Search

Energy production in the Williston Basin, located in the Prairie Pothole Region of central North America, has increased rapidly over the last several decades. Advances in recycling and disposal practices of saline wastewaters (brines) co-produced during energy production have reduced ecological risks, but spills still occur often and legacy practices of releasing brines into the environment caused persistent salinization in many areas. Aside from sodium and chloride, these brines contain elevated concentrations of metals and metalloids (lead, selenium, strontium, antimony and vanadium), ammonium, volatile organic compounds, hydrocarbons, and radionuclides. Amphibians are especially sensitive to chloride and some metals, increasing potential effects in wetlands contaminated by brines. We collected bed sediment and larval amphibians (Ambystoma mavortium, Lithobates pipiens and Pseudacris maculata) from wetlands in Montana and North Dakota representing a range of brine contamination history and severity to determine if contamination was associated with metal concentrations in sediments and if metal accumulation in tissues varied by species. In wetland sediments, brine contamination was positively associated with the concentrations of sodium and strontium, both known to occur in oil and gas wastewater, but negatively correlated with mercury. In amphibian tissues, selenium and vanadium were associated with brine contamination. Metal tissue concentrations were higher in tadpoles that graze compared to predatory salamanders; this suggests frequent contact with the sediments could lead to greater ingestion of metal-laden materials. Although many of these metals may not be directly linked with energy development, the potential additive or synergistic effects of exposure along with elevated chloride from brines could have important consequences for aquatic organisms. To effectively manage amphibian populations in wetlands contaminated by saline wastewaters we need a better understanding of how life history traits, species-specific susceptibilities and the physical-chemical properties of metals co-occurring in wetland sediments interact with other stressors like chloride and wetland drying.

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September-October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.

BackgroundCommercial manufacturers have formulated diets to promote not only weight reduction but also to reduce risks of chronic diseases. The objective of this study is to determine if these formulations satisfy requirements for essential nutrients.MethodsWe have selected two established commercial diets, one low fat, high carbohydrate (diet 1) and the other, high fat, low carbohydrate (diet 2) and determined “representative meals” through use of recipes suggested in the manufacturer’s manuals. Nutrition Data System for Research (NDSR) software has been used to perform the most extensive nutrient analysis to date of these diets. Tables report macronutrients (energy), vitamins, minerals, essential amino acids, essential lipids and nutrient-related components for a total of 62entries.ResultsDiet 1 satisfied requirements for 50 of these (81%) with only vitamin B12, vitamin D, and essential fatty acids not reaching recommended levels, while fiber and glycemic load exceeded suggested values. Diet 2 satisfied requirements for 46 of the components (71%) but had excess percentage of fat, especially saturated fat, sodium and cholesterol as well as decreased percentage of carbohydrate resulting in suboptimal intake of B-complex vitamins (B1, niacin and total folate) as well as fiber.ConclusionsNeither diet satisfied adequacy for all reported nutrients. However, based on nutrient content alone diet 1, if supplemented or modified, could be sustained over the long term whereas diet 2 should not be encouraged for long term adaptation

A biomembrane sample system where millimolar changes of cations induce reversible large scale (≥ 200 Å) changes in the membrane-to-surface distance is described. The system composes of a free-floating bilayer (FFB), formed adjacent to a self-assembled monolayer (SAM). To examine the membrane movements, differently charged FFBs in the presence and absence of Ca2+ and Na+, respectively, were examined using neutron reflectivity (NR) and quartz crystal microbalance (QCM) measurements, alongside molecular dynamics (MD) simulations. In NR the variation of Ca2+ and Na+ concentration enabled precision manipulation of the FFB-to-surface distance. Simulations suggest that Ca2+ ions bridge between SAM and bilayer whereas the more diffuse binding of Na+, especially to bilayers, is unable to fully overcome the repulsion between anionic FFB and anionic SAM. Reproduced NR results with QCM demonstrate the potential of this easily producible sample system to become a standard analysis tool for e.g. investigating membrane binding effects, endocytosis and cell signalling.

The development of high-field superconducting magnets and of Fourier transform techniques has made possible the application of solution Nuclear magnetic resonance (NMR) to humic materials from soils, composts, aquatic environments, sediments and coals. Although humic acid and fulvic acid constitute soluble fractions, and are defined by their solubility in base, and at all pH values, respectively, they are often not soluble to the 50-100 mg/ml. levels desirable for C NMR. Solution C NMR can provide unparalleled structural information on humic substances from many different sources. The H NMR spectra of humic materials also have features indicating a wide range of chemical structures. A combination of chemical and NMR techniques can be used to extract more information from C spectra.

15 páginas, 6 figuras, 1 tabla, Microorganisms strongly influence and are required to generate the selective substrate that provides nutrients and support for fungal growth, and ultimately to induce mushroom fructification under controlled environmental conditions. In this work, the fungal and bacterial microbiota living in the different substrates employed in a commercial crop (compost phase I, II and III, flush 1 and 2, and casing material on day 1, 6 and 8 after compost casing and during flush 1 and 2) have been characterized along the different stages of cultivation by metataxonomic analysis (16S rRNA and ITS2), analysis of phospholipid fatty acid content (PLFAs) and RT-qPCR. Additionally, laccase activity and the content of lignin and complex carbohydrates in compost and casing have been quantified. The bacterial diversity in compost and casing increased throughout the crop cycle boosted by the connection of both substrates. As reflected by the PLFAs, the total living bacterial biomass appears to be negatively correlated with the mycelium of the crop. Agaricus bisporus was the dominant fungal species in colonized substrates, displacing the pre-eminent Ascomycota, accompanied by a sustained increase in laccase activity, which is considered to be a major product of protein synthesis during the mycelial growth of champignon. From phase II onwards, the metabolic machinery of the fungal crop degrades lignin and carbohydrates in compost, while these components are hardly degraded in casing, which reflects the minor role of the casing for nourishing the crop. The techniques employed in this study provide a holistic and detailed characterization of the changing microbial composition in commercial champignon substrates. The knowledge generated will contribute to improve compost formulations (selection of base materials) and accelerate compost production, for instance, through biotechnological interventions in the form of tailored biostimulants and to design environmentally sustainable bio-based casing materials, The project was funded by the H2020 programme under the MarieSklodowska-Curie grant agreement no. 742966 and also funded bythe ADER project grant no. 2016-IDD-00013

