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Ringed seals Pusa hispida are reliant on snow and sea ice for denning, and a better understanding of ringed seal habitat selection and timing of emergence from snow dens (also called lairs) is needed to quantify and predict effects of climate change in the Arctic. We used generalized additive models to assess relationships between ringed seal counts, from spring aerial surveys in the Bering Sea (2012 and 2013) and Chukchi Sea (2016), and spatiotemporal covariates including survey date, remotely sensed snow and sea-ice values, and short-term weather data. We produced separate models for total ringed seal counts and for pup counts within each region. Our models showed that in both areas, total ringed seal counts increased over the course of the spring, especially after 15 May, indicating emergence from lairs and/or the onset of basking behavior. For the more northerly Chukchi Sea, we found a substantial unimodal effect of snow melt progression and a positive effect of snow depth on total ringed seal counts. In contrast, Bering Sea total ringed seal counts and pup counts in both regions were affected much more strongly by date than by habitat variables. Overall, our findings demonstrate that snow depth and melt play an important role in the timing of ringed seal den emergence, particularly in the Chukchi Sea, and suggest that ringed seal denning may be affected by continued shifts in melt and snow depth associated with climate change.

Polar bears are of international conservation concern due to climate change but are difficult to study because of low densities and an expansive, circumpolar distribution. In a collaborative U.S.-Russian effort in spring of 2016, we used aerial surveys to detect and estimate the abundance of polar bears on sea ice in the Chukchi Sea. Our surveys used a combination of thermal imagery, digital photography, and human observations. Using spatio-temporal statistical models that related bear and track densities to physiographic and biological covariates (e.g., sea ice extent, resource selection functions derived from satellite tags), we predicted abundance and spatial distribution throughout our study area. Estimates of 2016 abundance (N ^ *) ranged from 3,435 (95% CI: 2,300-5,131) to 5,444 (95% CI: 3,636-8,152) depending on the proportion of bears assumed to be missed on the transect line during Russian surveys (g(0)). Our point estimates are larger than, but of similar magnitude to, a recent estimate for the period 2008-2016 (N ^ * = 2 , 937; 95% CI 1,522-5,944) derived from an integrated population model applied to a slightly smaller area. Although a number of factors (e.g., equipment issues, differing platforms, low sample sizes, size of the study area relative to sampling effort) required us to make a number of assumptions to generate estimates, it establishes a useful lower bound for abundance, and suggests high spring polar bear densities on sea ice in Russian waters south of Wrangell Island. With future improvements, we suggest that springtime aerial surveys may represent a plausible avenue for studying abundance and distribution of polar bears and their prey over large, remote areas.

Medial arterial calcification (MAC) is a common outcome in diabetes and chronic kidney disease (CKD). It occurs as linear mineral deposits along the degraded elastin lamellae and is responsible for increased aortic stiffness and subsequent cardiovascular events. Current treatments for calcification, particularly in CKD, are predominantly focused on regulating the mineral disturbance and other risk factors. Ethylene diamine tetraacetic acid (EDTA), a chelating agent, can resorb mineral deposits, but the systemic delivery of EDTA may cause side effects such as hypocalcemia and bone resorption. We have developed elastin antibody conjugated albumin nanoparticles that target only degraded elastin in vasculature while sparing healthy tissues. In this study, we tested a targeted nanoparticle-based EDTA chelation therapy to reverse CKD-associated MAC. Renal failure was induced in Sprague-Dawley rats by a high adenine diet supplemented by high P and Ca for 28 days that led to MAC. Intravenous delivery of DiR dye-loaded nanoparticles confirmed targeting to vascular degraded elastin and calcification sites within 24 hours. Next, EDTA-loaded albumin nanoparticles conjugated with an anti-elastin antibody were intravenously injected twice a week for two weeks. The targeted nanoparticles delivered EDTA at the site of vascular calcification and reversed mineral deposits without any untoward effects. Systemic EDTA injections or blank nanoparticles were ineffective in reversing MAC. Reversal of calcification seems to be stable as it did not return after the treatment was stopped for an additional four weeks. Targeted EDTA chelation therapy successfully reversed calcification in this adenine rat model of CKD. We consider that targeted NP therapy will provide an attractive option to reverse calcification and has a high potential for clinical translation.

Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S -transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc70 in facilitating MHV68 lytic replication. IMPORTANCE Latency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68, this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral replication.

