Exploring Redox Modulation of Plant UDP-Glucose Pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

Integration of social and ecological sciences for natural resource decision making: challenges and opportunities.

The last 25 years have witnessed growing recognition that natural resource management decisions depend as much on understanding humans and their social interactions as on understanding the interactions between non-human organisms and their environment. Decision science provides a framework for integrating ecological and social factors into a decision, but challenges to integration remain. The decision-analytic framework elicits values and preferences to help articulate objectives, and then evaluates the outcomes of alternative management actions to achieve these objectives. Integrating social science into these steps can be hindered by failing to include social scientists as more than stakeholder-process facilitators, assuming that specific decision-analytic skills are commonplace for social scientists, misperceptions of social data as inherently qualitative, timescale mismatches for iterating through decision analysis and collecting relevant social data, difficulties in predicting human behavior, and failures of institutions to recognize the importance of this integration. We engage these challenges, and suggest solutions to them, helping move forward the integration of social and biological/ecological knowledge and considerations in decision-making.

Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies.

UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 µm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 µm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

The Arabidopsis LRR-RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF-PXY/TDR-WOX4 signaling pathway.

BACKGROUND: Although a number of leucine-rich repeat receptor-like kinase-encoding genes (LRR-RLKs) have been identified in plants, a functional role has been determined for only a few. Recent studies have demonstrated that an LRR-RLK, PXY/TDR, is important for the process of secondary vascular development. Other studies have indicated that PXY/TDR is unlikely to be the sole LRR-RLK involved in this complex process. RESULTS: In this study, in silico analyses led to the identification of three Arabidopsis LRR-RLK genes (PXY-correlated; PXC1, 2, 3) with transcript accumulation profiles that correlated strongly with several key regulators of vascular development, including PXY/TDR, HB-8, REV, and CLE41. Expression profiling using qPCR and promoter:reporter lines indicated that all three PXC genes are associated with the vasculature. One in particular, PXC1 (At2g36570), had a strong correlation with PXY/TDR. Shifting pxc1 mutants from long-days to short-days showed that loss of the gene led to a dramatic reduction in secondary wall formation in xylem fibers. Transcript analysis of mutants for a variety of secondary cell wall-associated genes, including PXY/TDR indicated that the pathways mediated by PXC1 connect with those mediated by the TDIF-PXY/TDR-WOX4 system. CONCLUSIONS: The data indicate that the LRR-RLK, PXC1 is involved in secondary cell wall formation in xylem fibers. Whereas further study is needed to identify the ligands and mode of action of the PXC1 protein, it is clear from this work that similarly to the shoot apical meristem (SAM), secondary vascular development requires contributions from a number of LRR-RLKs.

Holmium laser endourethrotomy and litholapaxy of an occult prostatic utricle calculus.

Prostatic utricle (PU) stones are rare and the surgical treatment of such PU anomalies has proved challenging given their location adjacent to the posterior urethra and proximity to important fertility and continence structures. We report the case of a 58-year-old male with hypospadias, microphallus, and irritative lower urinary tract symptoms (LUTS) found to have a large PU stone. He underwent successful complete endoscopic removal of the stone via transurethral unroofing and holmium laser litholapaxy. We conclude that it is an effective alternative to other surgical techniques once size of the stone and the patient's individual anatomy are considered.

Effects of Magnesium, Pyrophosphate and Phosphonates on Pyrophosphorolytic Reaction of UDP-Glucose Pyrophosphorylase.

UDP-glucose pyrophosphorylase (UGPase) carries a freely reversible reaction, using glucose-1-P and UTP to produce UDP-glucose (UDPG) and pyrophosphate (PPi), with UDPG being essential for glycosylation reactions in all organisms including, e.g., synthesis of sucrose, cellulose and glycoproteins. In the present study, we found that free magnesium (Mg2+) had profound effects on the reverse reaction of purified barley UGPase, and was absolutely required for its activity, with an apparent Km of 0.13 mM. More detailed analyses with varied concentrations of MgPPi allowed us to conclude that it is the MgPPi complex which serves as true substrate for UGPase in its reverse reaction, with an apparent Km of 0.06 mM. Free PPi was an inhibitor in this reaction. Given the key role of PPi in the UGPase reaction, we have also tested possible effects of phosphonates, which are analogs of PPi and phosphate (Pi). Clodronate and etidronate (PPi analogs) had little or no effect on UGPase activity, whereas fosetyl-Al (Pi analog), a known fungicide, acted as effective near-competitive inhibitor versus PPi, with Ki of 0.15 mM. The data are discussed with respect to the role of magnesium in the UGPase reaction and elucidating the use of inhibitors in studies on cellular function of UGPase and related enzymes.

