Efficient in vivo regulation of cytidine deaminase expression in the haematopoietic system using a doxycycline-inducible lentiviral vector system.

Regulated transgene expression may reduce transgene-specific and genotoxic risks associated with gene therapy. To prove this concept, we have investigated the suitability of doxycycline (Dox)-inducible human cytidine deaminase (hCDD) overexpression from lentiviral vectors to mediate effective myeloprotection while circumventing the lymphotoxicity observed with constitutive CDD activity. Rapid Dox-mediated transgene induction associated with a 6-17-fold increase in drug resistance was observed in 32D and primary murine bone marrow (BM) cells. Moreover, robust Dox-regulated transgene expression in the entire haematopoietic system was demonstrated for primary and secondary recipients of hCDD-transduced R26-M2rtTA transgenic BM cells. Furthermore, mice were significantly protected from myelosuppressive chemotherapy as evidenced by accelerated recovery of granulocytes (1.9±0.6 vs 1.3±0.3, P=0.034) and platelets (883±194 vs 584±160 10(3) per µl, P=0.011). Minimal transgene expression in the non-induced state and no overt cellular toxicities including lymphotoxicity were detected. Thus, using a relevant murine transplant model our data provide conclusive evidence that drug-resistance transgenes can be expressed in a regulated fashion in the lymphohaematopoietic system, and that Dox-inducible systems may be used to reduce myelotoxic side effect of anticancer chemotherapy or to avoid side effects of high constitutive transgene expression.

MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy.

Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.

Formation and ripening of alginate-like exopolymer gel layers during and after membrane filtration.

The properties of biofilm EPS are determined by the multiple interactions between its constituents and the surrounding environment. Because of the high complexity of biofilm EPS, its constituents' characterisation is still far from thorough, and identification of these interactions cannot be done yet. Therefore, we use gels of bacterial alginate-like exopolysaccharides (ALEs) as a model component for biofilm EPS in this work. These gels have been examined for their cohesive properties as a function of CaCl2 and KCl concentration. Hereto, ALE gel layers were formed on membranes by dead-end filtration of ALE solutions. Accumulation of the cations Ca2+ and K+ in the gels could be well predicted from a Donnan equilibrium model based on the fixed negative charges in the ALE. This suggests that there is no specific binding of Ca2+ to the ALE and that on the time scale of the experiments, the Ca2+ ions can distribute freely over the gel and the surrounding solution. The concentration of fixed negative charges in the ALE was estimated around 1 mmol/g VSS (volatile suspended solids, organic mass) from the Donnan equilibrium. Moreover, an accumulation of H+ was predicted. Gels with more CaCl2 in the supernatant were more compact and bore a higher osmotic pressure than those with less CaCl2, revealing the role of Ca2+ ions in the network crosslinking. It is hypothesised that this mechanism later transitions into a rearrangement of the ALE molecules, which eventually leads to a fibrous network structure with large voids.

Rheological characterisation of alginate-like exopolymer gels crosslinked with calcium.

Bacterial alginate-like exopolymers (ALE) gels have been used in this work as a model for the extracellular polymeric matrix of biofilms. Aim was to relate the mechanical properties and strength of this matrix that make biofilms as persistent to cleaning as they are, to the complex cohesive molecular interactions involved. Mechanical properties of the gels as a function of CaCO3 concentration were investigated using dynamic and static rheology. Gels with relatively low CaCO3 concentrations, between 100 µmol and 300 µmol per g ALE, were found to exhibit similar viscoelastic behaviour as real biofilms, with elastic moduli between 50 Pa and 100 Pa and dissipation factors between 0.2 and 0.3. Increasing CaCO3 concentrations resulted in an increase of the elastic modulus up to 250 Pa, accompanied by an increase in brittleness. At a CaCO3 concentration of 1250 µmol per g ALE this trend stopped, probably due to disturbance of the continuous ALE network by precipitation of salts. Therefore, overdosing of Ca salts can be an adequate approach for the removal of biofouling. All gels exhibited permanent strain hardening under medium strain, and their mechanical properties showed dependency on their strain history. Even after application of an oscillatory strain with 200% amplitude that caused the gel structure to collapse, the gels recovered 65 to 90% of their original shear modulus, for the major part within the first 20 s. Recovery was slightly less for gels with high CaCO3 concentration. In creep tests fitted with a Burgers model with multiple Kelvin elements at least three different interactions in the ALE gels could be distinguished with characteristic retardation times in the range of 10, 100 and 1000 s. Further identification of the mechanisms underlying the gel mechanics will allow the development of targeted strategies to undermine the mechanical strength of biofouling and aid the cleaning process.

Preliminary studies on the chemical properties of the toxic principle from Diamphidia nigroornata larvae, a source of Bushman arrow poison.

The Bushmen of southern Africa use the expressed contents of beetle larvae (Diamphidia, Lebistina and Polyclada species) as arrow poison. an aqueous extract of Diamphidia nigroornata larvae was fractionated by gel filtration on Sephadex G-50. Two fractions were obtained: one (I) of high molecular weight which contains a protein of 60 000 daltons, and a low molecular weight fraction (II) of non-protein nature. Both fractions proved to be lethal to mice: an LD50 of 0.5 - 0.95 (I) and 3.2 - 3.5 (II) mg/kg (intraperitoneal injection), respectively, was determined. The toxic principle of fraction I could be partly separated from the protein by ammonium sulfate precipitation followed by gel filtration. That of fraction II was further resolved into several subfractions by gel filtration of Sephadex G-10; however, the lethal activity was completely lost during purification. In thin-layer chromatography the low molecular weight toxin(s) did not react with reagents for steroids, alkaloids, sugars or terpenes, but showed a positive ninhydrin reaction. It is concluded that the toxic principle of the Bushman arrow poison is a highly labile, low molecular weight compound which is closely attached or bound to a protein protecting it from inactivation.

27-day cycles in human mortality: Traute and Bernhard Düll.

This tribute to her parents by one co-author (NDP) is the fruit of a more than a decade-long search by the senior author (FH) for the details of the lives of Bernhard and Gertraud ("Traute") Düll. These pioneers studied how space/terrestrial weather may differentially influence human mortality from various causes, the 27-day mortality pattern being different whether death was from cardiac or respiratory disease, or from suicide. FH is the translator of personal information about her parents provided by NDP in German. Figuratively, he also attempts to "translate" the Dülls' contribution in the context of the literature that had appeared before their work and after their deaths. Although the Dülls published in a then leading journal, among others (and FH had re-analyzed some of their work in a medical journal), they were unknown to academies or libraries (where FH had inquired about them). The Dülls thoroughly assembled death certificates to offer the most powerful evidence for an effect of solar activity reflected in human mortality, as did others before them. They went several steps further than their predecessors, however. They were the first to show possibly differential effects of space and/or Earth weather with respect to suicide and other deaths associated with the nervous and sensory systems vs. death from cardiac or respiratory disease as well as overall death by differences in the phase of a common 27-day cycle characterizing these mortality patterns. Furthermore, Bernhard Düll developed tests of human visual and auditory reaction time to study effects of weather and solar activity, publishing a book (his professorial dissertation) on the topic. His unpublished finding of an increased incidence of airplane crashes in association with higher solar activity was validated after his death, among others, by Tatiana Zenchenko and A. M. Merzlyi.

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures.

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280-320 nm) spectral range. However, it is also controlled by phytochrome. A Pfr/Ptot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of Pfr in order to be fully effective.