The remote pack ice of the arctic and subarctic seas is challenging to access, yet extremely important to understand and monitor. The pack ice holds the key to understanding ecosystem responses to climate change and is vital habitat for many species including ice-associated seals. Unoccupied aircraft systems (UAS) are a new class of tools that may overcome the traditional challenges associated with expansive offshore surveys. We conducted UAS flights over the pack ice during a spring 2009 National Oceanic and Atmospheric Administration (NOAA) cruise to the Bering Sea to determine whether advances in UAS technology can enable effective large-scale, systematic ship-based surveys for seals in the seasonal ice of the Bering, Beaufort, and Chukchi Seas. A fixed-wing ScanEagle UAS was successfully launched and recovered from the NOAA ship McArthur II to conduct small-scale transect surveys up to 5 nautical miles (M) from the ship's position. More than 27 000 images were collected from 10 flights over the Bering Sea pack ice and seals were identified in 110 of these images. Review of the images indicated a marked reduction in disturbance to seals when compared to images collected from occupied, low-altitude helicopter surveys. These results suggest that large-scale UAS surveys of arctic and subarctic habitat in United States airspace will be possible with improvements in technology, reduced operational costs, and the establishment of inclusive airspace regulations.

Technological advances have facilitated collection of vast quantities of photographic data from aerial surveys of marine mammals. However, when it is difficult to distinguish species from a distance, reliable identification from aerial images can often be challenging. This is the case for ice-associated seals, species for which global climate change has motivated intensive monitoring efforts in recent years. We assess species and age class identification from aerial images of four ice seal species (bearded seals, Erignathus barbatus; ribbon seals, Histriophoca fasciata; ringed seals, Pusa hispida; spotted seals, Phoca largha) in the Bering Sea. We also investigate the specific phenomenological and behavioral traits commonly associated with species identification and observer confidence. We generally found species and age class misidentification occurred at relatively low levels, but only 83% of spotted seals tended to be correctly identified (with 11% mistaken as ribbon seals). We also found certain traits were strong predictors for observed species, age class, or observer confidence. Our findings add to the growing body of evidence that species misidentification is pervasive in passive sampling of animal populations. Even low levels of misidentification have been demonstrated to induce substantial biases in estimators of species distribution and abundance, and it is important that statistical models account for such errors.

Background Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus. This clock is synchronized to environmental day/night cycles by photic input, which is dependent on the presence of mature brain-derived neurotrophic factor (BDNF) in the SCN. Mature BDNF is produced by the enzyme plasmin, which is converted from plasminogen by the enzyme tissue-type plasminogen activator (tPA). In this study, we evaluate circadian function in mice lacking functional tPA. Results tPA−/− mice have normal circadian periods, but show decreased nocturnal wheel-running activity. This difference is eliminated or reversed on the second day of a 48-h fast. Similarly, when placed on daily cycles of restricted food availability the genotypic difference in total wheel-running activity disappears, and tPA−/− mice show equivalent amounts of food anticipatory activity to wild type mice. Conclusions These data suggest that tPA regulates nocturnal wheel-running activity, and that tPA differentially affects SCN-driven nocturnal activity rhythms and activity driven by fasting or temporal food restriction. Electronic supplementary material The online version of this article (10.1186/s12899-018-0036-0) contains supplementary material, which is available to authorized users.

National Oceanic and Atmospheric Administration (NOAA) has adopted an integrated ecosystem research approach to understand climate effects on fish, seabirds, and marine mammals in the Arctic. The integrated ecosystem approach combines traditional oceanography, fisheries, and mammal research techniques to improve scientific understanding of how ecosystems function as a whole. Innovative technologies are being developed to aid in this effort. Two new technologies that NOAA deploys in the Arctic are an advanced thermal imaging technology used to survey ice seal abundance and a high-resolution, nonlethal technology, which integrates cameras and trawls to sample pelagic fishes (CamTrawl). The ice seal surveys relied on thermal imagery to detect warm seal bodies hauled out on cold sea ice. Compared to observer-based surveys, thermal detection surveys require fewer personnel and less postsurvey processing time, can be flown at a higher altitude (reducing disturbance of seals), and yield higher rates of seal detection. The CamTrawl is a self-contained stereo-camera system fitted to the aft end of a trawl at the cod end (i.e., capture bag), which can be left open. By integrating a camera system in the aft portion of a trawl, the CamTrawl concentrates marine organisms and presents this captive group to the cameras without having to recover them for onboard counting, as is done in traditional trawl tows. Compared to traditional survey methods, the CamTrawl more precisely places marine organisms spatially in their environment, which is useful because of small-scale variation in the composition and distribution of fish schools.