Linking knowledge to action in collaborative conservation.

Authors have documented a "research-implementation gap" in conservation. Research intended to inform conservation practice often does not, and practice often is not informed by the best science. We used the literature on policy learning (i.e., literature attributing policy change to learning) to structure a study of how practice is informed by science in collaborative conservation. We studied implementation by U.S. states of state wildlife action plans. On the basis of 60 interviews with government and nongovernmental organization representatives, we identified 144 implementation initiatives for State Wildlife Action Plans that were collaborative. We conducted case studies of 6 of these initiatives, which included interviews of key individuals and analysis of written documents. We coded interview transcripts and written documents to identify factors that influence availability and use of scientific information. We integrated these factors into a model of collaborative conservation. Although tangible factors such as funding and labor directly affected the availability of scientific information, practitioners' ability and willingness to use the information depended on less tangible factors such as the quality of interpersonal relationships and dialogue. Our work demonstrates empirically that relationships and dialogue led to: (1) the sharing of resources, such as funding and labor, that were needed to carry out research and produce information and (2) agreement among researchers and practitioners on conservation objectives, which was necessary for that new information to inform action. Our findings can be understood in the context of broader concepts articulated in the policy-learning literature, which establishes that social learning (improving relationships and dialogue) provides the foundation for conceptual learning (setting objectives) and technical learning (determining how to achieve these objectives).

Resumen: Diferentes autores han documentado un "vacío de investigación-implementación" en la conservación. La investigación que intenta informar a la práctica de la conservación a menudo no lo hace, y la práctica a menudo no es informada por la mejor ciencia. Utilizamos la literatura sobre aprendizaje de políticas (i.e., literatura que atribuye cambios en políticas al aprendizaje) para estructurar un estudio de cómo la práctica es informada por la ciencia en conservación colaborativa. Estudiamos la implementación de planes de acción para vida silvestre en estados de E.U.A. Con base en 60 entrevistas con representantes de organizaciones gubernamentales y no gubernamentales, identificamos 144 iniciativas de implementación de Planes de Acción Estatales para Vida Silvestre que fueron colaborativas. Realizamos estudios de caso de 6 de estas iniciativas, que incluían entrevistas a individuos clave y el análisis de documentos escritos. Codificamos las transcripciones de las entrevistas y los documentos escritos para identificar factores que influyen en la variabilidad y uso de la información científica. Integramos estos factores en un modelo de investigación colaborativa. Aunque factores tangibles, como el financiamiento y labor, directamente afectaron la disponibilidad de información científica, la habilidad y disponibilidad de practicantes para utilizar la información dependió de factores menos tangible como la calidad de relaciones interpersonales y de diálogo. Nuestro trabajo demuestra empíricamente que las relaciones y el diálogo llevaron a: (1) compartir recursos, como el financiamiento y la labor, que fueron necesarios para llevar a cabo el proyecto y producir información y (2) acuerdos sobre objetivos de conservación entre investigadores y practicantes, lo cual fue necesario para que la información nueva informe a la acción. Nuestros hallazgos pueden ser entendidos en el contexto de conceptos más amplios articulados en la literatura de aprendizaje de políticas, que establece que el aprendizaje social (mejora de relaciones y diálogo) proporciona el fundamento del aprendizaje conceptual (fijar objetivos) y el aprendizaje técnico (determinación de cómo alcanzar esos objetivos).

222 nm Photo-induced radical reactions in silazanes. A combined laser photolysis, EPR, GC-MS and QC Study.

