A tRNA modifying enzyme as a tunable regulatory nexus for bacterial stress responses and virulence.

Post-transcriptional modifications can impact the stability and functionality of many different classes of RNA molecules and are an especially important aspect of tRNA regulation. It is hypothesized that cells can orchestrate rapid responses to changing environmental conditions by adjusting the specific types and levels of tRNA modifications. We uncovered strong evidence in support of this tRNA global regulation hypothesis by examining effects of the well-conserved tRNA modifying enzyme MiaA in extraintestinal pathogenic Escherichia coli (ExPEC), a major cause of urinary tract and bloodstream infections. MiaA mediates the prenylation of adenosine-37 within tRNAs that decode UNN codons, and we found it to be crucial to the fitness and virulence of ExPEC. MiaA levels shifted in response to stress via a post-transcriptional mechanism, resulting in marked changes in the amounts of fully modified MiaA substrates. Both ablation and forced overproduction of MiaA stimulated translational frameshifting and profoundly altered the ExPEC proteome, with variable effects attributable to UNN content, changes in the catalytic activity of MiaA, or availability of metabolic precursors. Cumulatively, these data indicate that balanced input from MiaA is critical for optimizing cellular responses, with MiaA acting much like a rheostat that can be used to realign global protein expression patterns.

Acute Effects of Cheddar Cheese Consumption on Circulating Amino Acids and Human Skeletal Muscle.

Cheddar cheese is a protein-dense whole food and high in leucine content. However, no information is known about the acute blood amino acid kinetics and protein anabolic effects in skeletal muscle in healthy adults. Therefore, we conducted a crossover study in which men and women (n = 24; ~27 years, ~23 kg/m2) consumed cheese (20 g protein) or an isonitrogenous amount of milk. Blood and skeletal muscle biopsies were taken before and during the post absorptive period following ingestion. We evaluated circulating essential and non-essential amino acids, insulin, and free fatty acids and examined skeletal muscle anabolism by mTORC1 cellular localization, intracellular signaling, and ribosomal profiling. We found that cheese ingestion had a slower yet more sustained branched-chain amino acid circulation appearance over the postprandial period peaking at ~120 min. Cheese also modestly stimulated mTORC1 signaling and increased membrane localization. Using ribosomal profiling we found that, though both milk and cheese stimulated a muscle anabolic program associated with mTORC1 signaling that was more evident with milk, mTORC1 signaling persisted with cheese while also inducing a lower insulinogenic response. We conclude that Cheddar cheese induced a sustained blood amino acid and moderate muscle mTORC1 response yet had a lower glycemic profile compared to milk.

Reduced Physical Activity Alters the Leucine-Stimulated Translatome in Aged Skeletal Muscle.

Periods of inactivity experienced by older adults induce nutrient anabolic resistance creating a cascade of skeletal muscle transcriptional and translational aberrations contributing to muscle dysfunction. The purpose of this study was to identify how inactivity alters leucine-stimulated translation of molecules and pathways within the skeletal muscle of older adults. We performed ribosomal profiling alongside RNA sequencing from skeletal muscle biopsies taken from older adults (n = 8; ~72 years; 6 F/2 M) in response to a leucine bolus before (Active) and after (Reduced Activity) 2 weeks of reduced physical activity. At both visits, muscle biopsies were taken at baseline, 60 minutes (early response), and 180 minutes (late response) after leucine ingestion. Previously identified inactivity-related gene transcription changes (PFKFB3, GADD45A, NMRK2) were heightened by leucine with corresponding changes in translation. In contrast, leucine also stimulated translational efficiency of several transcripts in a manner not explained by corresponding changes in mRNA abundance ("uncoupled translation"). Inactivity eliminated this uncoupled translational response for several transcripts, and reduced the translation of most mRNAs encoding for ribosomal proteins. Ingenuity Pathway Analysis identified discordant circadian translation and transcription as a result of inactivity such as translation changes to PER2 and PER3 despite unchanged transcription. We demonstrate inactivity alters leucine-stimulated "uncoupled translation" of ribosomal proteins and circadian regulators otherwise not detectable by traditional RNA sequencing. Innovative techniques such as ribosomal profiling continues to further our understanding of how physical activity mediates translational regulation, and will set a path toward therapies that can restore optimal protein synthesis on the transcript-specific level to combat negative consequences of inactivity on aging muscle.

Maize leaf PPDK regulatory protein isoform-2 is specific to bundle sheath chloroplasts and paradoxically lacks a Pi-dependent PPDK activation activity.