Estimating the abundance of seals inhabiting sea ice is complicated because the areas are large, the ice area and distribution may change rapidly, and it is impractical to detect and count a (typically unknown) portion of the population that is in the water, rather than hauled out on the ice. We propose a method for resolving these issues by using a series of daily estimates that are imprecise by themselves, but yield an acceptable estimate when they are combined. Population surveys of spotted seals, ribbon seals and bearded seals were conducted over 279,880 km 2 of the Bering Sea between 13 April and 26 May 2007. During that period, the sea-ice conditions and spatial distribution of seals changed dramatically. We surveyed 2748 km 2 using line transect methods from a helicopter deployed from the US Coast Guard icebreaker Healy . Corrections for incomplete availability of seals used a generalized linear mixed model for seal haul-out probability using sensors on seals with data transmitted by satellite. We accounted for incomplete detection through standard distance-sampling methods along with a double-observer model. The availability and detection models were combined in a data model for local abundance in space and time. To accommodate the shifting ice and seal movements, we then developed a hierarchical spatially-autocorrelated regression model using remotely sensed sea ice concentration data to predict abundance at each survey date. While abundance estimation was very imprecise for each date, we were able to combine them to obtain good estimates of overall population abundance even though the population was spatially dynamic. The proposed hierarchical model combined submodels and accounted for their sources of uncertainty. Spotted seals were most abundant within the study area (233,700, 95% CI 137,300–793,100), followed by bearded seals (61,800, 95% CI 34,900–171,600) and ribbon seals (61,100, 95% CI 35,200–189,300).

Summary Automated detection systems employing advanced technology (e.g. infrared imagery, auditory recording systems, pattern recognition software) are compelling tools for gathering animal abundance and distribution data since investigators can often collect data more efficiently and reduce animal disturbance relative to surveys using human observers. Even with these improvements, analysing animal abundance with advanced technology can be challenging because of potential for incomplete detection, false positives and species misidentification. We argue that double sampling with an independent sampling method can provide the critical information needed to account for such errors. We present a hierarchical modelling framework for jointly analysing automated detection and double sampling data obtained during animal population surveys. Under our framework, observed counts in different sampling units are conceptualized as having arisen from a thinned log-Gaussian Cox process subject to spatial autocorrelation (where thinning accounts for incomplete detection). For multispecies surveys, our approach handles incomplete species observations owing to (i) structural uncertainties (e.g. in cases where the automatic detection data do not provide species observations) and (ii) species misclassification; the latter requires auxiliary information on the misclassification process. As an example of combining an automated detection system and a double sampling procedure, we consider the problem of estimating animal abundance from aerial surveys that use infrared imagery to detect animals, and independent, high-resolution digital photography to provide information on species composition and thermal detection accuracy. We illustrate our approach by analysing simulated data and data from a survey of four ice-associated seal species in the eastern Bering Sea. Our analysis indicated reasonable performance of our hierarchical modelling approach, but suggested a need to balance model complexity with the richness of the data set. For example, highly parameterized models can lead to spuriously high predictions of abundance in areas that are not sampled, especially when there are large gaps in spatial coverage. We recommend that ecologists employ double sampling when enumerating animal populations with automated detection systems to estimate and correct for detection errors. Combining multiple data sets within a hierarchical modelling framework provides a powerful approach for analysing animal abundance over large spatial domains.

Ecologists often use transect surveys to estimate the density and abundance of animal populations. Errors in species classification are often evident in such surveys, yet few statistical methods exist to properly account for them. In this paper, we examine biases that result from species misidentification when ignored, and we develop statistical models to provide unbiased estimates of density in the face of such errors. Our approach treats true species identity as a latent variable and requires auxiliary information on the misclassification process (such as informative priors, experiments using known species, or a double-observer protocol). We illustrate our approach with simulated census data and with double-observer survey data for ice-associated seals in the Bering Sea. For the seal analysis, we integrated misclassification into a model-based framework for distance-sampling data. The simulated data analysis demonstrated reliable estimation of animal density when there are experimental data to inform misclassification rates; double-observer protocols provided robust inference when there were "unknown" species observations but no outright misclassification, or when misclassification probabilities were symmetric and a symmetry constraint was imposed during estimation. Under our modeling framework, we obtained reasonable apparent densities of seal species even under considerable imprecision in species identification. We obtained more reliable inferences when modeling variation in density among transects. We argue that ecologists should often use spatially explicit models to account for differences in species distributions when trying to account for species misidentification. Our results support using double-observer sampling protocols that guard against species misclassification (i.e., by recording uncertain observations as "unknown").