Essential oils are volatile plant compounds that are biologically active and play an important role in natural plant protection. There are currently ∼3000 essential oils known, of which over 300 are commercially important for a variety of industries. In fact, the essential oil global market has been estimated to reach 13.94 billion USD with a demand of over 370,000 tons by 2024. These compounds have a wide variety of applications in agriculture, food and beverage, cosmetics, medicine, amongst other industries. A promising application of essential oils is as antimicrobials to target the vast number of diseases affecting crops. However, their volatile nature limits their effective use as free agents. To overcome this, we have investigated the use of mesoporous silica nanoparticles to protect essential oils from evaporation and degradation and to enhance their antimicrobial activity against bacterial phytopathogens. Silica nanoparticles were used due to their potential to be produced at an industrial scale and their biocompatibility. As a proof of concept, we evaluated 41 essential oils against bacterial phytopathogen Pseudomonas syringae pv. pisi, causative agent of pea bacterial blight. Additionally, we compared the effect of such essential oils against Pectobacterium carotovorum subsp. carotovorum and Pseudomonas fluorescens. Two of the most effective antimicrobials, cinnamon (Cinnamomum zeylanicum) and mustard (Brassica nigra) oils, were able to inhibit bacterial growth after 24 h at a concentration as low as 0.016% (v/v). Besides efficacy, the species-specificity of essential oils was demonstrated with >67% of oils tested displaying specificity towards pathogenic P. syringae pv. pisi over non-pathogenic Pseudomonas fluorescens. Furthermore, the encapsulation of essential oils into mesoporous silica nanoparticles (MSNPs) as a means of extending and improving their antimicrobial effect was found to enable a 10-fold increase in potency compared to the free essential oil. Cinnamaldehyde immobilised onto MSNPs proved to be the most effective antimicrobial, eliminating >99.8%, >99.9%, and >95% bacterial growth of P. fluorescens, P. syringae pv. pisi and P. carotovorum subsp. carotovorum, respectively. This system has the potential to be used to treat and prevent bacterial infections in crops and to enable a more controlled and effective exploitation of volatile compounds as antimicrobials.

The radionuclide composition of, and emitted radiation in, spent nuclear fuel from the future MYRRHA facility have been studied using depletion simulations to understand potential consequences for safeguards verification using non-destructive assay. The simulations show that both the gamma-ray and neutron emission rates in spent MYRRHA assemblies are lower than in spent PWR UO2 and MOX assemblies. In addition, gamma-ray emission rates from 134Cs and 154Eu are considerably lower, and the total neutron emission rate in MYRRHA fuel is much less sensitive to fuel burnup and cooling time. The main reason is that the fast neutron spectrum in MYRRHA affects the radionuclide production in the fuel. One result is that 244Cm, the main contributor to the neutron emission in spent light water reactor fuel, has a limited production in MYRRHA. Consequently, neutron-detection techniques could be used to more directly assay the plutonium content of spent MYRRHA fuel.

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

Glycosylation goes back and forth Plants produce receptors that recognize fragments of microbial flagellin, thus monitoring for infection by bacteria. Buscaill et al. studied how a flagellin fragment is made accessible for recognition by host glycosidases, which degrade the glycosylations shielding the peptide that triggers the immune response. The pathogen, in turn, evades detection by altering flagellin glycosylation and inhibiting the host glycosidase. This aspect of plant defense against infection plays out in the apoplast, the extracellular space within plant tissues. Science , this issue p. eaav0748

In the metal hyperaccumulator plant Noccaea caerulescens, zinc may provide a defence against pathogens. However, zinc accumulation is a variable trait in this species. We hypothesize that this variability affects the outcome of interactions between metal accumulation and the various constitutive and inducible defences that N. caerulescens shares with non-accumulator plants. We compare zinc concentrations, glucosinolate concentrations and inducible stress responses, including reactive oxygen species (ROS) and cell death, in four N. caerulescens populations, and relate these to the growth of the plant pathogen Pseudomonas syringae , its zinc tolerance mutants and Pseudomonas pathogens isolated from a natural population of N. caerulescens. The populations display strikingly different combinations of defences. Where defences are successful, pathogens are limited primarily by metals, cell death or organic defences; there is evidence of population-dependent trade-offs or synergies between these. In addition, we find evidence that Pseudomonas pathogens have the capacity to overcome any of these defences, indicating that the arms race continues. These data indicate that defensive enhancement, joint effects and trade-offs between different forms of defence are all plausible explanations for the variation we observe between populations, with factors including metal availability and metal-tolerant pathogen load probably shaping the response of each population to infection.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis. Preclinical studies and early phase clinical trials suggest that combined suppression of VEGF and platelet-derived growth factor-BB (PDGF-BB) provides better outcomes than suppression of VEGF alone, due to more frequent regression of neovascularization (NV) and suppression of subretinal fibrosis. We generated a dual variable domain immunoglobulin molecule, ABBV642 that specifically and potently binds and neutralizes VEGF and PDGF-BB. ABBV642 has been optimized for treatment of exudative AMD based on the following design characteristics: 1) high affinity binding to all VEGF-A isoforms and both soluble and extracellular matrix (ECM)-associated PDGF-BB; 2) potential for extended residence time in the vitreous cavity to decrease the frequency of intraocular injections; 3) rapid clearance from systemic circulation compared with molecules with wild type Fc region for normal FcRn binding, which may reduce the risk of systemic complications; and 4) low risk of potential effector function. The bispecificity of ABBV642 allows for a single injection of a single therapeutic agent, and thus a more streamlined development and regulatory path compared with combination products. In a mouse model of exudative AMD, ABBV642 was observed to be more effective than aflibercept. ABBV642 has potential to improve efficacy with reduced injection frequency in patients with exudative AMD, thereby reducing the enormous disease burden for patients and society.

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K(+) , that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca(2+) , Fe(2/3+) Mg(2+) , sucrose, β-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.

We measured changes in mass and in carbon (C), nitrogen (N) and phosphorus (P) concentrations and content of surface-placed and buried wood blocks decomposing over 12 years at 21 sites across Canada to evaluate the influence of the environment on C, N, and P dynamics. Carbon decomposition over time was best described using a sigmoidal fit, which was slightly better than a negative exponential function. Decomposition was slow at cold and wetland sites, with less than 15 % of the original C content lost after 12 years for 4 upland and 3 wetland sites. Decomposition rates were faster for buried than for surface blocks, except in wetlands and in a few upland sites that had high annual precipitation. Nitrogen was gained during the early stages of decomposition, followed by a net N loss once an average C:N mass ratio of 165 was reached for all upland surface-placed blocks, and 200 for upland buried blocks. Critical C:N values were weakly correlated with C:N ratios in the surface organic horizon, suggesting an influence of forest floor properties on decomposition dynamics with N release occurring sooner on more fertile sites. Critical values for N and P mineralization were greater than those reported for foliar litters.

