4)(2-). The effluent typically exhibits 6-12 toxic units (TUs) when tested with C. dubia. Phase I and II toxicity identification evaluations (TIEs) indicated Ca(2+) and SO(4)(2-) contributed only ∼4 TUs of toxicity, but this was likely an underestimate due to problems with simulating the supersaturated CaSO(4) concentrations in the effluent. Treatment of the effluent with BaCO(3) to precipitate Ca(2+) and SO(4)(2-) revealed that these ions contribute ∼6 TUs of the observed toxicity, but the remaining source(s) of toxicity (up to 6 TUs) remained unidentified. Subsequent investigations identified thiocyanate (SCN(-)) in the effluent at 100-150 μM. Toxicity tests reveal that C. dubia are sensitive to SCN(-) with an estimated IC25 of 8.3 μΜ for reproduction in moderately hard water suggesting between 12 and 18 TUs of toxicity in the effluent. Additional experiments demonstrated that SCN(-) toxicity is reduced in the high TDS matrix of the mining effluent. Testing of a mock effluent simulating the major ion and SCN(-) concentrations resulted in 10.4 TUs, suggesting that Ca(2+), SO(4)(2-) and SCN(-) are the three toxicants present in this effluent. This research suggests SCN(-) may be a more common cause of toxicity in mining effluents than is generally recognized., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Previous studies have indicated that salmonid fertilization success may be very sensitive to elevated concentrations of total dissolved solids (TDS) with effects at concentrations as low as 250mgl(-1) being reported. However, interpretation of these studies is complicated by poor control performance and variable concentration response relationships. To address this, a series of experiments were performed to evaluate TDS effects on Arctic Grayling (Thymallus arcticus) and Dolly Varden (Salvelinus malma) fertilization success and identify possible mechanisms for previously observed test variability and any observed effects of TDS. Results indicate that some of the experiments reported here were likely confounded by extended milt holding times prior to experiment initiation. Milt holding times >6h were shown to significantly reduce control fertilization and corresponding concentration response relationships were variable. When milt holding time was minimized during fertilization experiments, consistent control performance with >90% control fertilization was achieved and consistent concentration response relationships were observed for both species examined. Experiments performed under these conditions indicate that Arctic Grayling and Dolly Varden fertilization success is not sensitive to elevated TDS with EC20s (concentration causing 20% effect) of >2782 and >1817mgl(-1) (the highest concentrations tested), respectively. However, TDS was shown to significantly affect embryo water absorption during the water hardening phase immediately following fertilization. The lowest observable effect concentrations (LOECs) for this endpoint were 1402 and 964mgl(-1) for Arctic Grayling and Dolly Varden, respectively. The effect of reduced embryo turgidity, due to impaired water absorption, on resistance to mechanical damage under real world conditions needs further investigation in order to understand the implications of this observed effect.

Zinc perfluoro-fluoroalkyl-phthalocyanine, synthesized in high yield, does not exhibit electron loss, does not aggregate in solution, is photostable and produces (1)O(2) in very high quantum yields. Aerobic photo-oxygenation of an external substrate occurs without catalyst self-oxidation. The encapsulation of a metal center in a refractory organic environment could guide the design of other viable catalysts for oxygenation of substrates either for synthesis or for oxidative destruction of organic or biological molecules, under reaction conditions that include the use of only air and light.

The present paper presents original data and a review of the copper (Cu) toxicity literature for estuarine and marine environments. For the first time, acute Cu toxicity across the full salinity range was determined. Killifish, Fundulus heteroclitus, eggs were hatched in freshwater (FW), 2.5, 5, 10, 15, 22 and 35 ppt (seawater, SW) and juveniles were allowed to acclimate for 7 days prior to acute toxicity testing. Sensitivity was highest in FW (96 h LC50: 18 microg/l), followed by SW (96 h LC50: 294 microg/l) with fish at intermediate salinities being the most tolerant (96 h LC50 > 963 microg/l at 10 ppt). This approximately 50-fold, non-linear variation in sensitivity could not be accounted for by Cu speciation or competition among cations but can be explained by physiology. The relative Na(+) gradient from the blood plasma to the water is greatest in FW followed by SW and is smallest at 10 ppt. Regression of Cu toxicity versus the equilibrium potential for Na(+), which reflects the relative Na(+) gradient, revealed that 93% of the variation can be attributed to Na(+) gradients and thus osmoregulatory physiology. Examination of the existing literature on acute Cu toxicity in SW (defined as >25 ppt) confirmed that early life stages generally are most sensitive but this pattern may be attributable to size rather than developmental stage. Regardless of developmental stage and phylogeny, size clearly matters for Cu sensitivity. The existing literature on the influence of salinity on acute Cu toxicity as well as studies of mechanisms of Cu toxicity in fish and invertebrates are reviewed.

