Nitrogen-fixing bacterial communities differ between perennial agroecosystem crops.

Biocrusts, common in natural ecosystems, are specific assemblages of microorganisms at or on the soil surface with associated microorganisms extending into the top centimeter of soil. Agroecosystem biocrusts have similar rates of nitrogen (N) fixation as those in natural ecosystems, but it is unclear how agricultural management influences their composition and function. This study examined the total bacterial and diazotrophic communities of biocrusts in a citrus orchard and a vineyard that shared a similar climate and soil type but differed in management. To contrast climate and soil type, these biocrusts were also compared with those from an apple orchard. Unlike natural ecosystem biocrusts, these agroecosystem biocrusts were dominated by proteobacteria and had a lower abundance of cyanobacteria. All of the examined agroecosystem biocrust diazotroph communities were dominated by N-fixing cyanobacteria from the Nostocales order, similar to natural ecosystem cyanobacterial biocrusts. Lower irrigation and fertilizer in the vineyard compared with the citrus orchard could have contributed to biocrust microbial composition, whereas soil type and climate could have differentiated the apple orchard biocrust. Season did not influence the bacterial and diazotrophic community composition of any of these agroecosystem biocrusts. Overall, agricultural management and climatic and edaphic factors potentially influenced the community composition and function of these biocrusts.

CD4+ T Cell Responses to Toxoplasma gondii Are a Double-Edged Sword.

CD4+ T cells have been found to play critical roles in the control of both acute and chronic Toxoplasma infection. Previous studies identified a protective role for the Toxoplasma CD4+ T cell-eliciting peptide AS15 (AVEIHRPVPGTAPPS) in C57BL/6J mice. Herein, we found that immunizing mice with AS15 combined with GLA-SE, a TLR-4 agonist in emulsion adjuvant, can be either helpful in protecting male and female mice at early stages against Type I and Type II Toxoplasma parasites or harmful (lethal with intestinal, hepatic, and spleen pathology associated with a storm of IL6). Introducing the universal CD4+ T cell epitope PADRE abrogates the harmful phenotype of AS15. Our findings demonstrate quantitative and qualitative features of an effective Toxoplasma-specific CD4+ T cell response that should be considered in testing next-generation vaccines against toxoplasmosis. Our results also are cautionary that individual vaccine constituents can cause severe harm depending on the company they keep.

Comparison of Oxford Nanopore Technologies and Illumina MiSeq sequencing with mock communities and agricultural soil.

Illumina MiSeq is the current standard for characterizing microbial communities in soil. The newer alternative, Oxford Nanopore Technologies MinION sequencer, is quickly gaining popularity because of the low initial cost and longer sequence reads. However, the accuracy of MinION, per base, is much lower than MiSeq (95% versus 99.9%). The effects of this difference in base-calling accuracy on taxonomic and diversity estimates remains unclear. We compared the effects of platform, primers, and bioinformatics on mock community and agricultural soil samples using short MiSeq, and short and full-length MinION 16S rRNA amplicon sequencing. For all three methods, we found that taxonomic assignments of the mock community at both the genus and species level matched expectations with minimal deviation (genus: 80.9-90.5%; species: 70.9-85.2% Bray-Curtis similarity); however, the short MiSeq with error correction (DADA2) resulted in the correct estimate of mock community species richness and much lower alpha diversity for soils. Several filtering strategies were tested to improve these estimates with varying results. The sequencing platform also had a significant influence on the relative abundances of taxa with MiSeq resulting in significantly higher abundances Actinobacteria, Chloroflexi, and Gemmatimonadetes and lower abundances of Acidobacteria, Bacteroides, Firmicutes, Proteobacteria, and Verrucomicrobia compared to the MinION platform. When comparing agricultural soils from two different sites (Fort Collins, CO and Pendleton, OR), methods varied in the taxa identified as significantly different between sites. At all taxonomic levels, the full-length MinION method had the highest similarity to the short MiSeq method with DADA2 correction with 73.2%, 69.3%, 74.1%, 79.3%, 79.4%, and 82.28% of the taxa at the phyla, class, order, family, genus, and species levels, respectively, showing similar patterns in differences between the sites. In summary, although both platforms appear suitable for 16S rRNA microbial community composition, biases for different taxa may make the comparison between studies problematic; and even with a single study (i.e., comparing sites or treatments), the sequencing platform can influence the differentially abundant taxa identified.

Pig and Mouse Models of Hyperlipidemia and Atherosclerosis.

Atherosclerosis is a chronic inflammatory disorder that is the underlying cause of most cardiovascular disease. Resident cells of the artery wall and cells of the immune system participate in atherogenesis. This process is influenced by plasma lipoproteins, genetics, and the hemodynamics of the blood flow in the artery. A variety of animal models have been used to study the pathophysiology and mechanisms that contribute to atherosclerotic lesion formation. No model is ideal as each has its own advantages and limitations with respect to manipulation of the atherogenic process and modeling human atherosclerosis and lipoprotein profile. In this chapter we will discuss pig and mouse models of experimental atherosclerosis. The similarity of pig lipoprotein metabolism and the pathophysiology of the lesions in these animals with that of humans is a major advantage. While a few genetically engineered pig models have been generated, the ease of genetic manipulation in mice and the relatively short time frame for the development of atherosclerosis has made them the most extensively used model. Newer approaches to induce hypercholesterolemia in mice have been developed that do not require germline modifications. These approaches will facilitate studies on atherogenic mechanisms.