Natural biocides, such as cinnamon (Cinnamomum zeylanicum) bark essential oil, have enormous potential as antimicrobials but are limited by their volatility and rapid degradation. To counteract this and to prolong the efficacy of the biocide, cinnamaldehyde (CNAD, the main bioactive compound of cinnamon essential oil) was encapsulated into mesoporous silica nanoparticles (MSNPs). The synthesised CNAD-MSNPs can be used to tackle the issue of global crop loss; every year, more than 40% of global food production (estimated at $500 billion USD) is lost to diseases. This is despite the annual use of over two million tonnes of pesticides. To address seed borne diseases, CNAD-MSNPs were incorporated into a sodium alginate seed coating. As a proof of concept, this system was tested against Pseudomonas syringae pv. pisi, the causative agent of pea bacterial blight, and demonstrated to increase the number of symptomless plants by 143.58% twenty days after sowing. Additionally, the concentration of CNAD present in the alginate coating was estimated to be

Kalmia angustifolia is a boreal ericaceous shrub that can rapidly spread on black spruce forest cutovers in eastern Canada, where CPRS (i.e. Cutting with Protection of Regeneration and Soils") is practiced. The proliferation of Kalmia often coincides with a reduction in the growth rate of regenerating black spruce seedlings. We report on a study where we compared the local effects of Kalmia and black spruce seedling patches (i.e. two types of "Vegetation") on chemical and biochemical soil properties in CPRS cutovers within mesic spruce-moss and xeric spruce-lichen ecosystems, as well as in four mature spruce-moss forests (i.e. three "Site Types"). Results from 13C-CPMAS-NMR revealed lower O-alkyl C (i.e. carbohydrates), higher aromatic C (i.e. lignin and other phenolics) and higher carbonyl-C (i.e. amide-C and carboxyl groups) in spruce-moss than in spruce-lichen forest floors (F-horizon). In spite of these distinctions, we observed only a small number of Site Type x Vegetation interactions controlling soil properties. Vegetation had a significant effect on ten forest floor properties. Most notably, Kalmia patches had higher concentrations of condensed tannins and lower mineral N cycling. On the other hand, Site Type had a relatively greater effect on the deeper podzolic-B horizons, where mineral N and microbial activity were higher in mature spruce-moss forests than in the cutovers. Green and senescent Kalmia leaves collected at these sites had higher N, tannin and phenolic concentrations than green and senescent spruce needles. A 25 month litter bag study found lower decomposition of Kalmia leaf litter in spruce patches on spruce-lichen cutovers compared to spruce patches on spruce-moss cutovers, or to Kalmia patches on spruce-lichen cutovers. Given that black spruce seedlings obtain most of their nutrients from the forest floor, our results suggest that CPRS may have long-term negative effects on black spruce forest productivity if the spread of Kalmia is left unchecked.

Proximate analysis (PA) is widely used to assess foliar, litter, and wood quality. The acid-unhydrolyzable residue (AUR) of PA, originally known as Klason lignin from wood analysis, is often assumed to be entirely lignin-derived, but the AUR of much plant material also includes contributions from condensed tannins (CT) and cutin or suberin. To improve understanding of chemical changes throughout the PA procedure, we characterized seven foliar litters and their sequential PA fractions (nonpolar and hot-water extracted, AUR). Changes in total C and N, extractable and insoluble CT (as detected by butanol/HCl hydrolysis), δ13C values and solid-state 13C NMR spectra were consistent with loss of carbohydrates and protein after acid hydrolysis, and support previous studies that the AUR residue includes lignin, cutin and CT, all of which are depleted in δ13C. Hot-water extraction removed the bulk of extractable plus insoluble CT. Only trace levels were detected in the AUR, although 13C NMR shows that these are likely underestimates. The assumption of lignin-AUR equivalence still causes misinterpretation of PA results for many sample categories. It is time for the scientific community to limit use of “lignin” to chemically meaningful contexts?

Aims/hypothesis We investigated whether atorvastatin 10 mg daily lowered C-reactive protein (CRP) and whether the effects of atorvastatin on cardiovascular disease (CVD) varied by achieved levels of CRP and LDL-cholesterol. Methods CRP levels were measured at baseline and 1 year after randomisation to atorvastatin in 2,322 patients with type 2 diabetes (40–75 years, 69% males) in a secondary analysis of the Collaborative Atorvastatin Diabetes Study, a randomised placebo-controlled trial. We used Cox regression models to test the effects on subsequent CVD events (n = 147) of CRP and LDL-cholesterol lowering at 1 year. Results After 1 year, the atorvastatin arm showed a net CRP lowering of 32% (95% CI −40%, −22%) compared with placebo. The CRP response was highly variable, with 45% of those on atorvastatin having no decrease in CRP (median [interquartile range, IQR] per cent change −9.8% [−57%, 115%]). The LDL-cholesterol response was less variable, with a median (IQR) within-person per cent change of −41% (−51%, −31%). Baseline CRP did not predict CVD over 3.8 years of follow-up (HRper SD log 0.89 [95% CI 0.75, 1.06]), whereas baseline LDL-cholesterol predicted CVD (HRper SD 1.21 [95% CI 1.02, 1.44]), as did on-treatment LDL-cholesterol. There was no significant difference in the reduction in CVD by atorvastatin, with above median (HR 0.57) or below median (HR 0.52) change in CRP or change in LDL-cholesterol (HR 0.61 vs 0.50). Conclusions/interpretation CRP was not a strong predictor of CVD. Statin efficacy did not vary with achieved CRP despite considerable variability in CRP response. The use of CRP as an indicator of efficacy of statin therapy on CVD risk in patients with type 2 diabetes is not supported by these data. Trial registration NCT00327418 Electronic supplementary material The online version of this article (doi:10.1007/s00125-015-3586-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Factors regulating the persistence of lignin in terrestrial ecosystems are highly debated, and recently lignin has been proposed to have a low residence time in soils. The catalytic efficiency and activation energy of the oxidoreductase enzymes regulate the residence time of lignin in soils. We characterized the spatial and seasonal changes in the apparent Michaelis–Menten kinetics and activation energy of soil peroxidases in three ecosystems of differing litter chemistries- pine forest, deciduous forest, and agricultural ecosystem. Peroxidases rarely follow true Michaelis–Menten kinetics, hence we measured the apparent K m ( App K m ) and apparent V max ( App V max ) of soil peroxidases under potentially H2O2 non-limiting conditions using 3,3′,5,5′-tetramethylbenzidine as the reducing substrate. The App V max and App K m measured in this study, when used independently, exhibited only a weak relationship with total quantity and composition of lignins. Hence, we adopted the ratio of these two parameters ( App V max / App K m ) to define the apparent catalytic efficiency ( App CE) of peroxidases. Across the three ecosystems and seasons, the App CE of peroxidase was ecosystem specific, and exhibited a strong correlation with the monolignol composition of the resident plant species. The App CE of peroxidase was higher in agricultural soils that had lower lignin content. Pine soils with a higher relative proportion of vanillyl units that contribute to more recalcitrant inter-unit linkages in lignins exhibited the highest App V max and App K m , resulting in the lowest App CE. This decoupling of App CE of peroxidases from chemistry of the native lignin observed in our assay-system could indicate a relatively longer persistence of lignin in ecosystems receiving recalcitrant litter inputs. Even though the apparent activation energy ( App Ea) of peroxidases varied by depth and seasons across ecosystems, the App Ea did not relate to the chemistry or quantity of lignins, probably due to the substrate-saturated assay conditions. Our study captures the apparent kinetics of peroxidases in soils, which was ecosystem specific, and in part regulated by the composition of monolignols, thus providing a preliminary linkage between lignin chemistry and enzyme properties in natural systems.

