RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells

Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound more efficiently to the surface of human lung carcinoma cells (A549), known to contain alphaV integrins, as compared to WT baculovirus. In addition, the binding pattern of the RGD-displaying baculovirus showed extensive clustering. This most likely represents clustering of the integrin molecules on the cell surface, induced by binding of the RGD-displaying baculovirus. Finally, the transduction efficiency of an RGD-representing virus increased by almost three-fold as monitored by light emission measurements. In conclusion, these results suggest that the RGD-motif is functional on the surface of baculovirus and thereby these tropism-modified viruses bind more efficiently as well as enhance the transduction efficiency of human cancer cells expressing alphaV integrins.     

Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.     

Insect cells for antibody production: evaluation of an efficient alternative

In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic?" insect cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary cells. Using "High Five" cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG? heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" cells, high levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.     

The subnival–nival vascular plant species of Iran: a unique high-mountain flora and its threat from climate warming

This study provides a first country-wide overview of the vertical distribution patterns and the chorology of vascular plant species that occur in the uppermost elevation zones in Iran. The current distribution patterns are discussed with respect to potential warming-induced species losses. Iran’s subnival and nival vegetation zones are found at elevations above 3600–3900 m in a highly fragmented distribution across Alborz, Zagros, and NW-Iran. Based on literature research and on field observations, all vascular plant species living in the subnival–nival zone of Iranian mountains were identified (151 species) and classified into three altitudinal groups: Group A comprises species that occur mainly in subnival–nival habitats (51 species). Group B are species being common in subnival–nival areas but are equally present in the alpine zone (56 species). Group C are species that can reach to subnival areas but also grow in alpine, subalpine and sometimes lower altitudes (44 species). The chorological patterns differ among the three groups. The percentage of species being endemic to Iran decreases from group A, to B and C, with 68, 53 and 20%, respectively. A narrow altitudinal distribution at high elevations is clearly related to a small-scaled geographical distribution range. The outstanding rate of high-altitude endemism appears to result mainly from orographic isolation of the country’s highly scattered cold areas and by the absence of extensive Pleistocene glaciations. The narrow distribution of most of Iran’s cold-adapted mountain flora and the low potential of alternative cold habitats render it highly vulnerable to climate change.     

Insights into the completely annotated genome of Lactobacillus buchneri CD034, a strain isolated from stable grass silage

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.     

ACES: a machine learning toolbox for clustering analysis and visualization

Background

Studies that aim at explaining phenotypes or disease susceptibility by genetic or epigenetic variants often rely on clustering methods to stratify individuals or samples. While statistical associations may point at increased risk for certain parts of the population, the ultimate goal is to make precise predictions for each individual. This necessitates tools that allow for the rapid inspection of each data point, in particular to find explanations for outliers.

Results

ACES is an integrative cluster- and phenotype-browser, which implements standard clustering methods, as well as multiple visualization methods in which all sample information can be displayed quickly. In addition, ACES can automatically mine a list of phenotypes for cluster enrichment, whereby the number of clusters and their boundaries are estimated by a novel method. For visual data browsing, ACES provides a 2D or 3D PCA or Heat Map view. ACES is implemented in Java, with a focus on a user-friendly, interactive, graphical interface.

Conclusions

ACES has been proven an invaluable tool for analyzing large, pre-filtered DNA methylation data sets and RNA-Sequencing data, due to its ease to link molecular markers to complex phenotypes. The source code is available from https://github.com/GrabherrGroup/ACES.

     

Spatial pattern in dry grassland communities of the Central Alps and its ecophysiological significance

The spatial pattern of species and microhabitats in a dry grassland mosaic in a Central Alpine valley was studied by a variance/mean analysis. In addition, the water-usage strategies of the dominant species were investigated. A significant correlation between species pattern and microhabitat pattern was found for the grass species. Grasses, with their homorhizal root system, are unable to exploit sources of water in deep rock crevices, and are therefore dependent on the water-storage capacity of the particular soil layer. Dwarf shrubs and succulents (with one exception) are independent of this latter water source. The common chamaephytes exhibit contradictory types of stomatal behaviour.Artemisia alba is characterized by a water-conservation strategy, whereasHelianthemum nummularium spp.obscurum andTeucrium chamaedrys are not. The careful usage of the water reserves byArtemisia alba would seem to represent an adaptation to dry grassland conditions. The other dwarf shrubs, in contrast, with their ‘water-wasting’ strategy, have a root system like that ofArtemisia and are unable to compensate for the higher rate of water loss. Thus, the observed ecophysiological differences cannot be interpreted as being ‘different solutions to the same problem’, as a result of divergent evolution. Two alternative interpretations are: a) the stomatal behaviour of the chamaephytes is a characteristic of their respective genera and which is not particularly adaptive here; b) the dry grasslands studied are the remnants of a former vegetation type, which had a low level of competition.     

Picea abies andAbies alba forests of the austrian alps: Numerical classification and ordination

A TWINSPAN classification of a representative set of 3026 relevés of spruce and fir forests from the Eastern Alps (Austria) is presented. Ecological features of relevé clusters and species groups are described by means of Ellenberg indicator values, site factors and stand characteristics. The most important floristic discontinuity in the data set separates acidophilous communities on mostly silicate substrates from basiphilous communities on mostly carbonate substrates. Further divisions reflect a combined gradient of temperature, nutrient regime and shading. This is supported by the correlation of average Ellenberg values of sample plots with DCA axes. A qualitative comparison between TWINSPAN clusters and a syntaxonomic system widely used in the Austrian Alps is drawn. The two ordersPiceetalia excelsae andAthyrio-Piceetalia largely coincide with the clusters of the first level of divisions. Alliances are partly reproduced by TWINSPAN. Clusters on the fifth and fourth level of division mostly correspond to associations. However, a considerable portion of the lower level clusters is of a transitional type. Out of thirteen fir and spruce associations described for the Austrian Alps, five associations are not reproduced by TWINSPAN, i.e.Bazzanio-Piceetum, Veronico-Piceetum, Adenostylo alliariae-Abietetum, Asplenio-Piceetum andCarici-Piceetum. Three associations are split on the second level of division, i.e.Larici-Piceetum, Luzulo nemorosae-Piceetum andCalamagrostio variae-Piceetum.