A lysosome-targeted DNA nanodevice selectively targets macrophages to attenuate tumours.

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Electrostatic Method to Remove Particulate Organic Matter from Soil.

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Phytostabilization of acidic mine tailings with biochar, biosolids, lime, and locally-effective microbes: Do amendment mixtures influence plant growth, tailing chemistry, and microbial composition?

Abandoned mine lands present persistent environmental challenges to ecosystems and economies; reclamation an important step for overcoming these challenges. Phytostabilization is an elegant and cost-effective reclamation strategy, however, establishing plants on severely degraded soils is problematic, often requiring soil amendment additions. We evaluated whether amendment mixtures composed of lime, biochar, biosolids, and locally effective microbes (LEM) could alleviate the constraints that hinder phytostabilization success. We hypothesized that 1) plants grown in tailings amended with lime, biochar, and biosolids (LBB) would establish faster and grow larger than plants grown in tailings amended with lime only, and 2) the LEM source would influence microbial community function and structure in amended mine tailings. We conducted a greenhouse study that simulated in situ conditions to measure the influence of LBB-LEM amendment blends on plant growth, plant nutrients, metal concentrations, microbial function, and microbial community structure. Blue wildrye [Elymus glaucus Buckley ssp. Jepsonii (Burtt Davy) Gould] was grown in tailings collected from the Formosa mine site amended with various combinations of LBB-LEM. The above and below ground biomass of plants grown in mine tailings amended with LBB was 3 to 4 times larger than the biomass of plants grown in tailings amended only with lime. Although the LEM addition did not influence immediate plant growth, it did affect nutrient content and altered the rhizosphere community membership. As such, it is not yet clear if LEM-driven alterations in microbial membership will advance mine reclamation strategies by improving long-term growth.

Baseline and Temporal Changes in Sensitivity of Zymoseptoria tritici Isolates to Benzovindiflupyr in Oregon, U.S.A., and Cross-Sensitivity to Other SDHI Fungicides.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), a disease of wheat (Triticum aestivum) that results in significant yield loss worldwide. Z. tritici's life cycle, reproductive system, effective population size, and gene flow put it at high likelihood of developing fungicide resistance. Succinate dehydrogenase inhibitor (SDHI) fungicides (FRAC code 7) were not widely used to control STB in the Willamette Valley until 2016. Field isolates of Z. tritici collected in the Willamette Valley at dates spanning the introduction of SDHI (2015 to 2017) were screened for sensitivity to four SDHI active ingredients: benzovindiflupyr, penthiopyrad, fluxapyroxad, and fluindapyr. Fungicide sensitivity changes were determined by the fungicide concentration at which fungal growth is decreased by 50% (EC50) values. The benzovindiflupyr EC50 values increased significantly, indicating a reduction in sensitivity, following the adoption of SDHI fungicides in Oregon (P < 0.0001). Additionally, significant reduction in cross-sensitivity among SDHI active ingredients was also observed with a moderate and significant relationship between penthiopyrad and benzovindiflupyr (P = 0.0002) and a weak relationship between penthiopyrad and fluxapyroxad (P = 0.0482). No change in cross-sensitivity was observed with fluindapyr, which has not yet been labeled in the region. The results document a decrease in SDHI sensitivity in Z. tritici isolates following the introduction of the active ingredients to the Willamette Valley. The reduction in cross-sensitivity observed between SDHI active ingredients highlights the notion that careful consideration is required to manage fungicide resistance and suggests that within-group rotation is insufficient for resistance management.

Atherosclerosis: cell biology and lipoproteins.

PURPOSE OF REVIEW: Lipoproteins have significant role in both the promotion and prevention of atherosclerosis. This brief review will focus on recent reports on relationship between HDL and HDL subclasses and their composition and function, the role of apoC-III in metabolism of triglyceride-rich lipoproteins, the impact of Lipoprotein (a) (Lp(a)) on endothelial cells, and the mechanism of uptake of aggregated LDL by macrophages. RECENT FINDINGS: The complexity of the protein and lipid content of murine and human HDL and their relationship to its cholesterol efflux capacity have been examined. HDL has also been shown to have both antiatherogenic and proatherogenic properties. The relationship between apoC-III and LPL activity, apoprotein E mediated clearance of triglyceride-rich lipoproteins and the potential importance of apoC-III in the increased risk of cardiovascular disease in type 1 diabetics has been investigated. Oxidized phospholipid in Lp(a) promotes endothelial cells inflammatory and glycolytic responses. TLR4 participates in the uptake of aggregated LDL to contribute to foam cell formation. SUMMARY: These studies contribute to our mechanistic understanding of how lipoproteins contribute to atherogenesis and identify potential therapeutic targets.

CD1d Selectively Down Regulates the Expression of the Oxidized Phospholipid-Specific E06 IgM Natural Antibody in Ldlr-/- Mice.

