Holistic assessment of the microbiome dynamics in the substrates used for commercial champignon (Agaricus bisporus) cultivation

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.     

Biodiversity of sweetpotato (iPomoea batatas,Convolvulaceae) in Southern Mexico

Between 1987 and 1992 the phytogeographic region of southern Mexico was explored during three collecting trips made in search of cultivated and wild germplasm of the sweetpotato (Ipomoea batatas). The first trip was made in 1987, when we collected wild species in Ipomoea section Batatas found in the southeastern and southwestern regions of Mexico. A second trip was made in 1990, when we collected accessions of the cultivated species as well as wild species in the southeast, southwest and northeast. The third and final trip was oriented at identification, characterization and collecting seeds in the ecological niches ofI.tabascana andI.umbraticola. As a result of the three trips we collected 165 accessions of cultivated and wild germplasm with populations dispersed in 147 localities in 15 states of the Mexican region: Veracruz, Tabasco, Campeche, Chiapas, Oaxaca, Yucatán, Guerrero, Michoacán, San Luis Potosí, Hidalgo, Querétaro, Tamaulipas, Guanajuato, Puebla and México. Of the total accessions some 64 (38.3%) were of the cultivated species including nine accessions of feral material, and 103 accessions (61.7%) were of wild species made up of 59 accessions of seven species in the section Batatas, 37 of other species in the family Convolvulaceae, and seven yet to be determined. We have identified the largest genetic biodiversity in six localities of five states: Tabasco, Oaxaca, Michoacán, San Luis Potosí, and Puebla. Biodiversity maintenance in this region is associated with the day-of-the-dead festivities.     

Differential effects of interleukin-7 and interleukin-15 on NK cell anti-human immunodeficiency virus activity

The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3(-) CD16(+) CD56(+)) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01; P = 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior.     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

Chemiluminescence of the Fenton reaction and a dihydroxybenzene-driven Fenton reaction

A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex.     

Spider assemblages in the overstory, understory, and ground layers of managed stands in the western boreal mixedwood forest of Canada

Logging is the main human disturbance in the boreal forest; thus, understanding the effects of harvesting practices on biodiversity is essential for a more sustainable forestry. To assess changes in spider composition because of harvesting, samples were collected from three forest layers (overstory, understory, and ground) of deciduous and conifer dominated stands in the northwestern Canadian boreal mixedwood forest. Spider assemblages and feeding guild composition were compared between uncut controls and stands harvested to 20% retention. In total, 143 spider species were collected, 74 from the ground, 60 from the understory, and 71 from the overstory, and species composition of these three pools differed considerably among layers. Distinctive spider assemblages were collected from the canopy of each forest cover type but these were only slightly affected by harvesting. However, logging had a greater impact on the species composition in the understory and ground layers when compared with unharvested controls. Guild structure differed among layers, with wandering and sheet-weaving spiders dominant on the ground while orb-weaving and ambush spiders were better represented in the understory and overstory, respectively. Given the ecological importance of spiders and the expectation of faunal changes with increased harvesting, further efforts toward the understanding of species composition in higher strata of the boreal forest are needed.     

Sterol carrier protein-2-facilitated intermembrane transfer of cholesterol- and phospholipid-derived hydroperoxides

Sterol carrier protein-2 (SCP-2) facilitates cholesterol (Ch) and phospholipid (PL) transfer/exchange between membranes and appears to play a key role in intracellular lipid trafficking. Whether SCP-2 can also facilitate lipid hydroperoxide (LOOH) transfer between membranes and thereby potentially enhance dissemination of peroxidative damage was examined in this study. Transfer kinetics of photochemically generated cholesterol hydroperoxide (ChOOH) species (5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH) families (PCOOH, PEOOH, PSOOH) were determined, using HPLC with electrochemical detection for peroxide analysis. LOOH donor/acceptor pairs employed in transfer experiments included (i) all liposomes (e.g., agglutinable SUVs/ nonagglutinable LUVs); (ii) photoperoxidized erythrocyte ghosts/SUVs or vice versa; and (iii) SUVs/mitochondria. In a SUV/ghost system at 37 degrees C, the rate constant for total ChOOH spontaneous transfer was approximately 8 times greater than that for unoxidized Ch. Purified bovine liver and human recombinant SCP-2 exhibited an identical ability to stimulate overall ChOOH transfer, 0.5 unit/mL (based on [(14)C]Ch transfer) increasing the first-order rate constant (k) approximately 7-fold. SCP-2-enhanced translocation of individual ChOOHs increased with increasing hydrophilicity in the following order: 6beta-OOH < 6alpha-OOH < 5alpha-OOH < 7alpha/7beta-OOH. Likewise, SCP-2 stimulated PCOOH, PEOOH, or PSOOH transfer approximately 6-fold, but the net k was 1/5 that of 5alpha-OOH and 1/10 that of 7alpha/7beta-OOH. Donor membrane properties favoring SCP-2-enhanced LOOH transfer included (i) increasing PL unsaturation and (ii) increasing net negative charge imposed by phosphatidylserine. Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates 7alpha-OOH transfer from SUVs to isolated mitochondria and that this enhances peroxide-induced loss of the mitochondrial membrane potential. On the basis of these findings, we postulate that SCP-2, by trafficking ChOOHs and PLOOHs in addition to parent lipids, might exacerbate cell injury under oxidative stress conditions.     

New Application of the Comet Assay: Chromosome�CComet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.