In C4 plants, the pyruvate phosphate dikinase regulatory protein (PDRP) regulates the C4 pathway enzyme pyruvate phosphate dikinase (PPDK) in response to changes in incident light intensity. In maize (Zea mays) leaves, two distinct isoforms of PDRP are expressed, ZmPDRP1 and ZmPDRP2. The properties and C4 function of the ZmPDRP1 isoform are well understood. However, the PDRP2 isoform has only recently been identified and its properties and function(s) in maize leaves are unknown. We therefore initiated an investigation into the maize PDRP2 isoform by performing a side by side comparison of its enzyme properties and cell-specific distribution with PDRP1. In terms of enzyme functionality, PDRP2 was found to possess the same protein kinase-specific activity as PDRP1. However, the PDRP2 isoform was found to lack the phosphotransferase activity of the bifunctional PDRP1 isoform except when PDRP2 in the assays is elevated 5- to 10-fold. A primarily immuno-based approach was used to show that PDRP1 is strictly expressed in mesophyll cells and PDRP2 is strictly expressed in bundle sheath strand cells (BSCs). Additionally, using in situ immunolocalization, we establish a regulatory target for PDRP2 by showing a significant presence of C4 PPDK in BSC chloroplasts. However, a metabolic role for PPDK in this compartment is obscure, assuming PPDK accumulating in this compartment would be irreversibly inactivated each dark cycle by a monofunctional PDRP2.

Leucine Differentially Regulates Gene-Specific Translation in Mouse Skeletal Muscle.

Background: Amino acids, especially leucine, are particularly effective in promoting protein synthesis. Leucine is known to increase the rate of protein synthesis in skeletal muscle through the mechanistic target of rapamycin complex 1-dependent, as well as -independent, signaling pathways. However, the overall translation program is poorly defined, and it is unknown how the activation of these pathways differentially controls the translation of specific mRNAs.Objective: Ribosome profiling and RNA sequencing were used to precisely define the translational program activated by an acute oral dose of leucine.Methods: Adult male C57BL/6 mice were deprived of food overnight before the delivery of an acute dose of l-leucine (9.4 mg) (n = 6) or vehicle (n = 5) and tissues collected 30 min later. Ribosome footprints and total RNA were isolated and subjected to deep sequencing. Changes in gene-specific mRNA abundance and ribosome occupancy were determined between the leucine-treated and control groups by aligning sequence reads to Reference Sequence database mRNAs and applying statistical features of the Bioconductor package edgeR.Results: Our data revealed mRNA features that confer translational control of skeletal muscle mRNAs in response to an acute dose of leucine. The subset of skeletal muscle mRNAs that are activated consists largely of terminal oligopyrimidine mRNAs (false discovery rate: <0.05), whereas those with reduced translation had 5' untranslated regions with increased length. Only the small nuclear RNAs, which are required for ribosome biogenesis, were significantly altered in RNA abundance. The inferred functional translational program activated by dietary leucine includes increased protein synthesis capacity and energy metabolism, upregulation of sarcomere-binding proteins, modulation of circadian rhythm, and suppression of select immune components.Conclusions: These results clarify the translation program acutely stimulated by leucine in mouse skeletal muscle and establish new methodologies for use in future studies of skeletal muscle disease or aging and further examination of downstream effects of leucine on gene expression.

Factors influencing evidence-based practice for community nurses.

Factors influencing the development of evidence-based nursing practice (EBNP) were examined in Prince Edward Island, Canada. An adapted electronic questionnaire was distributed to practicing registered nurses and nurse practitioners (n=68). An analysis of variance revealed a significant difference between nurses' clinical practice setting and the EBNP scale. Significant differences were also found between age and education level when compared with the EBNP subscales where novice nurses were less likely to rely on experience and intuition, and expert nurses with a higher level of education reported being more skilful at synthesising and applying information from research findings into their nursing practice.

Senescence, dormancy and tillering in perennial C4 grasses.

Perennial, temperate, C4 grasses, such as switchgrass and miscanthus have been tabbed as sources of herbaceous biomass for the production of green fuels and chemicals based on a number of positive agronomic traits. Although there is important literature on the management of these species for biomass production on marginal lands, numerous aspects of their biology are as yet unexplored at the molecular level. Perenniality, a key agronomic trait, is a function of plant dormancy and winter survival of the below-ground parts of the plants. These include the crowns, rhizomes and meristems that will produce tillers. Maintaining meristem viability is critical for the continued survival of the plants. Plant tillers emerge from the dormant crown and rhizome meristems at the start of the growing period in the spring, progress through a phase of vegetative growth, followed by flowering and eventually undergo senescence. There is nutrient mobilization from the aerial portions of the plant to the crowns and rhizomes during tiller senescence. Signals arising from the shoots and from the environment can be expected to be integrated as the plants enter into dormancy. Plant senescence and dormancy have been well studied in several dicot species and offer a potential framework to understand these processes in temperate C4 perennial grasses. The availability of latitudinally adapted populations for switchgrass presents an opportunity to dissect molecular mechanisms that can impact senescence, dormancy and winter survival. Given the large increase in genomic and other resources for switchgrass, it is anticipated that projected molecular studies with switchgrass will have a broader impact on related species.