Clinical performance monitoring data on processes of care from a 3-year period were used to assess whether preventive foot care was associated with improved health outcomes in diabetes mellitus patients. Preventive foot care as well as sensory and pedal-pulse examinations were associated with reduced rates of Lower extremity amputation. It is believed that an administrative focus, resource direction, and improvement in process monitoring will lead to better patient outcomes. External review measures can be used by administrators and cLinicians to determine trends in quality of care and patient outcomes andto provide feedback on prevention efforts.

Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.

In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Microsomes isolated from excised shoots of 3-day-old. dark grown, grain sorghum [Sorghum bicolor (L.) Moench, Funk G522DR and DK 41Y] and corn seedlings [Zea mays (L.), Pioneer 3245] metabolized the sulfonylurea herbicide prosulfuron (CGA-152005). Corn microsomes predominantly formed a single major metabolite that resulted from hydroxylation of the phenyl ring at the C5 position. However, sorghum microsomes formed two major metabolites in an approximate 1:1 ratio. One was the 5-hydroxyphenyl metabolite, whereas the second metabolite resulted from ö-demethylation at C4 of the triazine ring. Metabolite identity was established by mass spectrometry and co-chromatography with authentic standards. Metabolism in both corn and sorghum was greatly enhanced by pretreatment of the seed with naphthalic anhydride and by subirrigation with 2.5% ethanol 24 h prior to harvest. Metabolism required a reduced pyridine nucleotide and was affected by several cytochrome P450 monooxygenase inhibitors (carbon monoxide, tetcyclacis, piperonyl butoxide, 1 aminobenzotriazole, and SKF-525A). The inhibitors differentially affected metabolism of prosulfuron. Microsomal oxidations from both untreated and inducer-treated tissue responded similarly to the inhibitors. In exploratory studies, microsomes isolated from shoots of wheat [Triticum aestivum L., Pioneer 2548], barley [Hordeum vulgare L., Boone], oats [Avena sativa L., Southern States 76-30-P242] and rice [Oryza sativa L„ Gulfmont], and room ripened avocado [Persea americana, Mill., Hass] mesocarp tissue also primarily formed the 5-hydroxyphenyl metabolite. Titration of seven different avocado microsomal preparations with prosulfuron provided typical type I difference spectra from which an average binding constant (Ks) of 187 ± 35 μm was obtained

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies.

Microsomes isolated from excised cotyledons of 3-day-old, dark-grown, mung bean ( Vigna radiata , L., cv Berken) seedlings metabolized two endogenous substrates (cinnamic acid and lauric acid), three organophosphate insecticides (diazinon, isazofos, and methidathion), three acetamide herbicides (metolachlor, CGA-24704, and alachlor), and bentazon. Cinnamic acid was aryl hydroxylated forming p -coumaric acid. Lauric acid was primarily hydroxylated at the terminal carbon (ω-hydroxylation). The three α-chloroacetamides were O-demethylated. With all three organophosphate insecticides, the phosphorothionate sulfur was oxidized to the corresponding oxon and the phosphoroester oxygen was cleaved in both diazinon and isazofos. Bentazon was aryl hydroxylated forming the 6-hydroxy derivative. The concentration of cytochrome P450 in the microsomal preparations was marginally enhanced by pretreatment of the seed with naphthalic anhydride (NA), but was markedly increased by subirrigation of NA-treated seed with ethanol and was additionally increased with the combination of NA, clofibrate, and ethanol. The extent of metabolism of only lauric acid paralleled the increases in cytochrome P450 content. The various seed/seedling treatments, however, did approximately double the rate of metabolism of the three organophosphates, the three chloroacetamides, and bentazon. Metabolism required a reduced pyridine nucleotide and was affected by several cytochrome P450 monooxygenase inhibitors (carbon monoxide, tetcyclacis, piperonyl butoxide, 1-aminobenzotriazole, and SKF-525A). The inhibitors differentially affected metabolism of the substrates. Microsomal oxidations from both untreated and inducer-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that metabolism may involve several monooxygenase isoforms.

2H-1,4-Benzoxazin-3(4H)-one has been identified as an intermediate in the biosynthesis of cyclic hydoxamic acids in maize by showing that the deuterium labelled compound is incorporated into DIMBOA with high retention of deuterium and high efficiency, by trapping radioactivity from [ 14 C] anthranilic acid in a pool of unlabelled benzoxazinone administered to maize shoots, and by showing that benzoxazinone is oxidized to DIBOA by maize microsomes in an NADPH- and oxygen-dependent reaction.

