Compounds Exuded by \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e That Induce a Modification of \u3cem\u3eRhizobium etli\u3c/em\u3e Lipopolysaccharide

Exudates released from germinating seeds and roots of a black-seeded bean (Phaseolus vulgaris cv. Midnight Black Turtle Soup) induce an antigenic change in the lipopolysaccharide (LPS) of Rhizobium etli CE3. By spectroscopic analyses and chromatographic comparisons with derived standards, the chemical structures of the aglycone portions of the major inducing molecules from seed exudate were deduced, and they were identified as delphinidin, cyanidin, petunidin, and malvidin. These anthocyanidins were present in seed exudate mainly as glycosides, the chief inducer being delphinidin 3-glucoside. Also present were 3-glucosides of petunidin and malvidin and glycosides of cyanidin and delphinidin. Seed exudate from a bean variety deficient in anthocyanins did not induce the LPS conversion. The ability of root exudate to induce an antigenic change in the LPS was due to compounds other than anthocyanins

Genetic Locus Required for Antigenic Maturation of \u3cem\u3eRhizobium etli\u3c/em\u3e CE3 Lipopolysaccharide

Rhizobium etli modifies lipopolysaccharide (LPS) structure in response to environmental signals, such as low pH and anthocyanins. These LPS modifications result in the loss of reactivity with certain monoclonal antibodies. The same antibodies fail to recognize previously isolated R. etli mutant strain CE367, even in the absence of such environmental cues. Chemical analysis of the LPS in strain CE367 demonstrated that it lacked the terminal sugar of the wild-type O antigen, 2,3,4-tri-O-methylfucose. A 3-kb stretch of DNA, designated as lpe3, restored wild-type antigenicity when transferred into CE367. From the sequence of this DNA, five open reading frames were postulated. Site-directed mutagenesis and complementation analysis suggested that the genes were organized in at least two transcriptional units, both of which were required for the production of LPS reactive with the diagnostic antibodies. Growth in anthocyanins or at low pH did not alter the specific expression of gusA from the transposon insertion of mutant CE367, nor did the presence of multiple copies of lpe3 situated behind a strong, constitutive promoter prevent epitope changes induced by these environmental cues. Mutations of the lpe genes did not prevent normal nodule development on Phaseolus vulgaris and had very little effect on the occupation of nodules in competition with the wild-type strain

MicroRNAs are exported from malignant cells in customized particles

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body

Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids

MicroRNA Detection in Plasma Samples

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Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease

Demography and Dispersal Ability of a Threatened Saproxylic Beetle: A Mark-Recapture Study of the Rosalia Longicorn (Rosalia alpina)

The Rosalia longicorn or Alpine longhorn (Coleoptera: Cerambycidae) is an endangered and strictly protected icon of European saproxylic biodiversity. Despite its popularity, lack of information on its demography and mobility may compromise adoption of suitable conservation strategies. The beetle experienced marked retreat from NW part of its range; its single population survives N of the Alps and W of the Carpathians. The population inhabits several small patches of old beech forest on hill-tops of the Ralska Upland, Czech Republic. We performed mark-recapture study of the population and assessed its distribution pattern. Our results demonstrate the high mobility of the beetle, including dispersal between hills (up to 1.6 km). The system is thus interconnected; it contained ∼2000 adult beetles in 2008. Estimated population densities were high, ranging between 42 and 84 adult beetles/hectare a year. The population survives at a former military-training ground despite long-term isolation and low cover of mature beech forest (∼1%). Its survival could be attributed to lack of forestry activities between the 1950s and 1990s, slow succession preventing canopy closure and undergrowth expansion, and probably also to the distribution of habitat patches on conspicuous hill-tops. In order to increase chances of the population for long term survival, we propose to stop clear-cuts of old beech forests, increase semi-open beech woodlands in areas currently covered by conifer plantations and active habitat management at inhabited sites and their wider environs

Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs

Plant Signals and Bacterial Components Induce Modifications in Rhizobium Lipopolysaccharide

The symbiosis of the soil microbe Rhizobium with leguminous plants is a model system of bacterial interaction with higher eukaryotes. In nature, symbiotic rhizobia contribute to the nitrogen cycle. Through nitrogen fixation, they allow legumes to thrive in nitrogen-poor soil. Symbiosis begins by Rhizobium eliciting root nodule development on the roots of its eukaryotic host. At the same time, the bacteria infect the nodule through an infection thread. The most abundant surface molecule of Gram-negative bacteria, lipopolysaccharide, is essential for this process. In R.etli, the most distal portion of the LPS, the 0-antigen (0-Ag) is necessary for successful infection. The infection process and various environmental cues such as low pH or oxygen tension, or the presence of seed exudate induce antigenic modifications of the 0-Ag. In addition, growth at low pH induces a shift in the electrophoretic LPS profile. R.etli\u27s proficiency in sensing environmental changes, and modulating its surface structure in response may be important for persistence in nature or in symbiosis. This study characterizes the antigenic and electrophoretic LPS modification. The plant-released inducers of the LPS changes, the biochemical basis of the LPS modifications, bacterial genetics pertaining to this change and the physiological consequences of the LPS modifications were assessed

Plant signals and bacterial components induce modifications in Rhizobium lipopolysaccharide

The soil bacterium Rhizobium etli infects its host common bean (Phaseolus vulgaris ) during the establishment of symbiosis. The most abundant molecule of the rhizobial outer membrane, lipopolysaccharide (LPS) is an essential factor for the infection. Compounds exuded from the seed and the root, and plant-induced acidification of the immediate environment, each trigger distinct modifications (LPM) in the LPS. The major inducing compounds of LPM from seed exudate were different than those released from the root and were found to be delphinidin 3-glucoside and glycosides of cyanidin, petunidin, and malvidin. The LPS modifications were detected by loss of antigenicity of monoclonal antibodies (mAbs). A R.etli genetic locus, lpe3 , was isolated and shown to be required for the synthesis of the epitopes of these mAbs. lpe3 coded for at least four genes, lpeABCD. lpeA was not transcriptionally regulated during LPM, and constitutive expression of lpeABCD did not prevent LPM. However, genes encoded by lpe3 were required for an additional LPS property induced by low pH (LPP). Sequences of these genes had similarity to sequences of genes involved in LPS synthesis in other bacteria. Functions encoded by lpe3 are required for wild-type antigenicity of R.efli LPS but may not be involved in LPM. Several mutants of R.efli deficient in LPM were isolated. Mutants CE397 and CE440 had truncated LPSs and lacked LPM induced by anthocyanins. Strain CE440, but not CE397, lacked LPM induced by low pH. Both mutants had symbiotic deficiencies. These studies indicated that LPM induced by anthocyanins required a more complete LIPS structure than detection of LPM by low pH. Growth directly on the seed, or in seed exudate alone, was also found to induce R.etli to produce an extracellular matrix, whose major component appeared to be EPS. Thus, R.etli responds to plant-released signals by modifying the abundance or structures of its outer membrane and extracellular polysaccharides. In doing so, R.etli may generate a great array of functional diversity in its most abundant surface molecules essential for the interaction with P.vulgaris in the environment