It is widely accepted that the functional and morphological differentiation of cells is initiated and determined by the interaction of molecules of the extracellular matrix and adhesion molecules of the cell membrane. To assess the influence of the underlying matrix on the characteristics of cells, enterocyte-like Caco-2 cells were cultivated on substrates commonly used for cell culture as well as on glass coated with hydrophobic layers. Providing the same starting conditions for growth, the parameters investigated on preconfluent Caco-2 cells were the number of adhering cells, the proliferative activity and the degree of differentiation indicated by the expression of three brush border enzymes. Whereas tissue culture treated polystyrene elicited highest rates of adhesion, proliferation, and differentiation, even glass altered the pattern of brush border enzyme expression. The hydrophobic surfaces strongly decreased the adhesion and the proliferation but the surviving cells exhibited a pronounced higher degree of differentiation. Interestingly, each sub-type of hydrophobic matrix triggered a different pattern of brush border enzyme expression. Thus, the development of a certain phenotype of a cell can not only be triggered by certain components of the extracellular matrix but also by artificially prepared surface coatings of the underlying matrix. In the future it seems to be feasible that cells can be programmed by tailoring the surface of the underlying substrate.

Previous studies indicate that the hatching success of brine shrimp (Artemia franciscana) cysts is surprisingly sensitive to ambient metal concentrations. These studies estimated median effective concentrations (EC50s) of 7, 5, and 28 microg l-1 for Cd, Cu, and Zn, suggesting that the hatching end point for A. franciscana is the most sensitive tested to date for Cd and Zn in saline environments and comparable in sensitivity with the most sensitive tested to date for Cu. Furthermore, these data suggest that brine shrimp are at significant risk from Cu and Zn in Great Salt Lake (GSL), UT, where ambient concentrations as high as 10 and 14 microg l-1, respectively, have been measured. Given that brine shrimp appear to be successfully reproducing in GSL, we hypothesized that these toxicity values were either biased low as a result of an artifact of the test method used or that site-specific water-quality conditions in the lake had decreased metal bioavailability such that brine shrimp could successfully reproduce. To test these hypotheses, we initiated a step-wise series of experiments. First we investigated the effects of pretreatment of brine shrimp cysts with antibiotics on brine shrimp sensitivity to metals because previous investigators as part of their test methods have used antibiotics. Next we considered the effect of ionic composition of the artificial test media on sensitivity. Finally, we evaluated the effects of the site-specific water quality of the GSL on metal bioavailability and toxicity. Results indicate that pretreatment of cysts with antibiotics had no effect on sensitivity. However, we were unable to repeat the previous values for Cd and Zn, obtaining EC50s of 11,859 and 289 microg l-1 for Cd and Zn, respectively. For Cu, however, we estimated an EC50 of 12 microg l-1, so we conducted further testing on the artificial media, adjusting the media composition to better reflect the Ca2+ and HCO3- concentration of normal seawater. This increased the EC50 to 28 microg l-1. Finally we evaluated the toxicity of Cu in GSL water and obtained an EC50 of 68 microg l-1, suggesting that the increased dissolved organic carbon in GSL has a significant protective effect. Overall, the results of this study suggest that brine shrimp hatching success is not particularly sensitive to Cd and Zn, but it is sensitive to Cu. However, site-specific water-quality conditions ensure that brine shrimp cyst hatching success is not significantly affected by any of these metals at the normal background concentrations that occur in GSL (<15 microg l-1).

The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.