Injecting anti-tumor necrosis factor (TNF)α antibodies into patient joints at the site of inflammation, inter-articular (IA) delivery, has demonstrated modest success for treatment of Spondyloarthritis (SpA), Rheumatoid Arthritis (RA), and osteoarthritis. However, IA delivery is not the treatment route of choice due to rapid clearance from the site of administration. Elastin-like polypeptides (ELPs) are reported to undergo phase transition; forming reversible aggregates above their transition temperature, which when injected into IA space have a 25-fold longer half-life compared to the soluble form. Here, we fused an ELP repeat to the C-terminus of each heavy chain of an anti-TNFα monoclonal antibody (mAb) and provide detailed characterization of the fusion IgG molecule. Expression and purification yielded homogenous protein confirmed by gels, hydrophobic-interaction chromatography, Capilary Electrophoresis (CE), Mass Spectrometry (MS), and analytical ultracentrifugation. The ELPs altered hydrophobicity and pI of the parent mAb and new elastic properties were imparted to the molecule; forming large stable complexes with a hydrodynamic radius of 40 nm above 39°C that dissociated into soluble, active monomer below 37°C. The fusion mAb retained its affinity and ability to neutralize TNFα as determined by surface plasmon resonance and cell-based assay, respectively, with equal potency to unmodified anti-TNFα mAb. Differential-scanning calorimetry studies show stabilization of adjacent CH2 and CH3 domains in the fusion molecule and the aggregated molecule was found to be fully functional after 7 days at 37°C. For the first time, we reveal architecture of an ELP-fusion mAb and binding to antigen using single-particle-transmission-electron microscopy. Unstructured ELP was visualized at the C-terminus and binding to antigen was shown at the N-terminus. Collectively, these studies indicate that it is possible to impart elastic properties to a monoclonal antibody while retaining purity, stability, and ability to effectively bind and neutralize antigen.

Wildfires release substantial quantities of carbon (C) into the atmosphere but they also convert part of the burnt biomass into pyrogenic organic matter (PyOM). This is richer in C and, overall, more resistant to environmental degradation than the original biomass, and, therefore, PyOM production is an efficient mechanism for C sequestration. The magnitude of this C sink, however, remains poorly quantified, and current production estimates, which suggest that ~1-5% of the C affected by fire is converted to PyOM, are based on incomplete inventories. Here, we quantify, for the first time, the complete range of PyOM components found in-situ immediately after a typical boreal forest fire. We utilized an experimental high-intensity crown fire in a jack pine forest (Pinus banksiana) and carried out a detailed pre- and postfire inventory and quantification of all fuel components, and the PyOM (i.e., all visually charred, blackened materials) produced in each of them. Our results show that, overall, 27.6% of the C affected by fire was retained in PyOM (4.8 ± 0.8 t C ha(-1)), rather than emitted to the atmosphere (12.6 ± 4.5 t C ha(-1)). The conversion rates varied substantially between fuel components. For down wood and bark, over half of the C affected was converted to PyOM, whereas for forest floor it was only one quarter, and less than a tenth for needles. If the overall conversion rate found here were applicable to boreal wildfire in general, it would translate into a PyOM production of ~100 Tg C yr(-1) by wildfire in the global boreal regions, more than five times the amount estimated previously. Our findings suggest that PyOM production from boreal wildfires, and potentially also from other fire-prone ecosystems, may have been underestimated and that its quantitative importance as a C sink warrants its inclusion in the global C budget estimates.

Plant pathogens such as fungi, oomycetes, viruses and bacteria infect important crops and account for significant economic losses worldwide. Therefore, it is critical to gain insights into plant-pathogen interactions at the cellular and molecular level. The outcome of the interaction between plants and pathogens greatly differs depending on the species, strains and cultivars involved as well as environmental factors, yet typically results in stress for the plant, the pathogen or both. These biotic-induced stresses can be monitored using a wide range of techniques, of which some of the most commonly used techniques are outlined in this chapter. One widely observed feature of biotic stress in plants is the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-). We describe the quantification of hydrogen peroxide by 3,3'-diaminobenzidine (DAB) staining and luminol-based assays, and of superoxide by nitroblue tetrazolium (NBT) staining. Other techniques detailed here include measurement of callose deposition by aniline blue staining, evaluation of cell death by trypan blue staining and analysis of the loss of membrane integrity by monitoring electrolyte leakage.

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis. The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods: In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro. In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain. This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest.

Environmental stresses not only influence production of plant metabolites but could also modify their resorption during leaf senescence. The production-resorption dynamics of polyphenolic tannins, a class of defense compound whose ecological role extends beyond tissue senescence, could amplify the influence of climate on ecosystem processes. We studied the quantity, chemical composition, and tissue-association of tannins in green and freshly-senesced Quercus rubra leaves exposed to different temperature (Warming and No Warming) and precipitation treatments (Dry, Ambient, Wet) at the Boston-Area Climate Experiment (BACE) in Massachusetts, USA. Climate influence not only the quantity of tannins, but also their molecular composition and cell-wall associations. Irrespective of climatic treatments, tannin composition in Q. rubra was dominated by condensed tannins (CTs, proanthocyanidins). When exposed to Dry conditions, Q. rubra produced higher quantities of tannins that were less polymerized. In contrast, under favorable conditions (Wet), tannins were produced in lower quantities, but the CTs were highly polymerized. Further, even as the overall tissue tannin content declined, the content of hydrolysable tannins (HTs) increased under Wet treatments. The molecular composition of tannins influenced their content in senesced litter. Compared to the green leaves, the content of HTs decreased in senesced leaves across treatments, whereas the CT content was similar between green and senesced leaves in Wet treatments that produced highly polymerized tannins. The content of total tannins in senesced leaves was higher in Warming treatments under both dry and ambient precipitation treatments. The more polymerized tannins in leaves produced under Wet treatments could withstand leaching loss and could potentially decrease the decomposition of these tissues. Our results suggest that, though climate directly influenced the production of tannins in green tissues (and similar patterns were observed in the senesced tissue), the influence of climate on tannin content of senesced tissue was partly mediated by the effect on the chemical composition of tannins. These different climatic impacts on leaves over the course of a growing season may alter forest dynamics, not only in decomposition and nutrient cycling dynamics, but also in herbivory dynamics.