Microsomes isolated from excised shoots of 3-day-old, dark-grown, corn (Zea mays L., Pioneer Hybrid 3245) seedlings metabolized two endogenous substrates (cinnamic acid and lauric acid), one insecticide (diazinon), and six herbicides (metolachlor, bentazon, CGA-152005, triasulfuron, primisulfuron, and nicosulfuron). Pretreatment of the seed with the safener naphthalic anhydride resulted in enhanced metabolism of all substrates except cinnamic acid. Metabolism required NADPH and was affected by several cytochrome P450 monooxygenase inhibitors (tetcyclacis, piperonyl butoxide, 1-aminobenzolriazole, SKF-525A, and tridiphane). The inhibitors differentially affected metabolism of the substrates. Tetcyclacis and piperonyl butoxide strongly inhibited the metabolism of all substrates except cinnamic acid. Microsomal oxidations from both unsafened and safener-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that metabolism may involve several monooxygenase isoforms.

Otto Warburg, the father of cellular bioenergetics, seems to have been the first investigator to report on inhibition of a plant biochemical reaction by a progenitor of a selective herbicide. The year was 1920 and the compound was phenylurethane (ethyl N-phenylcarbam ate or EPC). Warburg found that it strongly inhibited photosynthesis in Chlorella. EPC did not develop into a commercial herbicide, but the isopropyl derivatives (propham and chlorpropham) which were introduced in the late 1940s became selective herbicides. The phenylureas (monuron and diuron) were introduced in the early 1950s and shortly thereafter, interference with the Hill reaction by both phenylureas and phenylcarbamates was reported. During the latter part of the 1950s, into the 1960s, and even now, additional herbicidal chemistry was and is being announced that interferes with the Hill reaction. Duysens, in 1963, identified the site of action of diuron, i.e., on the acceptor side of PS II. Corwin Hansch, in 1966 introduced the SAR or QSAR concept in which inhibitory action of Hill inhibitors was related to various chemical and physical parameters. Because of differential responses to partial, thylakoid-associated reactions, the Hill inhibitors were subsequently divided into two groups: pure electron transport inhibitors (phenylureas, s-triazines, triazinones, and uracils) and inhibitory uncouplers (acylanilides, dinitrophenols, benzimidazoles, dinitroanilines, and benzonitriles). The inhibitory uncouplers (dinoseb-types), unlike the diuron-types, uncoupled photophosphorylation by interacting with the coupling factor complexes in both chloroplasts and intact mitochondria. Additionally, the bi-pyridyliums were shown to be reduced by PS I, hence, diverted electrons from the native acceptor. Field observations of triazine resistance were reported in 1970 and resistance was subse­ quently demonstrated at the thylakoid level. Application of the techniques of genetic engineering and biotechnology resulted in identification of the 32 kD a herbicide-binding protein and determination of its amino acid sequence. Crystallization and X-ray examination of the photosynthetic reaction center from Rhodopseudomonas by Michel et al. in the mid-1980s provided new models to account for interactions of herbicides with the D -1 protein. During the 1980s, herbicides were identified that interfered with biochemical machinery in chloroplasts that is not involved in electron transport and light harvesting: inhibition of lipid biosynthesis by aryloxyphenoxypropionates and cyclohexanediones, aromatic amino acid bio­ synthesis by glyphosate, branched chain amino acid biosynthesis by sulfonylureas and imidazolinones, carotenoid biosynthesis by pyridazinones, and porphyrin biosynthesis by diphenylethers and oxadiazoles. The current status of research in most, if not all, of the above areas was reported through oral and poster presentations at this Omiya Symposium.

Microsomes isolated from excised shoots of 3-day-old, dark-grown, grain sorghum [ Sorghum bicolor (L.) Moench. Funk G522DR, and DK 41Y] seedlings metabolized cinnamic acid, lauric acid, metolachlor, bentazon, and diazinon. but did not metabolize triasulfuron or primisulfuron. Pretreatment of G522DR seed with safeners (naphthalic anhydride, dichlormid, flurazole, BAS 145138, oxabetrinil, fluxofenim, and benoxacor) resulted in enhanced metabolism of lauric acid, bentazon, and diazinon. However, metabolism of cinnamic acid was not affected and that of metolachlor was depressed by safener treatments. Microsomes isolated from DK 41Y seedlings had higher endogenous levels of oxidative activity for lauric acid and bentazon than microsomes isolated from G522DR seedlings. Metabolism required NADPH and was affected by CO and other cytochrome P450 monooxygenase inhibitors (tetcyclacis, piperonyl butoxide, 1-aminobenzotriazole, SKF-525A, and tridiphane). The inhibitors differentially affected metabolism of the substrates. Only tetcyclacis strongly inhibited the metabolism of all substrates except cinnamic acid. Microsomal oxidations from both unsafened and safener-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that each substrate was probably metabolized by a different monooxygenase isoform.