The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at > or =2 degrees C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 10(1) to 10(5) CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7 degrees C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7 degrees C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148 degrees F (64.4 degrees C) for 17.5 s on milk inoculated with E. coli O157:H7 at 10(5) CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.

Although the California sea hare, Aplysia californica, is well known from neurobiological studies and is raised in the laboratory for this purpose, various aspects of its life history in the laboratory, such as aging dynamics, are unknown. Therefore we collected life history data on 4 cohorts of eggs from hatchery-reared animals and performed an actuarial analysis of mortality data. Temperature was controlled at 13 to 15 degrees C, the photoperiod was a 14:10-h light:dark cycle, and the seawater O2 concentration, pH, and salinity were held at optimized levels. The feeding protocol for 3 cohorts was unrestricted access to the red macroalga Gracilaria ferox, whereas the remaining cohort was fed standard hatchery rations of G. ferox 4 times per week. Growth was sigmoidal in each cohort and resulted in linear growth rates of 1.25 to 3.62 g/d during the exponential phase; these rates were not influenced by feeding level. Sexual maturity occurred at approximately 160 g, at ages ranging from 144 to 241 d. Egg production was highly variable in the different cohorts. Mean lifespan of cohorts fed ad libitum was approximately 228 d. In contrast, the cohort fed standard rations lived an average of 375 d and showed a lower initial mortality rate, suggesting that calorie restriction on a single-species diet prolongs lifespan in California sea hares.

We conducted a hatchery growth study to describe the variability in growth rates, spawning, and mortality of Aplysia californica in regard to rearing temperature. Animals were housed at a standard hatchery density of five animals per cage, at temperatures of 13, 15, 18, and 21 degrees Celsius. Animals reared at 13 or 15 degrees C grew as much as four times as large, lived twice as long, matured later, and spawned longer than did animals reared at 18 or 21 degrees C. At age 170 to 205 days the fastest growth rates occurred at 18 and 21 degrees C, and the slowest at 13 degrees C. As animals at 18 and 21 degrees C reached sexual maturity at ages 190 to 197 days, or approximately 60% through their lifespans, their growth rates slowed such that by age 260 days, the fastest growth rate was at 13 degrees C, and the slowest was at 21 degrees C. Animals reared at 13 and 15 degrees C reached sexual maturity at 242 and 208 days, respectively, or at approximately 40% of their life spans. Lifespan and maximum average animal weight were significantly inversely correlated with temperature (P

Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.

The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are demonstrated between oxidative modification of GAPDH and its subsequent S-glutathionylation. Similarly, a redox interaction between GSSG and GAPDH with Grx as the catalyst is shown, suggesting that the Grx molecule may participate in catalytic S-glutathionylation in intact cells. Furthermore, Grx itself can readily undergo S-glutathionylation, indicating the potential for regulation of this catalyst of the reversible S-glutathionylation of other proteins. The methodologies detailed in this work may provide a good reference point for other attempts to elucidate the mechanism of reversible S-glutathionylation of purified proteins in a manner that more closely resembles the situation arising in intact cells during the generation of oxidative stress.

To better understand the influence of kappa-opioid agonists on the effects of cocaine, rats were treated with daily injections of the selective kappa-opioid agonist U-69593 or bremazocine. In combination with 10 mg/kg cocaine, both compounds, at a dose of 0.32 mg/kg, greatly diminished locomotor activity, and these effects were maintained over a period of 5 days. In addition, the response to a challenge injection of 10 mg/kg cocaine several days after the end of kappa-Opioid agonist treatment with or without cocaine was markedly reduced. When naltrexone was given in combination with U-69593, it blocked the reduction in cocaine-induced locomotor activity after U-69593 treatment alone. However, a single injection of either kappa-opioid agonist alone had no effect on cocaine-induced locomotion several days later (i.e. no long-term effects), suggesting that multiple injections of the kappa-opioid agonist are needed to reduce the locomotor activating effects of cocaine other than acutely. In addition, treatment with the kappa-opioid agonist U-69593 (0.32 mg/kg) over a 5-day period decreased dopamine transporter densities in the caudate putamen, and this was also blocked by co-administration of naltrexone.