Background and Purpose— Established risk factors do not fully identify patients at risk for recurrent stroke. The SPARCL trial (Stroke Prevention by Aggressive Reduction in Cholesterol Levels) evaluated the effect of atorvastatin on stroke risk in patients with a recent stroke or transient ischemic attack and no known coronary heart disease. This analysis explored the relationships between 13 plasma biomarkers assessed at trial enrollment and the occurrence of outcome strokes. Methods— We conducted a case–cohort study of 2176 participants; 562 had outcome strokes and 1614 were selected randomly from those without outcome strokes. Time to stroke was evaluated by Cox proportional hazards models. Results— There was no association between time to stroke and lipoprotein-associated phospholipase A 2 , monocyte chemoattractant protein-1, resistin, matrix metalloproteinase-9, N-terminal fragment of pro-B-type natriuretic peptide, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1, or soluble CD40 ligand. In adjusted analyses, osteopontin (hazard ratio per SD change, 1.362; P P =0.0107), myeloperoxidase (hazard ratio, 1.177; P =0.0022), and adiponectin (hazard ratio, 1.207; P =0.0013) were independently associated with outcome strokes. After adjustment for the Stroke Prognostic Instrument-II and treatment, osteopontin, neopterin, and myeloperoxidase remained independently associated with outcome strokes. The addition of these 3 biomarkers to Stroke Prognostic Instrument-II increased the area under the receiver operating characteristic curve by 0.023 ( P =0.015) and yielded a continuous net reclassification improvement (29.1%; P P Conclusions— Osteopontin, neopterin, and myeloperoxidase were independently associated with the risk of recurrent stroke and improved risk classification when added to a clinical risk algorithm. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique Identifier: NCT00147602.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

In the Canadian boreal forest, jack pine stands generally have a thin forest floor and occupy sites with coarsetextured soils and good drainage. Black spruce occurs more often on poorly drained sites and develops a thick mossdominated forest floor, but the common attribution of this development to poor quality of black spruce foliar litter has not been tested. We determined needle, twig, cone, and bark litter inputs during 10 y for black spruce and jack pine along the Boreal Forest Transect Case Study in northern Saskatchewan and Manitoba. Analysis of C, N, total phenolics, condensed tannins, and solid-state 13C NMR spectra from years 1–3 showed only small differences between species, notably higher tannins in black spruce cones. There was similarly little difference between area-based inputs, including classes of C structures determined by NMR. Condensed tannin input for black spruce was approximately twice that for jack pine, but both were in the very low range of reported values. Similar anal...

In this paper, a molecular analytical approach for detecting hydrocarbonoclastic bacteria in water is suggested as a proxy measurement for tracking petroleum discharges in industrialized or pristine aquatic environments. This approach is tested for general application in cold marine regions (freezing to 5 °C). We used amplicon sequencing and qPCR to quantify 16S rRNA and GyrB genes from oleophilic bacteria in seawater samples from two different crude oil enrichments. The first experiment was conducted in a controlled environment using laboratory conditions and natural North Sea fjord seawater (NSC) at a constant temperature of 5 °C. The second was performed in the field with natural Arctic seawater (ARC) and outdoor temperature conditions from −7 °C to around 4 °C. Although the experimental conditions for NSC and ARC differed, the temporal changes in bacterial communities were comparable and reflected oil biotransformation processes. The common bacterial OTUs for NSC and ARC had the highest identity to Colwellia rossensis and Oleispira antarctica rRNA sequences and were enriched within a few days in both conditions. Other typical oil degrading bacteria such as Alcanivorax (n-alkane degrader) and Cycloclasticus (polycyclic aromatic hydrocarbons degrader) were rapidly enriched only in NSC conditions. Both the strong correlation between Oleispira SSU gene copies and oil concentration, and the specificity of the Oleispira assay suggest that this organism is a robust bioindicator for seawater contaminated by petroleum in cold water environments. Further optimization for automation of the Oleispira assay for in situ analysis with a genosensing device is underway. The assay for Colwellia quantification requires more specificity to fewer Colwellia OTUs and a well-established dose-response relationship before those taxa are used for oil tracking purposes.

Increased sedimentation and nutrient cycle changes in Prairie Pothole Region wetlands associated with agriculture threaten the permanence and ecological functionality of these important resources. To determine the effects of land use on sedimentation and nutrient cycling, soil cores were analyzed for cesium-137 (137Cs), lead-210 (210Pb), and potassium-40 (40K) activities; textural composition; organic and inorganic carbon (C); and total nitrogen (N) from twelve wetlands surrounded by cropland, Conservation Reserve Program (CRP) lands, or native prairie uplands. Separate soil cores from nine of these wetlands were also analyzed for phosphorus (P), nitrate (NO3), and ammonium (NH4) concentrations. Wetlands surrounded by cropland had significantly greater linear sediment accretion rates than wetlands surrounded by CRP or native prairie. Linear sediment accretion rates from wetlands surrounded by cropland were 2.7 and 6 times greater than wetlands surrounded by native prairie when calculated from the initial and peak occurrence of 137Cs, respectively, and 0.15 cm y−1 (0.06 in yr−1) greater when calculated from 210Pb. Relative to wetlands surrounded by CRP, linear sediment accretion rates for wetlands surrounded by cropland were 4.4 times greater when calculated from the peak occurrence of 137Cs. No significant differences existed between the linear sediment accretion rates between wetlands surrounded by native prairie or CRP uplands. Wetlands surrounded by cropland had increased clay, P, NO3, and NH4, and decreased total C and N concentrations compared to wetlands surrounded by native prairie. Wetlands surrounded by CRP had the lowest P and NO3 concentrations and had clay, NH4, C, and N concentrations between those of cropland and native prairie wetlands. We documented increased linear sediment accretion rates and changes in the textural and chemical properties of sediments in wetlands with cultivated uplands relative to wetlands with native prairie uplands. These findings demonstrate the value of the CRP at protecting wetland catchments to reduce sedimentation.

When plants are infected with pathogens they defend themselves via various processes. Once such process is the development of the hypersensitive response (HR), a kind of programmed cell death (PCD), in which localized cell death takes place in order to prevent pathogen spread to other part of tissue. The Arabidopsis and Pseudomonas syringae system is one of the best known examples to study the HR. Here we used the VisiSens™ oxygen-imaging system to investigate oxygen distributions in Arabidopsis leaves infected with an avirulent strain of P. syringae and undergoing the hypersensitive response. Using this method we observed a change in oxygen status at 6 h post-infection and a drop in oxygen levels at 24 h after infection.

CYP238A1, one of the two P450 enzymes in the genome of Pseudomonas putida KT2440, has been produced heterologously in Escherichia coli, purified, and found to bind acyclic and cyclic terpene alcohols such as farnesol, nerolidol, linalool, and terpineol. The other P450 enzyme in this organism (gene locus: PP1950) was also produced in E. coli but no substrate has been identified from a limited screen. A phthalate family oxygenase reductase (PFOR) encoded by the PP1957 gene, just downstream of the PP1955 gene for CYP238A1, accepts electrons from the reduced form of both nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate and is able to support monooxygenase activity of CYP238A1, both in vitro and in E. coli, in which both enzymes are produced. CYP238A1 oxidizes cis- and trans-nerolidol to the 9-hydroxy product, with no evidence of attack at the olefinic double bonds. The NADH turnover rate of 170 nmol(nmol-P450)⁻¹ Min⁻¹ for CYP238A1 with cis-nerolidol as substrate at a PP1957:CYP238A1 concentration ratio of 8:1 suggests that this PFOR could function as the physiological redox partner for CYP238A1. The physiological role of CYP238A1 may be related to the PP1955 gene being part of an island/cluster of inducible genes associated with energy metabolism and response to xenobiotics.