The initiation mechanism of the VUV-induced conversion of polyorganosilazanes into methyl-Si-O-Si networks was studied by means of model disilazane compounds. A combined experimental approach was chosen to determine the primary radicals and their properties (lifetimes, spectra) as well as the major final products. It was verified that both Si-N and Si-CH(3) cleavage occur in the condensed phase, the former with higher yield. The lifetime of the primary Si- and N-centred radicals in de-oxygenated n-hexane solution is less than

Vacuum-UV irradiation-based formation of methyl-Si-O-Si networks from poly(1,1-dimethylsilazane-co-1-methylsilazane).

The vacuum-UV (VUV)-induced conversion of commercially available poly(1,1-dimethylsilazane-co-1-methylsilazane) into methyl-Si-O-Si networks was studied using UV sources at wavelengths around 172, 185, and 222 nm, respectively. Time-of-flight secondary ion mass spectroscopy (TOF-SIMS), X-ray photo electron spectroscopy (XPS), and Fourier transform infrared (FTIR) measurements, as well as kinetic investigations, were carried out to elucidate the degradation process. First-order kinetics were found for the photolytically induced decomposition of the Si-NH-Si network, the subsequent formation of the methyl-Si-O-Si network and the concomitant degradation of the Si-CH(3) bond, which were additionally independent of the photon energy above a threshold of about 5.5 eV (225 nm). The kinetics of these processes were, however, dependent on the dose actually absorbed by the layer and, in the case of Si-O-Si formation, additionally on the oxygen concentration. The release of ammonia and methane accompanied the conversion process. Quantum-chemical calculations on methyl substituted cyclotetrasilazanes as model compounds substantiate the suggested reaction scheme. Layers <100 nm in thickness based on mixtures of poly(1,1-dimethylsilazane-co-1-methylsilazane) and perhydropolysilazane (PHPS) were coated onto polyethylene terephthalate (PET) foils by a continuous roll to roll process and cured by VUV irradiation by using wavelengths <200 nm and investigated for their O(2) and water vapor-barrier properties. It was found that the resulting layers displayed oxygen and water vapor transmission rates (OTR and WVTR, respectively) of <1 cm(3) m(-2) d(-1) bar(-1) and <4 g m(-2) d(-1), respectively.

The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases.

UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions.

Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.

Public understanding of One Health messages: The role of temporal framing.

Building on research in motivated reasoning and framing in science communication, we examine how messages that vary attribution of responsibility (human vs animal) and temporal orientation (now vs in the next 10 years) for wildlife disease risk influence individuals' conservation intentions. We conducted a randomized experiment with a nationally representative sample of US adults ( N = 355), which revealed that for people low in biospheric concern, messages that highlighted both human responsibility for and the imminent nature of the risk failed to enhance conservation intentions compared with messages highlighting animal responsibility. However, when messages highlighting human responsibility placed the risk in a temporally distal frame, conservation intentions increased among people low in biospheric concern. We assess the underlying mechanism of this effect and discuss the value of temporal framing in overcoming motivated skepticism to improve science communication.

Artelle et al. (2018) miss the science underlying North American wildlife management.

Artelle et al. (2018) conclude that "hallmarks of science" are largely missing from North American wildlife management based on a desk review of selected hunting management plans and related documents found through Internet searches and email requests to state and provincial wildlife agencies. We highlight three fundamental problems that compromise the validity of the conclusions posited: missing information to support selection of "hallmarks of science," confusion about the roles and nature of science and management, and failure to engage effectively with the scientists and managers actively managing wildlife populations in North America.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases.

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), ß-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not ß-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

WILDLIFE HEALTH AND PUBLIC TRUST RESPONSIBILITIES FOR WILDLIFE RESOURCES.