To assess the role of a Toyota production system (TPS) quality improvement (QI) intervention on appropriateness of perioperative antibiotic therapy and in length of hospital stay (LOS) among surgical patients.Pre-post quasi-experimental study using local and national retrospective cohorts.We used TPS methods to implement a multifaceted intervention to reduce nosocomial methicillin-resistant Staphylococcus aureus infections on a Veterans Affairs surgical unit, which led to a QI intervention targeting appropriate perioperative antibiotic prophylaxis. Appropriate perioperative antibiotic therapy was defined as selection of the recommended antibiotic agents for a duration not exceeding 24 hours from the time of the operation. The local computerized medical record system was used to identify patients undergoing the 25 most common surgical procedures and to examine changes in appropriate antibiotic therapy and LOS over time.Overall, 2550 surgical admissions were identified from the local computerized medical records. The proportion of surgical admissions receiving appropriate perioperative antibiotics was significantly higher (P.01) in 2004 after initiation of the TPS intervention (44.0%) compared with the previous 4 years (range, 23.4%-29.8%) primarily because of improvements in compliance with antibiotic therapy duration rather than appropriate antibiotic selection. There was no statistically significant decrease in LOS over time.The use of TPS methods resulted in a QI intervention that was associated with an increase in appropriate perioperative antibiotic therapy among surgical patients, without affecting LOS.

Two open, randomized, crossover bioavailability studies were carried out to assess the influence of concurrent antacid medication and food on the bioavailability of clopidogrel. A fed/fasting study was conducted in 12 elderly male subjects. Each subject took a single 75 mg dose of clopidogrel on two occasions-in the morning after an overnight fast, either during a standardized breakfast, or with breakfast delayed by 4 hours after dosing. A washout period of 7 days was observed between the two dosings. Twelve healthy male subjects participated in the antacid study. They fasted overnight and for 4 hours after dosing and took a single 75 mg dose of clopidogrel at 8:00 a.m. on two occasions separated by a washout period of 14 days. For one dose, Maalox 2 x 400 mg tablets were taken 1 hour before the clopidogrel dose. Pharmacokinetic parameters of SR26334, the main circulating metabolite of clopidogrel, were derived from plasma concentrations of the latter compound determined before and at regular intervals over 36 hours after dosing. For the fed/fasting study, mean Cmax values (+/-SD) were 2.7+/-0.62 mg/L and 2.1+/-0.96 mg/L for the fasting state and the fed state, respectively and the 90% CI of Cmax ratio was [0.57 - 0.97]. Mean AUC(0-obs) values (AUC to the last observed value) were 7.1+/-1.6 mg.h/L and 7.4+/-1.64 mg.h/L, respectively, and the 90% Cl of AUC ratios were [0.90 - 1.02] and [0.89 - 0.97], respectively. For the antacid study, mean Cmax values were 2.6+/-0.84 mg/L and 2.5+/-0.87 mg/L for the no-antacid regimen and the antacid regimen, respectively, and the 90% CI of Cmax ratio was [0.74 - 1.16]. Mean AUC(0-obs) values were 6.3+/-1.34 mg.h/L and 5.8+/-1.33 mg.h/L, respectively, and the 90% CI of AUC ratios was [0.89+/-0.97]. Thus, exposure to SR26334, and therefore net absorption of clopidogrel, was not significantly modified by food or by prior antacid ingestion.

Ethanol treatment of etiolated maize seedlings led to a marked enhancement in metabolism of the herbicides metolachlor and prosulfuron. cDNA clones representing eight genes that encode putative cytochrome P450 enzymes were isolated from maize. They fall into three families and are designated CYP71C5, CYP73A6, A7, and A8, and CYP81A1, A2, A3, and A4. Ethanol treatment induced the CYP81A subfamily at the mRNA level in both roots and shoots of etiolated seedlings.

Peer review of social work documentation has helped promote the uniformity and consistency with which services are delivered; and social workers are exposed to the professional practice of their colleagues, since all staff members participate in conducting peer reviews. The system also helps identify areas of weakness or deficiency common to staff members. The detection of such patterns is the responsibility of the peer review committee, which brings them to the attention of the social work service management and of the service's QA committee. Corrective action is then taken to remedy identified problems. The system also has the potential to help identify patient problems and needs not met by the existing system of health care delivery; such gaps in service can then be brought to the attention of the management of the medical center for consideration and possible resolution. By focusing on documentation elements as indicators of high-quality and appropriate social work intervention, the peer review system used by the social work service has helped enhance social worker performance and service delivery.

