Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta

Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

Control of pollen-mediated gene flow in transgenic trees

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.     

Silencing of the ADNP-family member, ADNP2, results in changes in cellular viability under oxidative stress

Activity-dependent neuroprotective protein (ADNP) 2 (KIAA0863; ZNF508) gene, a homeobox-profile containing gene, was identified in a screen for homologous proteins to ADNP. The human ADNP2 contains 1131 amino acid residues with a molecular weight of 122.8 KDa. In silico analysis indicated that ortholgs to ADNP2 exist in different phyla, suggesting that ADNP2 might be evolutionary conserved. Here, we began to explore the molecular and functional characterization of ADNP2. Results showed that the mouse ADNP2 mRNA is ubiquitously expressed in distinct normal tissues with increased expression in the brain, particularly in the cerebral cortex. During development, a relatively high level of ADNP2 gene expression was found in the embryonic mouse brain and was sustained throughout embryogenesis and adulthood. An increase in the mRNA was detected in differentiated P19 neuronal/glial-like cells as compared with the non-differentiated cells. To gain insight into ADNP2 function, ADNP2-deficient cell lines were established by the RNA silencing (small interfering RNA) technology. ADNP2 deficiency significantly changed the toxicity induced by hydrogen peroxide in P19 embryonic carcinoma cells, similar to what would be predicted for ADNP deficiency. These findings represent an initial characterization of ADNP2 and suggest that this gene product may have an important function in brain by playing a role in cellular survival pathways.     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.     

The Accelerated Failure Time Model Under Biased Sampling

Summary Chen (2009, Biometrics) studies the semi‐parametric accelerated failure time model for data that are size biased. Chen considers only the uncensored case and uses hazard‐based estimation methods originally developed for censored observations. However, for uncensored data, a simple linear regression on the log scale is more natural and provides better estimators.