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

Invariant Natural Killer T-Cells and Total CD1d Restricted Cells Differentially Influence Lipid Metabolism and Atherosclerosis in Low Density Receptor Deficient Mice.

Natural killer T (NKT) cells are a distinct subset of lymphocytes that bridge the innate and adaptive immune response and can be divided into type I invariant NKT cells (iNKT) and type II NKT cells. The objective of this study is to examine the effects of NKT cell on lipid metabolism and the initiation and progression of atherosclerosis in LDL receptor deficient (LDLR-/-) mice. Mice were fed an atherogenic diet for 4 or 8 weeks and plasma lipids, lipoproteins, and atherosclerosis were measured. The selective absence of iNKT cells in Jα18-/-LDLR-/- mice led to an increase in plasma cholesterol levels in female mice. Transgenic Vα14tg/LDLR-/- mice with elevated numbers of iNKT cells had increased late atherosclerosis of the innominate artery, though absence of either iNKT cells or all NKT cells and other CD1d expressing cells had varying effects on atherosclerotic lesion burden in the ascending aortic arch and aortic root. These studies not only highlight the potential modulatory role played by NKT cells in atherosclerosis and lipid metabolism, but also raise the possibility that divergent roles may be played by iNKT and CD1d restricted cells such as type II NKT cells or other CD1d expressing cells.

Mining the Drilosphere: Bacterial Communities and Denitrifier Abundance in a No-Till Wheat Cropping System.

Earthworms play important roles in no-till cropping systems by redistributing crop residue to lower soil horizons, providing macropores for root growth, increasing water infiltration, enhancing soil quality and organic matter, and stimulating nitrogen cycling. The soil impacted by earthworm activity, including burrows, casts, and middens, is termed the drilosphere. The objective of this study was to determine the effect of earthworms on soil microbial community composition in the drilosphere at different landscape slope positions. Soil cores (50 cm depth) were extracted from three landscape locations (top, middle, and bottom slope positions) on a sloping aspect of a no-till wheat farm. Soil was sampled at the bottom of the soil core from inside multiple earthworm (Lumbricus terrestris) channels (drilosphere) and from adjacent bulk soil. Bacterial communities were characterized for 16S rRNA gene diversity using high-throughput sequencing and functional denitrifier gene abundance (nirK, nirS, and nosZ) by quantitative PCR. Bacterial communities were structured primarily by the landscape slope position of the soil core followed by source (bulk versus drilosphere soil), with a significant interaction between core position and source. The families AKIW874, Chitinophagaceae, and Comamonadaceae and the genera Amycolatopsis, Caulobacter, Nocardioides, and Variovorax were more abundant in the drilosphere compared to the bulk soil. Most of the individual bacterial taxa enriched in the drilosphere versus bulk soil were members of Actinobacteria, including Micrococcales, Gaiellaceae, Solirubrobacterales, and Mycobacterium. In general, the greatest differences in communities were observed in comparisons of the top and bottom slope positions in which the bottom slope communities had significantly greater richness, diversity, and denitrifier abundance than the top slope position. Populations of denitrifiers (i.e., ratio of nirK+nirS to 16S rRNA) were more abundant in earthworm-impacted soils and there was a significant impact of L. terrestris on soil community composition which was observed only in the top landscape position. There were significant correlations between the abundance of nirK and nirS and taxa within Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia, and Chloroflexi, suggesting a broad diversity of denitrifying bacteria. Earthworms influence the soil microbial communities, but the impact depends on the slope location in a variable landscape, which likely reflects different soil characteristics.

Apoproteins E, A-I, and SAA in Macrophage Pathobiology Related to Atherogenesis.

Macrophages are core cellular elements of both early and advanced atherosclerosis. They take up modified lipoproteins and become lipid-loaded foam cells and secrete factors that influence other cell types in the artery wall involved in atherogenesis. Apoproteins E, AI, and SAA are all found on HDL which can enter the artery wall. In addition, apoE is synthesized by macrophages. These three apoproteins can promote cholesterol efflux from lipid-loaded macrophages and have other functions that modulate macrophage biology. Mimetic peptides based on the sequence or structure of these apoproteins replicate some of these properties and are potential therapeutic agents for the treatment of atherosclerosis to reduce cardiovascular diseases.

Apoprotein E and Reverse Cholesterol Transport.

Apoprotein E (apoE) is a multifunctional protein. Its best-characterized function is as a ligand for low-density lipoprotein (LDL) receptor family members to mediate the clearance of apoB-containing atherogenic lipoproteins. Among its other functions, apoE is involved in cholesterol efflux, especially from cholesterol-loaded macrophage foam cells and other atherosclerosis-relevant cells, and in reverse cholesterol transport. Reverse cholesterol transport is a mechanism by which excess cellular cholesterol is transported via lipoproteins in the plasma to the liver where it can be excreted from the body in the feces. This process is thought to have a role in the attenuation of atherosclerosis. This review summarizes studies on the role of apoE in cellular cholesterol efflux and reverse cholesterol transport and discusses the identification of apoE mimetic peptides that may promote these pathways.