A microsomal fraction isolated from the shoots of 3- to 4-day-old, dark-grown, grain sorghum (Sorghum bicolor cv. Funk G 522 D R ) seedlings was characterized. The preparations had a cytochrome P-450 content that varied from approximately 90 to 150 pmol P-450/mg protein with cytochrome P-420 varying from 0 to 3% of the P-450 content. Type I difference spectra were formed with cinnamic acid and metolachlor, and a type II spectrum was formed with tetcyclacis. In short-term assays with [14C]metolachlor as substrate, the preparations produced a single time-dependent product that separated on silica gel TLC plates developed in benzene/acetone (2:1, v/v). RF values for metolachlor and the metabolite were approximately 0.70 and 0.48, respectively. The microsomal reaction required N A D P H and oxygen, and was inhibited by carbon monoxide, with the inhibition being partially reversed by actinic light. Compounds known to inhibit the activity of cytochrome P-450 monooxygenases (piperonyl butoxide, tetcyclacis, and tridiphane) also prevented formation of the metabolite. Identity of the metabolite was confirmed by TLC and positive ion thermospray LC/MS to be 2-chloro-N-(2-ethyl-6- methylphenyl)-N-(2-hydroxy-l-methylethyl)acetamide. Hence, the reaction catalyzed by the sorghum microsomes involved O-demethylation of the methoxypropyl side chain of metolachlor.

The effects of luteolin, quercetin, and taxifolin on light induced phosphorylation and electron transport in isolated, greenhouse-grown, spinach (Spinacia oleracea L.) thylakoids were investigated. Luteolin and quercetin interacted with components associated with both the ATP-generating pathway and the electron-transport pathway. However, the action of taxifolin involved only the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of phosphorylation than oxygen uptake in coupled whole-chain electron transport, inhibition of the light-activated Mg2+-ATPase, and inhibition of the Ca2+-ATPase associated with CF1. The following order of decreasing inhibitory effectiveness was exhibited: luteolin > quercetin >>> taxifolin. On the electron-transport pathway, luteolin and quercetin interfered with the activity of the QB-protein complex as evidenced by inhibition of the partial reaction with diphenylcarbazide as the electron donor and 2,6-dichlorophenolindophenol as electron acceptor; alteration of the chlorophyll fluorescence transients; and competitive displacement of radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine].

Tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] cross-reacts as a synergist to both herbicides and insecticides. As reported herein, tridiphane inhibited the monooxygenase activity of one or more mouse hepatic cytochrome P450 isozymes. With hepatic microsomes isolated from mice pretreated with phenobarbitol (PB) as well as with purified P450 enzymes, tridiphane efficiently inhibited demethylase and deethylase activities catalyzed by PB-induced P450 enzymes, i.e., I 50 values of approximately 4.0 μ M . However, tridiphane was a weak inhibitor of enzyme activities catalyzed by hepatic microsomes isolated from 3-methylcholanthrene (3-MC)-pretreated mice. Type I binding spectra were obtained with hepatic microsomes isolated from uninduced mice ( K s = 12.6 μM ) and PB-induced mice ( K s = 1.0 μM ), and with a purified enzyme isolated from PB-induced mice ( K s = 0.12 μM ). However, tridiphane did not form difference spectra with microsomes isolated from 3-MC-treated mice or with a purified P450 enzyme isolated from 3-MC-treated mice. Inhibition of p -nitroanisole O -demethylase suggested that tridiphane acted as a competitive inhibitor of enzyme activity and produced a K m of 440 μ M with a K i between 0.72 and 1.04 μ M .

13C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoe tubiflora was used to observe Crassulacean acid metabolism in vivo. 13C signals from C-4 of malate were observed after overnight exposure of leaves to 13CO2. Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, 13C signals were detected in all four carbons of malate in the previously labeled leaves. The 13C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.

The effects of flavone and seven flavonols on the light-induced electron transport and phosphorylation of isolated spinach ( Spinacia oleracea L.) chloroplasts were investigated. With the exception of flavonol (3-hydroxyflavone), all of the compounds interacted with components of both the ATP-generating and electron transport pathways. Flavonol only interacted with the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of the phosphorylation reaction than coupled whole-chain electron transport, inhibition of cyclic phosphorylation, inhibition of the light-activated Mg 2+ -ATPase, and inhibition of the heat-activated Ca 2+ -ATPase associated with CF 1 . The overall decreasing order of effectiveness for inhibition of cyclic phosphorylation was: galangin > quercetin = kaempferol = myricetin = flavonol > fisetin > flavone > morin. On the electron transport pathway, all of the compounds, except flavonol, interacted with the Q B -protein complex as, evidenced by inhibition of uncoupled electron transport, alteration of chlorophyll fluorescence transients, and competitive displacement of previously bound radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- s -triazine]. The decreasing order of effectiveness for inhibition of uncoupled electron transport was: fisetin > quercetir = galangin > kaempferol > flavone > morin.

