Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

Chlamydia trachomatis Infection Induces Replication of Latent HHV-6

Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.     

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.     

Ectomycorrhizal fungi of the Seychelles: diversity patterns and host shifts from the native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia bijuga (Caesalpiniaceae) to the introduced Eucalyptus robusta (Myrtaceae), but not Pinus caribea (Pinaceae)

Ectomycorrhizal (ECM) fungi form highly diverse communities in temperate forests, but little is known about their community ecology in tropical ecosystems. Using anatomotyping and rDNA sequencing, ECM fungi were identified on root tips of the introduced Eucalyptus robusta and Pinus caribea as well as the endemic Vateriopsis seychellarum and indigenous Intsia bijuga in the Seychelles. Sequencing revealed 30 species of ECM fungi on root tips of V. seychellarum and I. bijuga, with three species overlapping. Eucalyptus robusta shared five of these taxa, whereas P. caribea hosted three unique species of ECM fungi that were likely cointroduced with containerized seedlings. The thelephoroid (including the anamorphic genus Riessiella), euagaric, boletoid and hymenochaetoid clades of basidiomycetes dominated the ECM fungal community of native trees. Two species of Annulatascaceae (Sordariales, Ascomycota) were identified and described as ECM symbionts of V. seychellarum. The low diversity of native ECM fungi is attributed to deforestation and long-term isolation of the Seychelles. Native ECM fungi associate with exotic eucalypts, whereas cointroduced ECM fungi persist in pine plantations for decades.     

Identification in the mould Hypocrea jecorina of a gene encoding an NADP(+): d-xylose dehydrogenase

A gene coding for an NADP(+)-dependent d-xylose dehydrogenase was identified in the mould Hypocrea jecorina (Trichoderma reesei). It was cloned from cDNA, the active enzyme was expressed in yeast and a histidine-tagged enzyme was purified and characterized. The enzyme had highest activity with d-xylose and significantly smaller activities with other aldose sugars. The enzyme is specific for NADP(+). The K(m) values for d-xylose and NADP(+) are 43 mM and 250 microM, respectively. The role of this enzyme in H. jecorina is unclear because in this organism d-xylose is predominantly catabolized through a path with xylitol and d-xylulose as intermediates and the mould is unable to grow on d-xylonic acid.     

Toward novel HIV-1 integrase binding inhibitors: molecular modeling, synthesis, and biological studies

The identification of a novel hit compound as integrase binding inhibitor has been accomplished by means of virtual screening techniques. A small family of structurally related molecules has been synthesized and biologically evaluated with one of the compounds showing an IC(50)=12 microM.     

Geographic variation in fitness‐related traits of the bladderwrack Fucus vesiculosus along the Baltic Sea‐North Sea salinity gradient

In the course of the ongoing global intensification and diversification of human pressures, the study of variation patterns of biological traits along environmental gradients can provide relevant information on the performance of species under shifting conditions. The pronounced salinity gradient, co‐occurrence of multiple stressors, and accelerated rates of change make the Baltic Sea and its transition to North Sea a suitable region for this type of study. Focusing on the bladderwrack Fucus vesiculosus, one of the main foundation species on hard‐bottoms of the Baltic Sea, we analyzed the phenotypic variation among populations occurring along 2,000 km of coasts subjected to salinities from 4 to >30 and a variety of other stressors. Morphological and biochemical traits, including palatability for grazers, were recorded at 20 stations along the Baltic Sea and four stations in the North Sea. We evaluated in a common modeling framework the relative contribution of multiple environmental drivers to the observed trait patterns. Salinity was the main and, in some cases, the only environmental driver of the geographic trait variation in F. vesiculosus. The decrease in salinity from North Sea to Baltic Sea stations was accompanied by a decline in thallus size, photosynthetic pigments, and energy storage compounds, and affected the interaction of the alga with herbivores and epibiota. For some traits, drivers that vary locally such as wave exposure, light availability or nutrient enrichment were also important. The strong genetic population structure in this macroalgae might play a role in the generation and maintenance of phenotypic patterns across geographic scales. In light of our results, the desalination process projected for the Baltic Sea could have detrimental impacts on F. vesiculosus in areas close to its tolerance limit, affecting ecosystem functions such as habitat formation, primary production, and food supply.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.