Water and nitrogen addition differentially impact plant competition in a native rough fescue grassland

We examined how water and nitrogen addition and water–nitrogen interactions affect root and shoot competition intensity and competition–productivity relationships in a native rough fescue grassland in central Alberta, Canada. Water and nitrogen were added in a factorial design to plots and root exclusion tubes and netting were used to isolate root and shoot competition on two focal species (Artemisia frigida and Chenopodium leptophyllum). Both water and nitrogen were limiting to plant growth, and focal plant survival rates increased with nitrogen but not water addition. Relative allocation to root biomass increased with water addition. Competition was almost entirely belowground, with focal plants larger when released from root but not shoot competition. There were no significant relationships between productivity and root, shoot, or total competition intensity, likely because in this system shoot biomass was too low to cause strong shoot competition and root biomass was above the levels at which root competition saturates. Water addition had few effects on the intensity of root competition suggesting that root competition intensity is invariant along soil moisture gradients. Contrary to general expectation, the strength of root competition increased with nitrogen addition demonstrating that the relationship between root competition intensity and nitrogen is more complex than a simple monotonic decline as nitrogen increases. Finally, there were few interactions between nitrogen and water affecting competition. Together these results indicate that the mechanisms of competition for water and nitrogen likely differ.     

Biomass accessibility analysis using electron tomography

Background

Substrate accessibility to catalysts has been a dominant theme in theories of biomass deconstruction. However, current methods of quantifying accessibility do not elucidate mechanisms for increased accessibility due to changes in microstructure following pretreatment.

Results

We introduce methods for characterization of surface accessibility based on fine-scale microstructure of the plant cell wall as revealed by 3D electron tomography. These methods comprise a general framework, enabling analysis of image-based cell wall architecture using a flexible model of accessibility. We analyze corn stover cell walls, both native and after undergoing dilute acid pretreatment with and without a steam explosion process, as well as AFEX pretreatment.

Conclusion

Image-based measures provide useful information about how much pretreatments are able to increase biomass surface accessibility to a wide range of catalyst sizes. We find a strong dependence on probe size when measuring surface accessibility, with a substantial decrease in biomass surface accessibility to probe sizes above 5–10 nm radius compared to smaller probes.

     

Detecting cellulase penetration into corn stover cell walls by immuno‐electron microscopy

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild‐, moderate‐ and high‐severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme‐ and polymer‐specific antibodies. Low severity dilute‐acid pretreatment (20 min at 100°C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120°C) allowed the enzymes to penetrate ～20% of the cell wall, and the high severity (20 min pretreatment at 150°C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute‐acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high‐resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes. Biotechnol. Bioeng. 2009;103: 480–489. © 2009 Wiley Periodicals, Inc.     

The role of parasite release in invasion of the USA by European slugs

Several species of European slugs are invasive in the USA, threatening native species and damaging agricultural and horticultural crops. One possible explanation for the success of these invaders is parasite release. To test this hypothesis we collected European slugs in part of their native range (United Kingdom) and in the USA and compared prevalence, distribution and species richness of their nematode parasites. All slugs were dissected and examined for the presence of nematodes. In the UK, nematodes were present at 93% of study sites and 16.4% of all slugs examined were associated with nematodes whereas in the USA the respective figures were 34% of sites and 5.4% of slugs. Nematode species richness was greater in the UK with 12 species being found, seven of which were thought to be truly parasitic as opposed to being phoretic or necromenic. Nine species of nematode were found in the USA, four of which were truly parasitic. Four of the ten European slug species examined in the USA, were entirely free of truly parasitic nematodes whereas all were infected by nematodes in some sites in the UK. There was a significant difference in the prevalence of truly parasitic nematodes in five of these species when comparing their home versus invasive range. A significant difference in parasite prevalence was observed when comparing native and introduced slug species in the USA, however, this was not significant in the UK. Our data support a role for parasite release during the invasion of the USA by European slugs.     

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.     

Redistribution of xylan in maize cell walls during dilute acid pretreatment

Developing processes for the conversion of biomass for use in transportation fuels production is becoming a critically important economic and engineering challenge. Dilute acid pretreatment is a promising technology for increasing the enzymatic digestibility of lignocellulosic biomass. However, a deeper understanding of the pretreatability of biomass is needed so that the rate of formation and yields of sugars can be increased. Xylan is an important hemicellulosic component of the plant cell wall and acts as a barrier to cellulose, essentially blocking cellulase action. To better understand xylan hydrolysis in corn stover, we have studied changes in the distribution of xylan caused by dilute acid pretreatment using correlative microscopy. A dramatic loss of xylan antibody signal from the center of the cell wall and an increase or retention of xylan at the plasma membrane interface and middle lamella of the cell were observed by confocal laser scanning microscopy (CLSM). We also observed a reduction in xylan fluorescence signal by CLSM that is generally consistent with the decrease in xylan content measured experimentally in the bulk sample, however, the compartmentalization of this xylan retention was not anticipated. Biotechnol. Bioeng. 2009;102: 1537–1543. © 2008 Wiley Periodicals, Inc.     

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.     

Cellular immune response to an engineered cell-based tumor vaccine at the vaccination site

The engineered expression of the immune co-stimulatory molecules CD80 and CD137L on the surface of a neuroblastoma cell line converts this tumor into a cell-based cancer vaccine. The mechanism by which this vaccine activates the immune system was investigated by capturing and analyzing immune cells responding to the vaccine cell line embedded in a collagen matrix and injected subcutaneously. The vaccine induced a significant increase in the number of activated CD62L(-) CCR7(-) CD49b(+) CD8 effector memory T cells captured in the matrix. Importantly, vaccine responsive cells could be detected in the vaccine matrix within a matter of days as demonstrated by IFN-gamma production. The substitution of unmodified tumor cells for the vaccine during serial vaccination resulted in a significant decrease in activated T cells present in the matrix, indicating that immune responses at the vaccine site are a dynamic process that must be propagated by continued co-stimulation.     

Recent and rapid population growth and range expansion of the Lyme disease tick vector,Ixodes scapularis,in North America

Migration is a primary force of biological evolution that alters allele frequencies and introduces novel genetic variants into populations. Recent migration has been proposed as the cause of the emergence of many infectious diseases, including those carried by blacklegged ticks in North America. Populations of blacklegged ticks have established and flourished in areas of North America previously thought to be devoid of this species. The recent discovery of these populations of blacklegged ticks may have resulted from either in situ growth of long‐established populations that were maintained at very low densities or by migration and colonization from established populations. These alternative evolutionary hypotheses were investigated using Bayesian phylogeographic approaches to infer the origin and migratory history of recently detected blacklegged tick populations in the Northeastern United States. The data and results indicate that newly detected tick populations are not the product of in situ population growth from a previously established population but from recent colonization resulting in a geographic range expansion. This expansion in the geographic range proceeded primarily through progressive and local migration events from southern populations to proximate northern locations although long‐distance migration events were also detected.