A series of meperidine analogues was synthesized and the binding affinities for the dopamine and serotonin transporters were determined. The substituents on the phenyl ring greatly influenced the potency and selectivity of these compounds for the transporter binding sites. In general, meperidine (3) and its analogues were more selective for serotonin transporter binding sites and the esters 9 were more potent than the corresponding nitriles 8. The 3,4-dichloro derivative 9e was the most potent ligand of the series for dopamine transporter binding sites while the 2-naphthyl derivative 9g exhibited the most potent binding affinity and was highly selective for serotonin transporter binding sites.

The metallic group XVIa elements selenium and tellurium possess remarkably similar chemical properties. However, unlike selenium, tellurium is not an essential micronutrient and, indeed, induces both acute and chronic toxicity in a variety of species. Despite this, very little is known of the molecular mechanisms of toxicity of tellurium, particularly with respect to potential chemical interactions with selenium-containing components in the cell. In this work we describe a novel interaction of inorganic tellurite with hepatocellular selenoproteins, particularly with selenium-dependent glutathione peroxidase. The accumulation of (121Te)-tellurite into cultured primary rat liver hepatocytes was shown to be much more rapid than that of (75Se)-selenite on a molar basis. Neither the uptake of (121Te)-tellurite nor of (75Se)-selenite was affected by a large molar excess of the unlabelled counterpart, respectively. Interestingly, separation of the hepatocellular proteins on continuous pH denaturing gels demonstrated clear binding of radiolabelled tellurium to a number of protein bands, including one at 23 and one at 58 kDa, which corresponded to proteins readily labelled in cells treated with (75Se)-selenite. The binding of (121Te) to these proteins was insensitive to reduction with mercaptoethanol and not affected by pre-treatment of the cells with cycloheximide. When purified selenium-dependent glutathione peroxidase was treated directly with (121Te)-tellurite, the protein became labelled in an analogous manner to that achieved in intact cells. This was not affected by coincubation of the enzyme with (121Te)-tellurite and one or both of its substrates. Additionally, incubation of the peroxidase with tellurite effectively inhibited its ability to catalyse glutathione-dependent reduction of hydrogen peroxide. These data suggest that inorganic tellurite delivers tellurium to the intracellular milieu in a form capable of binding to some intracellular selenoproteins and at least in the case of glutathione peroxidase, cause inhibition of catalytic activity. The nature of the binding seems not to be due to the insertion of tellurocysteine into the protein and the insensitivity to reductive cleavage with mercaptoethanol seems to preclude the formation of stable telluro-selenides in the proteins. These data may offer alternative explanations for the established toxicity of tellurium via disruption of selenoprotein function, particularly by the induction of intracellular oxidative stress by the inhibition of Se-dependent glutathione peroxidase.

In this report the protein human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been examined to clarify the roles of (a) direct oxidation and (b) thiol-disulphide exchange (with glutathione disulphide) on the modification of its catalytic activity. An in vitro system using purified human GAPDH and [35S]-GSSG (glutathione disulphide), has permitted clarification of these possibilities by showing that S-glutathionylation of GAPDH does not result in an inactivation of the enzyme. Rather, the direct oxidation of GAPDH with hydrogen peroxide is responsible for inhibition of the catalytic activity of the protein. Furthermore, pre-treatment of the enzyme with hydrogen peroxide enhances the formation of glutathione-GAPDH mixed disulphides in the presence of glutathione disulphide. This may serve as a molecular "switch" directing the protein to other reported functions in the cell. It is also shown that the efficiency of S-glutathionylation of either native or oxidised GAPDH is enhanced by the presence of recombinant glutaredoxin (thiol transferase) of either bacterial or human origin. Under the conditions of analysis the glutaredoxin itself is also shown to readily undergo S-glutathionylation external to its active site. Taken together, the data indicate the complexity of mechanisms likely to be involved in regulating cellular proteins during oxidative stress and implicate controlled enzyme-catalysed S-glutathionylation as a potential selectivity factor in the redox modification of protein function by glutathione., (Copyright 1998 Academic Press.)