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell-pilus-cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5alpha and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl(2), temperature of 22 degrees C, plasmid concentration of 0.8 ng/microl, P. putida UWC1 cell concentration of 2.5 x 10(9) CFU (colony forming unit)/ml and reaction volume of 500 microl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 +/- 2.3 x 10(-6) transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5alpha and P. fluorescens SBW25 with efficiencies of 1.16 +/- 0.13 x 10(-6) and 4.33 +/- 0.78 x 10(-6) transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.

Effective gene trapping and screening requires sensory and regulatory compatibility of both host and exogenous systems. The naturally competent bacterium Acinetobacter baylyi ADP1 is able to efficiently take up and integrate exogenous DNA into the chromosome, making it an attractive host system for a wide range of metagenomic applications. To test the ability of A. baylyi ADP1 to express the XylR-regulated Pu promoter from Pseudomonas putida mt-2, we have constructed and examined an A. baylyi ADP1 strain, ADPWH-Pu-lux-xylR. The Pu promoter in ADPWH-Pu-lux-xylR was specifically induced by toluene, m-, p- and o-xylene. The substrate-induced Pu promoter was highly dependent on the growth medium: it was repressed in rich media until stationary phase, but was immediately induced in minimal medium with glucose as the sole carbon source (MMG). However, the Pu promoter was repressed in MMG when it was supplemented with 5 g l(-1) yeast extract. Further investigation showed that the Pu promoter in MMG was repressed by 0.5 g l(-1) aspartic acid or asparagine, but not repressed by glutamine. Changing the carbon/nitrogen ratios by addition of ammonia did not significantly affect the Pu promoter activity but addition of nitrate did. These results show that A. baylyi ADP1 reproduced characteristics of the XylR-regulated Pu promoter observed in its original host. It demonstrates that A. baylyi could provide an excellent genetic host for a wide range of functional metagenomic applications.

Pseudomonas syringae and Erwinia chrysanthemi attack plants in seemingly dissimilar ways. E. chrysanthemi strains secrete multiple plant tissue-disintegrating pectic enzymes and cause extensive necrosis and maceration in a wide range of hosts. P. syringae strains possess considerable host specificity, have little pectolytic capacity, generally do not macerate host tissues, and cause only delayed necrosis in their hosts. Nevertheless, both pathogens possess hip genes, and they produce harpins - protein elicitors of the hypersensitive response (HR).

In P. syringae, the co-ordinated regulation of different systems required for pathogenicity and virulence seems logical but has not been established. This question was addressed in the present study by analysing production of the phytotoxin coronatine (COR) in defined hrp/hrc mutants of P. syringae pv. tomato DC3000. COR was produced in vitro by mutants of DC3000 defective in hrcC, which encodes an outer-membrane protein required for type III-mediated secretion. When inoculated in plants, hrcC mutants produced chlorotic regions indicative of COR production, but lacked the necrotic lesions produced by the wild-type DC3000. Furthermore, a DC3000 mutant containing a polar mutation in hrcC, which inactivates hrcC, hrpT and hrpV, produced significantly higher amounts of COR than the wild-type strain in vitro. This mutant was able to produce COR earlier and at lower cell densities than the wild-type. The results indicate that the hrp/hrc secretion system is not required for COR production, but mutations in this system may have regulatory effects on the production of virulence factors such as COR.

Extended Abstract The current lack of preventive and treatment measures available against bacterial diseases affecting crops is an important problem in agriculture. The use of antibiotics and copper compounds is restricted and can cause bacterial resistance and negative impacts on the environment [1, 2]. Nevertheless, other successful treatment methods have not been fully established. Promising natural compounds exist which have been proven to have strong antimicrobial properties, such as essential oils, and could be used to target bacterial phytopathogens. However, the volatile nature of these compounds limits their application and use in agriculture and a suitable delivery method is extremely desirable. The aim of this study is to evaluate the use of mesoporous silica nanoparticles (MSNPs) to encapsulate essential oils and be used as a preventive and control measure against Pseudomonas syringae pv. pisi, the causative agent of pea bacterial blight [3]. MSNPs allow for a targeted and controlled delivery of the essential oil in the presence of the bacterial pathogen. Essential oils are an environmentally-friendly and safe alternative to antibiotics and other antibacterial agents and the use of nanoparticles as a delivery vehicle allows the oils to be maintained in their active form for long periods of time until their release is needed. MSNPs can protect the loaded biocide from evaporation or degradation while improving their stability and miscibility in aqueous solutions. Additionally, silica is a safe, biodegradable and biocompatible material [4] that is naturally present in plant systems and has been shown to have mainly positive effects on plant growth and development [5, 6, 7, 8]. The present study evaluated the effect of 41 essential oils against P. syringae pv. pisi and compared it to the effect on two other bacterial strains; Pectobacterium carotovorum and Pseudomonas fluorescens. The effect was shown to be strainspecific and one of the most effective oils against P. syringae pv. pisi, ajwain oil, was selected for encapsulation into the MSNPs, which were then incorporated into an alginate seed coating to treat the seeds prior to sowing. Using a seed treatment as a biocide application method reduces the quantity of antimicrobials needed, as well as the amount of nanoparticles released to the environment, when compared to other methods such as soil or foliar applications. This decreases the risk of microbial resistance to biocides while increasing the safety and efficacy of the product. The effect of different alginate formulations on the plant’s germination, growth and development was studied to assess the influence of MSNPs and the use of this technology as a seed treatment against seed-borne pathogens. Peas coated with loaded MSNPs appeared to better resist infection and grow taller than control seeds. Further experiments will be performed to optimise the loading of the MSNPs and maximise the killing efficacy against the pathogens. Additionally, the applications of this study can be extended to both other pathogens as well as other host plants. To our knowledge, this is the first report of MSNPs loaded with a natural biocide and incorporated into an alginate seed coating to protect peas from bacterial phytopathogen Pseudomonas syringae pv pisi. The development of novel and safe biocide delivery methods is essential to optimise food production and achieve a sustainable agriculture in the future.

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition.

Tannins account for a significant proportion of plant biomass and are likely to contribute to the residues formed by incomplete biomass combustion (black carbon, BC). Nonetheless, the molecular properties of thermally modified tannins have not been investigated in laboratory charring experiments. We applied conventional analytical pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) and thermally assisted hydrolysis and methylation (THM-GC-MS) to investigate the effects of heat treatment with a muffle furnace on the properties of condensed tannins (CT) from Corsican pine (Pinus nigra) needles. Py-GC-MS showed a decrease in the relative abundance of the 1,2,3-trihydroxybenzenes (pyrogallols) at ≥300 °C and of the dihydroxybenzenes (mainly catechols) at ≥350 °C due to dehydroxylation of the CT B ring. Further dehydroxylation led to formation of monohydroxybenzenes (phenols), which showed a strong enrichment between 350 and 400 °C and, at higher temperatures, to a series of monocyclic and polycyclic aromatics [benzene, alkyl benzenes and polycondensed aromatic hydrocarbons (PAHs)]. Degradation of the A ring could not be recognized via Py-GC-MS, probably because of the poor chromatographic behavior of 1,3,5-trihydroxybenzenes (phloroglucinols). The progressive dehydroxylation and eventual polycondensation of the CT B ring was corroborated using THM-GC-MS. In addition, with THM-GC-MS the thermal rearrangement of CT A rings at 300 °C and higher was inferred from the relative abundance of 1,3,5-trimethoxybenzenes (methylated phloroglucinol derivatives). These compounds were observed at moderate/high temperature (up to 450 °C) and can not be produced from THM of lignin, suggesting that they may be markers of CT in natural BC samples. © 2012 Elsevier Ltd.