P39, a novel soybean protein allergen, belongs to a plant-specific protein family and is present in protein storage vacuoles.

Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide.

Reactive oxygen species, ABA and nitric oxide interactions on the germination of warm-season C4-grasses.

Hydrogen peroxide (H(2)O(2)) as a source of reactive oxygen species (ROS) significantly stimulated germination of switchgrass (Panicum virgatum L.) seeds with an optimal concentration of 20 mM at both 25 and 35 degrees C. For non-dormant switchgrass seeds exhibiting different levels of germination, treatment with H(2)O(2) resulted in rapid germination (<3 days) of all germinable seeds as compared to seeds placed on water. Exposure to 20 mM H(2)O(2) elicited simultaneous growth of the root and shoot system, resulting in more uniform seedling development. Seeds of big bluestem (Andropogon gerardii Vitman) and indiangrass [Sorghastrum nutans (L.) Nash] also responded positively to H(2)O(2) treatment, indicating the universality of the effect of H(2)O(2) on seed germination in warm-season prairie grasses. For switchgrass seeds, abscisic acid (ABA) and the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI) at 20 microM retarded germination (radicle emergence), stunted root growth and partially inhibited NADPH-oxidase activity in seeds. H(2)O(2) reversed the inhibitory effects of DPI and ABA on germination and coleoptile elongation, but did not overcome DPI inhibition of root elongation. Treatment with H(2)O(2) appeared to enhance endogenous production of nitric oxide, and a scavenger of nitric oxide abolished the peroxide-responsive stimulation of switchgrass seed germination. The activities and levels of several proteins changed earlier in seeds imbibed on H(2)O(2) as compared to seeds maintained on water or on ABA. These data demonstrate that seed germination of warm-season grasses is significantly responsive to oxidative conditions and highlights the complex interplay between seed redox status, ABA, ROS and NO in this system.

ABA, ROS and NO are Key Players During Switchgrass Seed Germination.

Seed dormancy and germination are complex physiological processes usually under hormonal control. Germination of seeds from many plants including switchgrass, are inhibited by ABA and promoted by NO or ROS. However, ABA apparently requires both ROS and NO as intermediates in its action, with ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide (ROS), but not NO will overcome ABA-imposed inhibition of germination. Stimulation of germination by external ROS can be partially blocked by NO-scavengers, suggesting that NO is required for seed germination in switchgrass as well as for ABA-induced inhibition of germination. Collectively, these data suggest that multiple mechanisms might be required to sense and respond to varying levels of ABA, NO and ROS in switchgrass seeds.

Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum. L).

This work examined cell composition gradients in maturing tillers of switchgrass (Panicum virgatum L.) with the aim of developing baseline information on this important forage and biomass crop. Flowering tillers were collected from plants raised from seeds in a greenhouse and field, harvested at soil level and separated into internodes beginning with the node subtending the peduncle. Internodes were analyzed using microscopy, by fiber digestion, high-performance liquid chromatography and by gas chromatography-mass spectrometry to obtain anatomical and compositional data. Microscopy demonstrated the development and maturation of cortical fibers which eventually became confluent with the fiber sheath surrounding vascular bundles in the lower internodes. Detergent fiber analysis indicated increasing cellulose and lignin contents and decreases in cell solubles and hemicelluloses with increasing distance of the internodes from the top of the plant. Soluble phenolics were greatest in amounts and complexity in top internodes. The lower internodes contained greater levels of wall-bound phenolic acids, principally as 4-coumarate and ferulate.

Chinch bug (Hemiptera: Blissidae) mouthpart morphology, probing frequencies, and locations on resistant and susceptible germplasm.

Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. Previous research has indicated that some grasses exhibit resistance to multiple chinch bug species, whereas others are resistant to only one species. The objectives of this research were to document differences in the probing frequencies and locations among Blissus species as well as differences in mouthpart morphology as a first step in understanding the differential responses of grasses to chinch bug feeding. Scanning electron microscopy detected differences in the total lengths of proboscises as well as individual mouthpart segments among the four species studied. Blissus occiduus Barber probed significantly more often on buffalograss, Buchloë dactyloides (Nuttall) Engelmann, than any other plant material. Probing locations of B. occiduus and Blissus leucopterus leucopterus (Say) were similar on both B. occiduus-resistant and susceptible buffalograsses and KS94 sorghum, Sorghum bicolor (L.) Moench (B. occiduus-resistant, B. l. leucopterus-resistant). However, on 'Wheatland' sorghum (B. occiduus-resistant, B. l. leucopterus-susceptible), stylet tracts of B. l. leucopterus most often terminated in the bundle sheath cells, whereas those of B. occiduus generally terminated in the vascular tissues.

Nitric oxide accelerates seed germination in warm-season grasses.

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25 degrees C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 microM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 microM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3-4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.