Twelve substituted 2,6-dinitroaniline herbicides were shown to interfere with electron transport and phosphorylation in isolated mung bean ( Phaseolus aureus Roxb.) mitochondria. Postulated sites of action involved complexes I and II, at or near the flavoprotein associated with the oxidation of exogenous NADH, the alternate (cyanide-insensitive) oxidase, and some step in the phosphorylation pathway. Differences among the various 2,6-dinitroanilines tested were generally quantitative rather than qualitative. All of the 2,6-dinitroanilines inhibited the rate of valinomycin-induced mitochondrial swelling in isotonic KCl within the same concentration range that caused inhibition of state 3 respiration. It is postulated that reduced membrane fluidity, or inhibition of chloride transport, may explain the effects on valinomycin-induced swelling. We propose that the 2,6-dinitroanilines partition into the inner mitochondrial membrane, thereby decrease membrane fluidity, and alter membrane permeability. The resulting perturbations to the structural organization of the electron transport components may be responsible for the apparent multiple-site inhibition of respiration.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.

Many herbicides inhibit chloroplast electron transport by interfering with a proteinaceous component of the Qв complex located in the appressed granal membrane. Certain of these herbicides, designated inhibitory uncouplers, also interfere with photophosphorylation and affect other chloroplast-mediated responses, some of which involve components located in the nonappressed granal membrane. The inhibitory uncouplers can be divided into dinoseb (phenolic) types which contain dissociable protons and dicryl (acylanilide) types which are nonionic. The dinoseb types can function as protonophores and shuttle protons across the thylakoid membrane at low concentrations and can alter the integrity of semipermeable membranes at higher concentrations. However, the dicryl types only alter the integrity of the membranes. The inhibitory uncouplers, but not the DCMU-types of electron transport inhibitors: stimulated electron trans- port from DPIPH2 to methyl viologen; inhibited valifiomycin-induced swelling of intact chloroplasts; increased the permeability of the chloroplast envelope to K+ in the absence of an ionophore; prevented energization of the thylakoid membrane by PS I; and increased the permeability of phosphatidyl choline liposomes to protons. Chlorination response patterns obtained with isomers of N-phenyl-2-methylpentanamides in the above reactions, in general, were similar for interference with the Qв complex, i.e., in all assays, dichlorination in the 3,4 or 3,5 positions was associated with maximum inhibitory potency, whereas substitution in an ortho position decreased inhibitory activity. With a series of 1-alkyl-3-(α,α,α-trifluoro-m-tolyl)ureas, maximum inhibition of electron transport was obtained with the butyl derivative, whereas maximum responses for uncoupling and membrane disturbances were obtained with the hexyl or octyl derivatives. Some of the interferences produced by inhibitory uncouplers may result from interactions with the lipoidal components of chloroplast membranes.

Characteristics of mitochondria isolated from genotypes differing in plant vigor were evaluated with divergent selections from a population of (L.) and selections within a broad-base population of random homozygous lines in (L.) Merr. These selections differed markedly in plant growth by the sixth leaf (or trifoliolate leaf) stage of growth. Cytoplasmic backgrounds were controlled so that variability under nuclear gene control was investigated primarily. Significant variability among mitochondria isolated from soybean genotypes was found for adenosine diphosphate-oxygen (ADP/O) ratios with malate. However, selection groups in both maize and soybean had similar ADP/O ratios. Mitochondria from vigorous selections had higher respiratory control ratios (RC) with malate and lower succinate to malate ratios (S/M) for ADP-activated (state-3) respiration than the nonvigorous selections in both maize and soybean material. No mitochondrial complementation between diverse soybean genotypes was found when realate, succinate, and nicotinamide adenine dinucleotide (NADH) were used as substrates. With α-ketoglutarate as substrate, no complementation for efficiency of phosphorylation was found between two pairs of soybean lines representing the extreme contrasts available. A highly significant reduction in state-3 oxidation rates occurred when the mitochondria were mixed; however, no differences in mitochondrial activities were found between vigorous and nonvigorous genotypes, a difference required if the complementation effect reflected a genotypic potential. Mitochondrial complementation effects did not appear to reflect productivity in soybeans.