Exposure of human umbilical vein endothelial cells to oxidants, such as hydrogen peroxide and diamide, has been shown to induce protein-specific S-thiolation of cellular proteins. In this study we have now identified glutathione (reduced form) as the major low molecular-weight thiol that is bound to protein substrates in human umbilical vein endothelial (HUVE) cells during oxidative stress and investigated the dose- and time-response relationship of diamide- and hydrogen peroxide-induced 8-thiolation of HUVE cell protein. Intact HUVE cells are able to rapidly reduce S-thiolated proteins with almost quantitative reappearance of reduced glutathione in the cells and protection from acute, lytic cytotoxicity. Additionally, studies were performed with detergent-solubilized cell extracts to determine the nature of the reductants operating in HUVE cells to maintain protein thiol homeostasis. The results clearly show the involvement of NADH- and NADPH-dependent systems. These data suggest that the reversible S-thiolation of proteins in these human endothelial cells may represent a significant cellular antioxidant and regulatory mechanism during oxidative stress.

The subacute toxic effects of dietary deoxynivalenol (DON) were examined in grower pigs during a 32 day feeding period. DON was incorporated into the feed at 0, 1, and 3 mg/kg, added as either the purified toxin (P) or as naturally contaminated corn (N). Growth performance and blood biochemical and haematological parameters were monitored throughout the study. At the higher toxin levels (diets 3P, 3N) significantly lower feed consumption and body weight gains were evident soon after the start of feeding, but while weight gains of pigs fed the pure DON diet (3P) recovered after several days, values for pigs fed the naturally contaminated diet (3N) remained depressed over the course of the study. It is possible that these observations reflected the presence of other unidentified toxic compounds in the naturally contaminated grain. Generally, blood chemistry parameters of pigs fed the contaminated diets were not different from controls, with the exception of alpha-globulin and possibly cortisol in animals receiving diets 3N or 3P. Data suggested that the effect of DON on the alpha-globulin fraction may have been independent of the feed refusal syndrome associated with this toxin. Alterations in several haematological measurements were noted to occur sporadically with the 3 ppm diets, including higher RBC count, haematocrit and platelet level, however these effects could not be separated from the influence of decreased feed intake and were of limited value in diagnosing the effects of low level dietary DON on swine.

Microcystin-YM a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa was radiolabeled with 125I, and used to investigate the uptake of the toxin by freshly isolated rat hepatocytes. The uptake was temperature dependent with apparent activation energy of 18 kcal/mole (77 kJ/mole) for the initial rate of uptake. Uptake of non-toxic (10-20 nM) doses of microcystin by hepatocytes continued with time, the intracellular to extracellular distribution ratio for the toxin was 70 at 60 min for 10(6) cells/ml. Uptake of higher doses of microcystin (100 nM and more) stopped when the cells blebbed: a toxic response of hepatocytes to microcystin. Uptake of microcystin by hepatocytes was inhibited 70-80% by the addition of 10 microM sodium deoxycholate or bromsulphthlein, compounds that protect hepatocytes from the toxic effects of microcystin.

The nature of the energy source for phosphate transport was studied in strains of Escherichia coli in which either one of the two major systems (PIT, PST) for phosphate transport was present. In the PIT system, phosphate transport is coupled to the proton-motive force. The energy source for the PST system appears to be phosphate-bond energy, as has been found in other systems involving binding proteins. High concentration gradients of phosphate (between 100 and 500) are established by both systems.

The nephrotoxicity of hexachloro-1,3-butadiene (HCBD) appears to depend on the initial formation of a glutathione (GSH) conjugate in the liver. In the present study we have examined the hepatic metabolism of HCBD using isolated hepatocytes and following in vivo administration. Exposure of isolated hepatocytes to HCBD resulted in a dose-dependent depletion of GSH. HPLC analysis of the incubation medium demonstrated the formation of two products. When isolated hepatocytes containing [3H]GSH were exposed to [14C]HCBD, coincident elution of 3H and 14C corresponding to the previously recognized HPLC peaks was observed. Both products were sensitive to treatment with gamma-glutamyl transpeptidase (gamma-GT), providing additional support for their identification as GSH conjugates. The ratio of 3H to 14C in the two peaks indicated the formation of both a mono- and a bis-substituted GSH conjugate of HCBD. The identification of the mono- and bis-GSH conjugates was further confirmed by the preparation of synthetic standards which displayed retention times by HPLC identical to the biological products. The production of the total and individual GSH conjugates displayed both dose and time dependence. The production of the total as well as the ratio of mono- to bis-conjugate was found to depend on the availability of GSH. At low HCBD exposure levels the bis-substituted conjugate accounted for more than 20% of the total conjugate produced by isolated hepatocytes. This value decreased at higher HCBD concentrations. Analysis of bile collected from rats following intraportal administration of [14C]HCBD revealed the presence of both the mono- and bis-substituted GSH conjugates of HCBD as well as additional 14C-containing metabolites. The results of the present study clearly demonstrate the production of both a mono- and a bis-substituted GSH conjugate of HCBD. The potential importance of this finding in terms of the nephrotoxicity of HCBD is discussed.