A significant development in wildlife management is the mounting concern of wildlife professionals and the public about wildlife health and diseases. Concurrently, the wildlife profession is reexamining implications of managing wildlife populations as a public trust and the concomitant obligation to ensure the quality (i.e., health) and sustainability of wildlife. It is an opportune time to emphasize the importance of wildlife health, specifically to advocate for comprehensive and consistent integration of wildlife health in wildlife management. We summarize application of public trust ideas in wildlife population management in the US. We argue that wildlife health is essential to fulfilling public trust administration responsibilities with respect to wildlife, due to the central responsibility of trustees for ensuring the well-being of wildlife species (i.e., the core resources of the trust). Because both health of wildlife and risk perceptions regarding threats posed by wildlife disease to humans and domestic animals are issues of growing concern, managing wildlife disease and risk communication vis-à-vis wildlife health is critical to wildlife trust administration. We conclude that wildlife health professionals play a critical role in protecting the wildlife trust and that current conditions provide opportunities for important contributions by wildlife health professionals in wildlife management.

The role of one health in wildlife conservation: a challenge and opportunity.

Numerous emerging infectious diseases (EIDs) have arisen from or been identified in wildlife, with health implications for both humans and wildlife. In the practice of wildlife conservation, to date most attention has focused on the threat EIDs pose to biodiversity and wildlife population viability. In the popular media and public eye, however, wildlife is often only portrayed as the cause of EIDs and resultant human health impacts. There is little coverage on the roles of human-induced habitat destruction or wildlife population stress in EID spread, nor the negative impacts of disease on wildlife. Here, we focus on a little-studied and seldom discussed concern: how real and perceived risks of wildlife-associated diseases for human and companion animal health might erode public support for wildlife conservation. We believe that wildlife-associated EIDs and public perceptions of these risks are among the most important threats to wildlife conservation. In light of this concern, we explore the challenges and opportunities for addressing this situation in a One Health context that emphasizes the interdisciplinary collaboration and the inextricable nature of human and animal health and disease.

Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase.

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

Understanding risk perceptions to enhance communication about human-wildlife interactions and the impacts of zoonotic disease.

Inclusion of wildlife in the concept of One Health is important for two primary reasons: (1) the physical health of humans, domesticated animals, and wildlife is linked inextricably through shared diseases, and (2) humans' emotional well-being can be affected by their perceptions of animal health. Although an explicit premise of the One Health Initiative is that healthy wildlife contribute to human health, and vice versa, the initiative also suggests implicitly that wildlife may pose threats to human health through zoonotic disease transmission. As people learn more about One Health, an important question surfaces: How will they react to communications carrying the message that human health and wildlife health are linked? In the absence of adequate relevant research data, we recommend caution in the production and dissemination of One Health messages because of possible unintended or collateral effects. Understanding how and why individuals perceive risks related to wildlife diseases is essential for determining message content that promotes public support for healthy wildlife populations, on the one hand, and, on the other, for identifying messages that might inadvertently increase concern about human health effects of diseased wildlife. To that end, we review risk perception research and summarize the few empirical studies that exist on perceived risk associated with zoonoses. We conclude with some research questions that need answering to help One Health practitioners better understand how the public will interpret their messages and thus how to communicate positively and without negative collateral consequences for wildlife conservation.

Pediatric hemorrhagic cystitis.

PURPOSE: To review the current literature as it pertains to hemorrhagic cystitis (HC) in the pediatric bone-marrow transplant (BMT) population. By reviewing the pathophysiology of the disease, preventive methods, and therapeutic options, urologists may be better equipped to manage this challenging clinical scenario. MATERIALS AND METHODS: The HC literature was reviewed using a MEDLINE/PubMed literature search, specifically focusing on the pediatric BMT population as it pertains to the incidence, pathophysiology, prevention, and treatment of HC. RESULTS: Conservative estimates of HC incidence in recent retrospective studies of pediatric BMT populations still approach 10-20%. Several high-volume pediatric BMT centers have reported contemporary data on their experience with HC providing increased insight into incidence and pathophysiology. Accumulating evidence linking BK virus to HC is a significant development warranting further investigation. Other contributing agents/risk factors need identification in the likely multifactorial etiology of HC. Preventive and therapeutic strategies have made modest advances, but certainly need further validation with prospective randomized studies. CONCLUSIONS: Pediatric BMT patients are susceptible for HC development despite preventive measures and improved insight into the pathophysiology. Unfortunately, there are no evidence-based treatment guidelines for this difficult clinical issue that frequently requires prolonged care and multiple treatment modalities necessitating judicious patience in the application of more aggressive interventions.