Managing second-growth forests for multiple benefits requires enhanced information on decomposition of woody debris generated during forestry operations. Experimental thinning and biomass sampling at the Shawnigan Lake Research Forest on southern Vancouver Island facilitated retrospective sampling of Douglas-fir ( Pseudotsuga menziesii (Mirb.) Franco) stem sections (4.9–21.5 cm diameter) from 0 to 23 years after cutting. A sigmoidal pattern of density loss was observed, while a single-exponential fit gave a decay rate (k) of 0.058 year–1. Initial N concentrations were higher in bark than in sapwood and heartwood, and all increased during decay, while C/N ratios declined to around 175 for wood and 70 for bark. Nitrogen contents initially increased but tended to decline after about 50% C loss. Solid-state 13C nuclear magnetic resonance spectroscopy showed little change in organic composition, consistent with white-rot fungal decay, with only a few 19-year samples showing large increases in lignin typical of extensive brown-rot decay. Hydrolyzable monosaccharides of wood and bark were dominated by glucose followed by mannose, xylose, galactose, and arabinose. For wood, the mole fraction of mannose decreased with decomposition, whereas those of glucose, arabinose, and galactose increased slightly and that of xylose remained constant for both brown and white rot. Our results support indications that decomposition of smaller diameter woody debris from managed conifer forests will likely be dominated by white-rot fungi, leaving residues high in cellulose rather than in lignin.

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.

We examined how tannin structure influences reactivity in tannin assays and carbon and nitrogen mineralization. Condensed tannins from the foliage of ten tree and shrub species and from pecan shells (Carya illinoensis) had different proportions of: (a) epicatechin (cis) and catechin (trans) isomers, (b) procyanidin (PC) and prodelphinidin (PD) monomers, and (c) different chain lengths. The response of each tannin to several widely used tannin assays was determined. Although there was some variation in response to proanthocyanidin (butanol/HCl) and Folin Ciocalteu assays, we did not deduce any predictable relationship between tannin structure and response to either assay. There was little variation in protein precipitation among the different tannins. To assess biological activity, six of the tannins were incubated with forest humus for 22 days. We determined that, while PC-based tannins remained at least partly extractable for the duration of the incubation, tannins with a high proportion of PD subunits rapidly became unextractable from soil. There was a positive correlation between net nitrogen mineralization and cis chemical structure. Carbon mineralization was enhanced initially by the addition of tannins to humus, but after 22 days, a negative correlation between the proportion of cis subunits and respiration was determined. Overall, we were not able to demonstrate consistent effects of structure on either microbial mineralization or reactivity to chemical assays; such relationships remain elusive.

Slowing or even cessation of litter decomposition with time is well-known, but there is insufficient understanding of the chemical changes that contribute to increasing recalcitrance. Samples from the Canadian Intersite Decomposition Experiment (CIDET) were used to determine 6-year chemical changes for all 11 litters from a site with rapid initial decomposition (Morgan Arboretum, MAR) and for three litters at three colder sites. Six-year mass remaining was 17–37% at MAR, with higher values at the colder sites. Atomic C/N ratios declined and phenolics and condensed tannins generally decreased to minimal values. However, for the three species compared across four sites, phenolics and tannins showed small increases for species with the lowest initial values and also tended to increase with increasing mass loss. For the foliar litters at MAR, there was an average increase in proportion of acid-unhydrolyzable residue (AUR) and decreases in proportions of acid-hydrolyzable (ACID) and extractable fractions, with final AUR/(ACID + AUR) ratios within 0.55–0.66. Principal component analysis showed that foliar litters (and to a lesser extent wood) became more alike after 6 years, decomposition being associated with increase of Fe, Al, N, and AUR concentrations and decrease of K, Mg, tannins, phenolics, and non-polar and water-soluble fractions. However, litters were also affected by site soil chemistry, with some high 6-year accumulations of Ca, Mg, Fe, Al, Mn, and Mg at two sites. Increasing recalcitrance likely arises from increasing dominance of complex, less-soluble organic structures, collectively represented by AUR, together with increases in heavy elements such as Al and Fe, which also specifically bind and stabilize organic matter.

There is still a poor understanding of how changes in the organic composition of litter contribute to slowing or even cessation of decomposition. Using 13C nuclear magnetic resonance (NMR) spectroscopy of samples from the Canadian Intersite Decomposition Experiment (CIDET), we asked whether increasing lignin per se could account for the well-known increase in acid-unhydrolyzable residue (AUR), and secondly, using three litters from four sites with different mean annual temperatures, whether changes in organic composition would follow similar trajectories with C mass loss. At 6 years, there was 16–39% C remaining for 10 foliar litters and wood blocks at a site with rapid initial decomposition, and higher amounts remaining for three species at three colder sites. 13C NMR spectra obtained with rapid cross-polarization (CP) mainly showed increasing similarity among the foliar litters, although wood showed little change in composition. Foliage generally showed loss of O- and di-O-alkyl C, mainly from carbohydrate, and increase in alkyl, aromatic, phenolic and carboxyl C. However, O-alkyl C loss was limited, especially for litters with slow initial decomposition, and many litters showed relatively small changes in intensity distribution. Quantitative 13C (“BD”) spectra showed similar trends, but even smaller changes in C composition, and 6-year CP difference spectra showed that C was lost across the whole range of structures. Changes in δ13C were small and variable, but could be correlated to some extent with loss of carbohydrates versus tannins. Lignin was not selectively preserved, and the increase of resistant structures derived from lignin, tannins, and cutin collectively accounts for increasing AUR. Compositional changes of NMR C fractions across sites with different temperatures were small and inconsistent, likely due to the influence of other site factors; however, changes in their contents did largely follow consistent trajectories with %C remaining.

Nitrilase enzymes (nitrilases) catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have a wide range of industrial and biotechnological applications, including the synthesis of industrially important carboxylic acids and bioremediation of cyanide and toxic nitriles. Nitrilases are produced by a wide range of organisms, including plants, bacteria and fungi, but despite their biotechnological importance, the role of these enzymes in living organisms is relatively underexplored. Current research suggests that nitrilases play important roles in a range of biological processes. In the context of plant-microbe interactions they may have roles in hormone synthesis, nutrient assimilation and detoxification of exogenous and endogenous nitriles. Nitrilases are produced by both plant pathogenic and plant growth-promoting microorganisms, and their activities may have a significant impact on the outcome of plant-microbe interactions. In this paper we review current knowledge of the role of nitriles and nitrilases in plants and plant-associated microorganisms, and discuss how greater understanding of the natural functions of nitrilases could be applied to benefit both industry and agriculture.