Isolated renal epithelial cells were used to study the mechanism of toxicity of pentachlorobutadienyl-glutathione (PCBG), a nephrotoxic glutathione conjugate of hexachlorobutadiene. The cytotoxicity of PCBG displayed a very steep dose-response relationship; at 10 microM PCBG no toxicity was observed whereas 25, 50, and 100 microM PCBG all resulted in a similar degree of toxicity. In all cases, loss of cell viability was observed only after a 30-min lag period and reached a plateau of 50 to 60% nonviable cells between 90 and 100 min. Toxic doses of PCBG also resulted in the depletion of cellular thiols. Blocking PCBG metabolism by inhibition of gamma-glutamyl transpeptidase [1-gamma-L-glutamyl-2-(2-carboxyphenyl)hydrazine (anthglutin), 2 mM] or renal cysteine conjugate beta-lyase (aminooxyacetic acid, 0.5 mM) resulted in complete protection against PCBG-induced cell damage. Exposure of isolated renal epithelial cells to 100 microM PCBG resulted in the rapid formation of plasma membrane blebs which appeared to be associated with a loss of Ca2+ from the mitochondrial compartment and an elevation of cytosolic Ca2+ concentration as measured by Quin-2. PCBG treatment also resulted in the inhibition of cell respiration and a marked depletion of cellular ATP content, indicating additional mitochondrial effects of the toxin. Our results support a role for renal cysteine conjugate beta-lyase in the metabolic activation of PCBG and suggest that PCBG-induced renal cell injury may be the result of selective effects on mitochondrial function.

Mutants of Escherichia coli K-12 were constructed such that each possessed one single major system for phosphate transport. A comparison of these strains showed that one of the systems (PIT) was fully constitutive, required no binding protein, and operated in spheroplasts. It permitted the complete exchange of intracellular phosphate with extracellular phosphate (or arsenate) and was completely inhibited by uncouplers. The other system, PST, was repressible by phosphate concentrations above 1 mM, required the phosphate-binding protein for full activity, and did not operate in spheroplasts. It catalyzed very little exchange between internal and external phosphate and was resistant to uncouplers. The maximal velocities attained by the two systems were approximately the same, but the affinity for phosphate in the PST system was greater by two orders of magnitude. In strains in which both systems were fully operative, the initial rates of uptake was nearly additive, and the systems appeared to interact with a common intracellular phosphate pool.

Reconstitution of phosphate transport in Escherichia coli was demonstrated. Conversion of E. coli K10 cells to spheroplasts decreased phosphate transport to about 2%. Addition of purified phosphate-binding protein at physiological levels to these spheroplasts caused a mean 14-fold increase in phosphate transport rate. Crude shock fluid fractions were also stimulatory but not if the shock fluid was obtained from mutants lacking phosphate-binding protein. The effect of the binding protein was abolished by its specific antibody. The phosphate was shown to have entered the cell, where it became esterified. Reconstitution was not possible with cold-shocked or osmotically shocked cells.

In phosphate-starved cells of Escherichia coli, the synthesis of alkaline phosphatase and some additional periplasmic proteins is derepressed. One of these proteins, which does not appear in a phoS- constitutive strain, has been identified as well the periplasmic phosphate-binding protein.

