Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

A new strategy to infer circularity applied to four new complete frog mitogenomes

We applied a novel strategy to infer sequence circularity and complete assembly of four mitochondrial genomes (mitogenomes) of the frog families Bufonidae (Melanophryniscus moreirae), Dendrobatidae (Hyloxalus subpunctatus and Phyllobates terribilis), and Scaphiopodidae (Scaphiopus holbrookii). These are the first complete mitogenomes of these four genera and Scaphiopodidae. We assembled mitogenomes from short genomic sequence reads using a baiting and iterative mapping strategy followed by a new ad hoc mapping strategy developed to test for assembly circularization. To assess the quality of the inferred circularization, we used Bowtie2 alignment scores and a new per‐position sequence coverage value (which we named “connectivity”). Permutation tests with 400 iterations per specimen and 1% or 5% chance of mutation at the ends of the putative circular sequences showed that the proposed method is highly sensitive, with a single nucleotide insertion or deletion being sufficient for circularity to be rejected. False positives comprised only 2% of all observations and possessed significantly lower alignment scores. The size, gene content, and gene arrangement of each mitogenome differed among the species but matched the expectations for their clades. We argue that basic studies on circular sequences can benefit from the results and bioinformatics procedures introduced here, especially when closely related references are lacking.     

Cell Wall Assembly and Intracellular Trafficking in Plant Cells Are Directly Affected by Changes in the Magnitude of Gravitational Acceleration

Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.     

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the sensitive determination of folates in rice

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC–MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC–MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC–MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 μg/100 g and 0.12 and 0.91 μg/100 g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 μg/100 g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material.     

Somatic embryogenesis of Cyclamen persicum in liquid medium

A method is described for the production of somatic embryos of Cyclamen persicum Mill. in liquid medium. Five steps are involved; initiation of embryogenic cell lines, proliferation of pro-embryogenic masses (PEMs) on auxin-containing medium, development of somatic embryos on hormone-free medium with high osmolarity, germination and subsequent plantlet formation. Cell lines were initiated by culturing the explant, the seedling tuber, directly in liquid medium. Three parameters were important for obtaining embryogenic cell lines; explant density, hormone concentrations and subculture regime. The rate of uptake of the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin influenced the formation of PEMs. Highly embryogenic cell lines were obtained only when PEMs had formed within 5–7 weeks. PEMs were proliferated for at least 24 months and could be isolated from each subculture for the production of somatic embryos. A high sucrose content (175 m M ) in the development medium without hormones ensured efficient embryo development from PEMs. A subsequent subculture in low sucrose concentration (58 m M ) induced the formation of a tuber, thus promoting germination. Arabinogalactan-proteins (AGPs) from carrot seeds and AGPs bound by the monoclonal antibody ZUM 18 increased the number of PEMs in a culture, showing that the activity of AGPs is not species specific.