Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5' and 3' mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.     

Exotic tree seedlings are much more competitive than natives but show underyielding when growing together

Aims Invasive species continue to be a worldwide threat to ecosystems mainly as a cause for biodiversity loss. Forest ecosystems, for example, are subject to a change in species composition due to the invasion of exotic species. Specifying the attributes that cause the strong competitiveness of several exotic species may improve the ability to understand and effectively manage plant invasions in the future. In this study the following hypotheses were tested: (1) biomass production of below- and aboveground plant components of the exotic tree species is higher than that of the natives, resulting in a higher competitiveness of the exotics; (2) the exclusion of root competition has a positive effect on the biomass production of the inferior native species; and (3) mixtures of native and exotic species yield a higher biomass production than the respective monocultures.Methods A pot experiment, containing about 2000 tree seedlings, was established. We investigated the biomass productivity and growth reactions of two native (Quercus robur L., Carpinus betulus L.) and two exotic tree species (Prunus serotina Ehrh., Robinia pseudoacacia L.) in different intra- and interspecific, competitive situations with and without the influence of root competition.Important findings The biomass production of both exotic species was significantly higher and led to a strong competitive advantage, resulting in a biomass decrease of the less competitive native species. The high belowground biomass of both exotic species had a negative effect on the biomass production. The competitive pressure of exotic tree seedlings on the native ones was largely driven by root competition. Furthermore, mixtures of native and exotic tree species had a higher productivity than their growth in monocultures would have predicted. Competition was lower for exotic species in mixtures with the less productive native species compared to the competition in monocultures or in mixture with the other highly productive exotic species. Accordingly, both highly competitive exotic species produced less biomass in mixture with each other compared to monocultures. Despite the significantly higher biomass of P. serotina in all mixtures and in monoculture, R. pseudoacacia seemed to be the dominating species. Due to its strong root competition, R. pseudoacacia significantly reduced the biomass production of P. serotina .     

New and fast method to quantify respiration rates of bacterial and plankton communities in freshwater ecosystems by using optical oxygen sensor spots

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 x 10(6) respiring cells ml(-1) of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20 degrees C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 x 10(6) respiring bacteria ml(-1)) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 micromol O(2) liter(-1) h(-1). The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.     

Native and introduced gastropods in laurel forests on Tenerife,Canary Islands

The introduction of non-native gastropods on islands has repetitively been related to a decline of the endemic fauna. So far, no quantitative information is available even for the native gastropod fauna from the laurel forests (the so-called Laurisilva) of the Canary Islands. Much of the original laurel forest has been logged in recent centuries. Based on vegetation studies, we hypothesized that densities and the number of introduced species decline with the age of the regrowth forests. We sampled 27 sites from which we collected thirty native and seven introduced species. Two introduced species, Milax nigricans and Oxychilus alliarius, were previously not reported from the Canary Islands. Assemblage composition was mainly structured by disturbance history and altitude. Overall species richness was correlated with slope inclination, prevalence of rocky outcrops, amounts of woody debris and leaf litter depth. Densities were correlated with the depth of the litter layer and the extent of herb layer cover and laurel canopy cover. Introduced species occurred in 22 sites but were neither related to native species richness nor to the time that elapsed since forest regrowth. One introduced slug, Lehmannia valentiana, is already wide-spread, with densities strongly related to herb cover. Overall species richness seemed to be the outcome of invasibility, thus factors enhancing species richness likely also enhance invasibility. Although at present introduced species contribute to diversity, the potential competition between introduced slugs and the rich native semi-slug fauna, and the effects of introduced predatory snails (Oxychilus spp. and Testacella maugei) warrant further monitoring.     

The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.