Jeppe Emmersen

DeepSAGE—digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples

Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.

Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras

The major potato tuber proteins of the Kuras cultivar, which is the dominant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis. The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanum tuberosum). Proteins were characterized using MS and scored against potato protein databases, derived from both ‘Kuras only’ and ‘all potato’ expressed sequence tags (EST) and full‐length cDNAs. Despite the existence of ∼ 180 000 ESTs, the currently available potato sequence data showed a severe under‐representation of genes or long transcripts encoding proteins > 50 kDa (3.5% of all) compared with the complete proteome of Arabidopsis thaliana (33% of all). We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras. Other proteins identified include annexin, glyoxalase I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants. Known S. tuberosum patatins cluster into three clades all represented in Kuras. S. tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPI K, found to date only in Kuras. Furthermore, protein abundance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber.

The potato tuber transcriptome: analysis of 6077 expressed sequence tags

This is the first report of the biosynthetic potential of a tuber storage organ investigated by expressed sequence tag sequencing. A cDNA library was generated from the mature tuber of field grown potato (Solanum tuberosum var. Kuras). Partial sequences obtained from 6077 clones were assembled into 828 clusters and 1533 singletons. The average read length was 592 bp, and 2254 clones were full length. 5717 clones showed homology to genes from other organisms. Genes involved in protein synthesis, protein destination and cell defense predominated in tuber compared to stolon, shoot and leaf organs. 1063 clones were unique to tuber. Transcripts of starch metabolizing enzymes showed similar relative levels in tuber and stolon.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark

OBJECTIVES The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). METHODS The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. RESULTS Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. CONCLUSIONS Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Transcript elongation factor TFIIS is involved in Arabidopsis seed dormancy.

Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

Hypoxia and adipose-derived stem cell-based tissue regeneration and engineering.

Introduction: Realization that oxygen is one of the key regulators of development and differentiation has a profound significance on how current cell-based and tissue engineering applications using adipose-derived stem cells (ASCs) can be further improved. Areas covered: The article provides an overview of mechanisms of hypoxic responses during physiological adaptations and development. Furthermore, a synopsis of the hypoxic responses of ASCs is provided, and this information is presented in context of their utility as a major source of stem cells across the regenerative applications explored to date. Expert opinion: The reader will obtain insight into a highly specific area of stem cell research focusing on ASCs and hypoxia. In order to enhance the level of comprehension, a broader context with other stem cell and experimental systems is provided. It is emphasized that the pericellular oxygen tension is a critical regulatory factor that should be taken into account when devising novel stem cell-based therapeutic applications along with other parameters, such as biochemical soluble factors and the growth substrates.

The expression level of the chromatin-associated HMGB1 protein influences growth, stress tolerance and transcriptome in Arabidopsis

High mobility group (HMG) proteins of the HMGB family are small and relatively abundant chromatin-associated proteins. As architectural factors, the HMGB proteins are involved in the regulation of transcription and other DNA-dependent processes. We have examined Arabidopsis mutant plants lacking the HMGB1 protein, which is a typical representative of the plant HMGB family. In addition, our analyses included transgenic plants overexpressing HMGB1 and mutant plants that were transformed with the HMGB1 genomic region (complementation plants), as well as control plants. Both the absence and overexpression of HMGB1 caused shorter primary roots and affected the sensitivity towards the genotoxic agent methyl methanesulfonate. The overexpression of HMGB1 decreased the seed germination rate in the presence of elevated concentrations of NaCl. The complementation plants that expressed HMGB1 at wild-type levels did not show phenotypic differences compared to the control plants. Transcript profiling by microarray hybridization revealed that a remarkably large number of genes were differentially expressed (up- and down-regulated) in plants lacking HMGB1 compared to control plants. Among the down-regulated genes, the gene ontology category of stress-responsive genes was overrepresented. Neither microscopic analyses nor micrococcal nuclease digestion experiments revealed notable differences in overall chromatin structure, when comparing chromatin from HMGB1-deficient and control plants. Collectively, our results show that despite the presence of several other HMGB proteins, the lack and overexpression of HMGB1 affect certain aspects of plant growth and stress tolerance and it has a marked impact on the transcriptome, suggesting that HMGB1 has (partially) specialized functions in Arabidopsis.

Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro.

In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers. These findings could have implications for the use of hibernating brown bears as a model to study disuse osteoporosis.

The Mycoplasma hominis vaa gene displays a mosaic gene structure

Mycoplasma hominis contains a variable adherence‐associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolates by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N‐terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequence boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.