Rehabilitation and reforestation of disused forest roads and landings can be facilitated by the incorporation of organic matter. The British Columbia forest industry creates residual woody materials, but they are nutrient poor and may leach phenolic compounds. We assessed the potential for wood wastes (chipped cedar wood waste, sort-yard waste, hogfuel) and co-composts with shellfish waste or municipal biosolids to provide inorganic N and release phenolics and condensed tannins, compared with natural forest floor and mineral soil. Initial concentrations of tannins and phenolics were low, and 13C cross-polarization and magic-angle spinning nuclear magnetic resonance spectroscopy showed that composts were still dominated by wood. During a 426-d laboratory leaching experiment, release of phenolics from woody amendments (other than cedar wood) was lower than from native forest floor. The pH levels of woody amendments and their leachates were also within the range of native forest floor and soil (except cedar wood, which was the most acidic material). Co-composts had higher total N and available P, greatly reduced tannins and phenolics, and negligible leaching of polyphenols. Uncomposted materials released very little N during the incubation. Hogfuel-biosolids compost released a large amount of nitrate, but only during the first 100 d. Shrimp-wood compost released moderate amounts of ammonium and nitrate throughout the incubation, had high available P and low tannin content, and released less polyphenols than did native forest floors. Our results indicate that appropriate use of these amendments does not pose an environmental risk with regard to the parameters measured in this study.

Summary • The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. • Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin–protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein–tannin precipitates. The purity of the precipitates was verified using solid-state 13C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin–protein complexes as the principal N source was also compared. • The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin–protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin–protein precipitates. • These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin–protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.

Previous studies suggested that late-successional tannin-rich plant communities increase the amount of dissolved organic N (DON) relative to dissolved inorganic N (DIN) in decomposing litter. We devised an experiment to test this hypothesis by adding varying proportions of black spruce (Picea mariana) and tannin-rich Kalmia angustifolia litter to forest floor samples collected on six black spruce cutovers. An increasing proportion of Kalmia litter increased condensed tannin and total phenolic concentrations over the course of a 46-week incubation period. Mineral N concentrations did not vary among treatments in spite of much higher total N concentrations in Kalmia litter. This was more likely due to the formation of protein–tannin complexes rather than microbial immobilization of N, as indicated by the decline in available C with increasing Kalmia litter on two of the five sampling dates. There was a significant positive linear trend between the proportion of Kalmia litter and the DON/DIN ratio on one sampling date (week 13) only. Results suggest that the DON/DIN ratio is controlled by confounding factors (e.g., tannins bonding with non-extractable humus particles) and has limited value for describing ecological succession.

Photosynthetically fixed CO 2 is converted into terrestrial bio(macro)molecules and sequestered as soil organic matter (SOM) by (bio)chemical and physical stabilization processes. SOM is generally divided in arbitrary pools for modeling SOM dynamics. Biochemically recalcitrant SOM fractions are enriched with alkyl carbon (C) structures and resist decomposition due to intrinsic molecular properties. The proportion of alkyl C and the mean age of SOM increase with increase in soil depth. Precursors of these recalcitrant bio(macro)molecules such as glycerides, waxes, and terpenoids occur in plants, microorganisms and animals. The intrinsic biochemical stability of naturally occurring recalcitrant aliphatic biomacromolecules may enhance the terrestrial storage of atmospheric CO 2 . Also, aliphatic macromolecules may be formed in soils upon non-enzymatic polymerization of low-molecular-weight lipids. In this review we propose that increasing the soil organic carbon (SOC) pool by land-use and management practices should also include strategies to increase the proportion of aliphatic compounds in the belowground biomass. Thus, collaborative research is needed to study the fate of plant-, microbial- and animal-derived aliphatic C as precursors for stabilized aliphatic SOC fractions, in particular in deeper soil horizons.

The carbon (C) cycle in boreal regions is strongly influenced by fire, which converts biomass and detrital C mainly to gaseous forms (CO2 and smaller proportions of CO and CH4), and some 1–3% of mass to pyrogenic C (PyC). PyC is mainly produced as solid charred residues, including visually-defined charcoal, and a black carbon (BC) fraction chemically defined by its resistance to laboratory oxidation, plus much lower proportions of volatile soot and polycyclic aromatic hydrocarbons (PAHs). All PyC is characterized by fused aromatic rings, but varying in cluster sizes, and presence of other elements (N, O) and functional groups. The range of PyC structures is often described as a continuum from partially charred plant materials, to charcoal, soot and ultimately graphite which is formed by the combination of heat and pressure. There are several reasons for current interest in defining more precisely the role of PyC in the C cycle of boreal regions. First, PyC is largely resistant to decomposition, and therefore contributes to very stable C pools in soils and sediments. Second, it influences soil processes, mainly through its sorption properties and cation exchange capacity, and third, soot aerosols absorb solar radiation and may contribute to global warming. However, there are large gaps in the basic information needed to address these topics. While charcoal is commonly defined by visual criteria, analytical methods for BC are mainly based on various measures of oxidation resistance, or on yield of benzenepolycarboxylic acids. These methods are still being developed, and capture different fractions of the PyC structural continuum. There are few quantitative reports of PyC production and stocks in boreal forests (essentially none for boreal peatlands), and results are difficult to compare due to varying experimental goals and methods, as well as inconsistent terminology. There are almost no direct field measurements of BC aerosol production from boreal wildfires, and little direct information on rates and mechanisms for PyC loss. Structural characterization of charred biomass and forest floor from wildfires generally indicates a low level of thermal alteration, with the bulk of the material having H/C ratios still >0.2, and small aromatic cluster sizes. Especially for the more oxidation-resistant BC fraction, a variety of mainly circumstantial evidence suggests very slow decomposition, with turnover on a millennial timescale (in the order of 5–7 ky), also dependent on environmental conditions. However, there is also evidence that some PyC may be lost in only tens to hundreds of years due to a combination of lower thermal alteration and environmental protection. The potential for long-term PyC storage in soil may also be limited by its consumption by subsequent fires. Degraded, functionalized PyC is also incorporated into humified soil organic matter, and is transported eventually to marine sediments in dissolved and particulate form. Boreal production is estimated as 7–17 Tg BC y−1 of solid residues and 2–2.5 Tg BC y−1 as aerosols, compared to global estimates of 40–240 and 10–30 Tg BC y−1, respectively. Primary research needs include basic field data on PyC production and stocks in boreal forests and peatlands, suitable to support C budget modeling, and development of standardized analytical methods and of improved approaches to assess the chemical recalcitrance of typical chars from boreal wildfires. To accomplish these goals effectively will require much greater emphasis on interdisciplinary cooperation.