Massively parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts, we analyzed mRNA from Arabidopsis (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 cDNA models indicated: (1) massively parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10%, but increased the overall sequence coverage by 50%. (2) Mapping of the ESTs to their predicted full-length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium, and long transcripts were equally represented. (3) Over 16,000 of the ESTs that mapped to the genome were not represented in the existing dbEST database. In some cases, the ESTs provide the first experimental evidence for transcripts derived from predicted genes, and, for at least 60 locations in the genome, pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together, the results indicate massively parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of nonmodel plants with less complete genomic information.For approximately 30 years, sequencing of DNA by the dideoxy terminator strategy introduced by Sanger (1977) has provided the basis for almost all available information about nucleotide sequences. Pyrosequencing is an alternative technology that detects the pyrophosphate released during DNA polymerase-catalyzed incorporation of nucleotides. The pyrophosphate liberated with each nucleotide addition can generate light in a reaction coupled to ATP sulfurylase and luciferase.

C 4 photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C 3 and C 4 leaves, and to identify candidates associated with the C 4 pathway, we used massively parallel mRNA sequencing of closely related C 3 (Cleome spinosa) and C 4 (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C 3 and C 4 leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C 4 leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C 3 and C 4 leaves differs, provides a blueprint for the NAD-malic enzyme C 4 pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.

Selective pressure exerted by a massive decline in atmospheric CO 2 levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C 4 photosynthesis. C 4 species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C 4 trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C 3 and C 4 species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C 3 to C 3 -C 4 intermediate to C 4 species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C 4 -associated characteristics. Single gene analysis defines the C 4 cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C 4 associated pathways. Molecular evidence for a photorespiratory CO 2 pump prior to the establishment of the C 4 cycle-based CO 2 pump is provided. Cluster analysis defines the upper limit of C 4 -related gene expression changes in mature leaves of Flaveria as 3582 alterations.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.

SummaryPVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles.

D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH (d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism. 2-Hydroxyglutarate (2HG3 ; 2-hydroxypentanedioic acid) is a five-carbon dicarboxylic acid with the hydroxy group on the ␣-carbon. D-2HG accumulates in humans in the inherited neurometabolic disorder 2-hydroxyglutaric aciduria (2HGA) due to a deficiency in D-2HG dehydrogenase (D-2HGDH) (1), which converts D-2HG to 2-oxoglutarate (2OG); the electron transfer flavoprotein (ETF); or the ETF-ubiquinone oxidoreductase (ETFQO) (2), both electron acceptors of D-2HGDH (1). The clinical symptoms encompass developmental retardation, neurological dysfunction, and cerebral atrophy (1). In addition to high levels of 2HG, patients with 2HGA also have high concentrations of TCA cycle intermediates. On the other hand, excess accumulation of D-2HG contributes to the formation and malignant progression of brain tumors (3). Mutations in the cytosolic enzyme IDH1 (isocitrate dehydrogenase 1) occur in ϳ80% of secondary brain cancer tumors and in nearly onetenth of acute myelogenous leukemia tumors. Normally, IDH1 catalyzes the conversion of isocitrate to 2OG. Cancer-associated mutations in IDH1 reduce the affinity of the enzyme for isocitrate and increase the affinity for NADPH and 2OG. This prevents the oxidative decarboxylation of isocitrate to 2OG and facilitates the conversion of 2OG to D-2HG. In this way, IDH1 mutations cause a gain of function, resulting in the production and accumulation of D-2HG (3).D-2HG occurs in mammals (i) in the conversion of 2OG to D-2HG through a hydroxy acid-oxoacid transhydrogenase with the concomitant conversion of ␥-hydroxybutyrate to succinic semialdehyde (4), (ii) as an intermediate in the succinate-glycine cycle between 2OG semialdehyde and 2OG (5), and (iii) in ...

The contribution of metabolism to heat stress may play a significant role in defining robustness and recovery of systems; either by providing the energy and metabolites required for cellular homeostasis, or through the generation of protective osmolytes. However, the mechanisms by which heat stress attenuation could be adapted through metabolic processes as a stabilizing strategy against thermal stress are still largely unclear. We address this issue through metabolomic and transcriptomic profiles for populations along a thermal cline where two seagrass species, Zostera marina and Zostera noltii, were found in close proximity. Significant changes captured by these profile comparisons could be detected, with a larger response magnitude observed in northern populations to heat stress. Sucrose, fructose, and myo-inositol were identified to be the most responsive of the 29 analyzed organic metabolites. Many key enzymes in the Calvin cycle, glycolysis and pentose phosphate pathways also showed significant differential expression. The reported comparison suggests that adaptive mechanisms are involved through metabolic pathways to dampen the impacts of heat stress, and interactions between the metabolome and proteome should be further investigated in systems biology to understand robust design features against abiotic stress.

Food processing wears down teeth, thus affecting tooth functionality and evolutionary success. Other than intrinsic silica phytoliths, extrinsic mineral dust/grit adhering to plants causes tooth wear in mammalian herbivores. Dental microwear texture analysis (DMTA) is widely applied to infer diet from microscopic dental wear traces. The relationship between external abrasives and dental microwear texture (DMT) formation remains elusive. Feeding experiments with sheep have shown negligible effects of dust-laden grass and browse, suggesting that intrinsic properties of plants are more important. Here, we explore the effect of clay- to sand-sized mineral abrasives (quartz, volcanic ash, loess, kaolin) on DMT in a controlled feeding experiment with guinea pigs. By adding 1, 4, 5, or 8% mineral abrasives to a pelleted base diet, we test for the effect of particle size, shape, and amount on DMT. Wear by fine-grained quartz (>5/<50 µm), loess, and kaolin is not significantly different from the abrasive-free control diet. Fine silt-sized quartz (∼5 µm) results in higher surface anisotropy and lower roughness (polishing effect). Coarse-grained volcanic ash leads to significantly higher complexity, while fine sands (130 to 166 µm) result in significantly higher roughness. Complexity and roughness values exceed those from feeding experiments with guinea pigs who received plants with different phytolith content. Our results highlight that large (>95-µm) external silicate abrasives lead to distinct microscopic wear with higher roughness and complexity than caused by mineral abrasive-free herbivorous diets. Hence, high loads of mineral dust and grit in natural diets might be identified by DMTA, also in the fossil record.