Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.     

Spatial and seasonal variation in amino compounds in the xylem sap of a mistletoe (Viscum album) and its hosts (Populus spp. and Abies alba)

In a field study, the composition and concentrations of amino compounds in the xylem sap of the mistletoe, Viscum album L., and in the xylem sap of two host species, an evergreen conifer (Abies alba Mill.) and a deciduous broad-leaved tree (Populus x euramericana), were analyzed. The xylem sap of both hosts and mistletoe contained large, but similar amounts of total organic nitrogen in low molecular weight amino compounds (TONLW). Nevertheless, individual amino compounds accumulated in the xylem sap of mistletoe relative to the host xylem sap, indicating selective uptake. In the xylem sap of Populus, major amino compounds (asparagine (Asn) and glutamine (Gln)) and the bulk parameters, TONLW and proteinogenic amino acids, showed significant seasonal variation. In Abies and in mistletoe on either host, variation of amino compounds in xylem sap was largely explained by inter-annual differences, not by seasonal variation. In both hosts, TONLW in the xylem sap was dominated by Gln. There was a steady decrease in relative abundance of Gln from the host xylem sap to the mistletoe xylem sap and to the stems and leaves of mistletoe. Simultaneously, the abundance of arginine (Arg) increased. Arginine was the predominant amino compound in the stems and leaves of mistletoe, occurring at concentrations previously observed only in leaves of trees exposed to excess nitrogen. We conclude that Gln (2 mol N mol(-1)) delivered by the host xylem sap is converted, in mistletoe, to Arg (4 mol N mol(-1)) and that the organic carbon liberated from Gln contributes significantly to the parasite's heterotrophic carbon gain. Statistical analyses of the data support this conclusion. Accumulation of Arg in mistletoe is an indication of excess N supply as a result of the uptake of amino compounds from the host xylem sap and a lack of phloem uploading.     

Identification of isopeptide bonds in heat-treated wheat gluten peptides

Results in this paper confirm heat-induced isopeptide bond formation in wheat gluten. Heating (24 h, 130 °C) of wheat gluten [moisture content 7.4%] decreased its extractability in sodium dodecyl sulfate containing buffer (pH 6.8), even after reduction of disulfide (SS) bonds. Thus, both SS bonds and non-SS bonds were responsible for the extractability loss. Cross-links of the lysinoalanine and lanthionine type were not present in the heated samples, but heat treatment reduced levels of available amino groups. Heating of purified and alkylated high molecular weight glutenin subunits (HMW-GS) under similar conditions also resulted in extractability loss, demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine. These observations indicated that heat treatment had induced isopeptide bond formation, resulting in larger and unextractable molecules. Heating HMW-GS lysine- and glutamine-containing peptides induced the formation of isopeptide bonds, thereby supporting the above hypothesis. The level of isopeptide bond formation increased with heating time.     

Gross nitrogen mineralisation and fungi-to-bacteria ratios are negatively correlated in boreal forests

In terrestrial ecosystems, gross nitrogen mineralisation is positively correlated to microbial biomass but negatively to soil organic matter C-to-N ratios; the influence of the microbial community structure is less well known. Here, we relate rates of gross N mineralisation to fungi-to-bacteria ratios in three natural forest types of contrasting N availability and in a long-term N-loading experiment in a boreal forest. We report, for the first time, a strong negative correlation between gross N mineralisation and the fungi-to-bacteria ratio ( = 0.91, P = 0.0005, N = 7). There was also a negative correlation between gross N mineralisation and the C-to-N ratio ( = 0.89, P = 0.001, N = 7), but a weaker positive correlation between gross N mineralisation and soil pH ( = 0.64, P = 0.019, N = 7). Our analysis suggests that soil fungi-to-bacteria and C-to-N ratios are interrelated and that they exert strong influences on soil N cycling in boreal forests.     

Evaluation of agricultural ecosystem services in fallowing land based on farmers’ participation and model simulation

Fallowing with green fertilizer can benefit agricultural ecosystem services (AES). Farmers in Taiwan do not implement fallow practices and plant green fertilizer because the current subsidy level (46,000 NT$ per ha) is too low to manage fallowing. This paper defines the objective of government agriculture policy or the farmer’s objective as maximization of farm productivity, approximated to the value of social welfare and AES. Farms, which do not follow proper fallowing practices, often have poorly maintained fallow land or left farmland abandoned. This results in negative environmental consequences such as cutworm infestations in abandoned land, which in turn can affect crops in adjacent farmlands. The objectives of this study are twofold. First, it determines the proper fallowing subsidy based on the concept of payment for ecosystem services to entice more farmers to participate in fallowing. Second, it simulates the benefit of planting green manure in fallow land to the supply of AES based on the rate of farmers who are willing to participate in fallow land practices and essential parameters that can affect soil fertility change. The approach involves a series of interviews and a developed empirical model. The value of AES when the rate of farmer participation is 100% represents a 1.5% increase in AES (448,317,000 NT$) over the value at the current participation rate of 14%. This study further concludes that the appropriate fallowing subsidy has a large positive impact on AES and social welfare (e.g., benefit from food and biofuel supplies) and is seen as a basis of ecological governance for sustainable agro-ecosystems.     

Foliar potassium status explains Douglas fir response to nitrogen fertilization in the inland Northwest,USA

Despite apparently inadequate N levels throughout the Inland Northwest of the United States, trees on some sites showed no increased growth 6 yr after N fertilization. Nor did higher N application rates consistently produce higher response. These facts indicated that other factors are limiting tree growth at these sites. Results suggest that K status is one important factor, influencing N fertilization response in the following general ways; (1) for stands with low pretreatment foliar K levels, the amount and duration of growth response are reduced, and higher N fertilizer rates produce less response; and (2) for all stands, growth response declines when foliar K decreases after N fertilization.