A crude mycelial protein extract from a 16-day-old culture of A. parasiticus, on purification, lost 50% of its ability to degrade aflatoxin B1. The addition of hydrogen peroxide increased this activity to 97% of that of the crude extract. Ducklings dosed orally with aflatoxin extracts from 14- and 20-day-old cultures containing 46 micrograms or more of aflatoxin B1 developed enlarged livers, haemorrhaged and died in less than 10 days, giving and LD50 of 17.5 and 17.1 micrograms aflatoxin B1 per 50 g body weight respectively for each extract. When pure aflatoxin B1 was mixed with either the crude or purified mycelial protein extract the aflatoxin B1 level was decreased by 29% as was the toxicity of the mixture. The main breakdown product of aflatoxin B1 was isolated and was shown to have an RF value of 0.34, was non-fluorescent, and was non-toxic for ducklings at oral doses as high as 400 micrograms per 50 g body weight. The mutagenic effect of aflatoxin B1 on Salmonella typhimurium was relative to its concentration. The main breakdown product of aflatoxin B1 was non-mutagenic.

The controversial dissension concerning the nature of the pentose cycle in liver is investigated. The metabolism of [2-14C]Glc and [1-14C]Rib in chronically perfused normal and regenerating rabbit liver and acutely perfused rat liver are used to test the mechanistic predictions and contribution of the F-type pentose cycle. 14C was traced in Glc, Glc 6-P, Fru 6-P, glycogen and Rib 5-P. None of the data complied with the critical theoretical limits set for the C-1/C-3 ratio (the identity badge of the F-type pentose cycle or pathway) for all values of F-type PC from 0-100%. Thus apparent F-type PC measurements using the Katz & Wood method gave a wide scatter of calculated values. The 14C distributions in Rib 5-P do not conform with the predictions of the F-type PC but are in agreement with the many previous results of similar experiments reported by Hiatt and co-workers. In perfused rat liver the C-1/C-3 constants in Glc 6-P and glycogen also failed to conform with F-PC theory following the metabolism of [2-14C]Glc. The metabolism of [5-14C]Glc and distribution of 14C in Glc 6-P and glycogen showed that L-type PC was 18%, in close agreement with a previous published value of 22% for rat hepatocytes. Metabolism of [6-14C]Glc and [4-14C]Glc (as [4,5,6-14C]Glc) showed that Pyruvate Recycling was active in perfused rat liver. None of the data from these comprehensive investigations can confirm the results of the recent study reported by the Landau laboratory on the pentose pathway metabolism of Glc and Rib in perfused rat liver.

Hepatic GSH conjugation is the initial step in the mammalian biotransformation of hexachloro-1,3-butadiene (HCBD) and analogous haloalkenes. The present paper reports an in vitro investigation of the glutathione-dependent conversion of HCBD to water-soluble products, i.e. the enzyme-catalyzed conjugation of HCBD with GSH. The method employed avoids artifacts due to the volatility, low solubility and hydrophobic nature of the chloro-carbon substrate. In order to assess the relative importance of membrane-bound and cytosolic glutathione S-transferase in the conjugation process, microsomal and cytosolic fractions from adult rat liver were tested separately for their ability to promote water solubilisation of the substrate. In addition, microsomal purified and liposomally reconstituted glutathione S-transferase, were tested. The reaction exhibited Michaelis-Menten kinetics, and conjugation rates were linear for at least 20 min. The hepatic microsomal fraction metabolized HCBD 116 times faster than the cytosolic fraction when substrate saturated. Both mono- and bis-substituted conjugates were formed by microsomal as well as by the cytosolic fraction. Treatment of animals with inducers and the use of specific inhibitors indicated absence of cytochrome P-450 involvement in the formation of water soluble HCBD metabolites and supported the view that microsomal glutathione S-transferase is more important in catalyzing GSH conjugation of this haloalkene than the cytosolic forms of transferases.

The freshwater, bloom forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin which causes death accompanied by liver necrosis. We show here that the time and dose-dependent blebbing of isolated hepatocytes is accompanied by the activation of phosphorylase a, with no changes in cyclic AMP levels, and by glutathione (acid-soluble thiols) depletion. These results suggest that the disruption of cytoskeletal structures is accompanied by disturbances in cellular calcium homeostasis and by decreased protection against oxidative damage to the cells.