Jonathan Peter Merrison

Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles

ABSTRACTPurposeThis work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice.MethodsAerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method.ResultsFlow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).ConclusionsThis work provides a technology platform for effective pulmonary delivery and gene silencing of RNAi therapeutics with potential use in preclinical studies of respiratory disease treatment.

Winds at the Phoenix landing site

[1] Wind speeds and directions were measured on the Phoenix Lander by a mechanical anemometer, the so-called Telltale wind indicator. Analysis of images of the instrument taken with the onboard imager allowed for evaluation of wind speeds and directions. Daily characteristics of the wind data are highly turbulent behavior during midday due to daytime turbulence with more stable conditions during nighttime. From L s ~77°-123° winds were generally ~4 m s -1 from the east, with 360° rotation during midday. From L s ~123°-148° daytime wind speeds increased to an average of 6-10 m s -1 and were generally from the west. The highest wind speed recorded was 16 m s -1 seen on L s ~147°. Estimates of the surface roughness height are calculated from the smearing of the Kapton part of the Telltale during image exposure due to a 3 Hz turbulence and nighttime wind variability. These estimates yield 6 ± 3 mm and 5 ± 3 mm, respectively. The Telltale wind data are used to suggest that Heimdal crater is a source of nighttime temperature fluctuations. Deviations between temperatures measured at various heights are explained as being due to winds passing over the Phoenix Lander. Events concerning sample delivery and frost formation are described and discussed. Two different mechanisms of dust lifting affecting the Phoenix site are proposed based on observations made with Mars Color Imager on Mars Reconnaissance Orbiter and the Telltale. The first is related to evaporation of the seasonal CO 2 ice and is observed up to L s ~95°. These events are not associated with increased wind speeds. The second mechanism is observed after L s ~111° and is related to the passing of weather systems characterized by condensate clouds in orbital images and higher wind speeds as measured with the Telltale.

Simulation of the Martian dust aerosol at low wind speeds

[1] Performing realistic simulations is crucial for developing, testing, and subsequently analyzing results of experiments sent to the surface of Mars. A wind tunnel has been constructed, in which the atmospheric conditions of pressure and wind speed are controlled to match those observed by the Pathfinder mission to Mars. Injection into the wind tunnel of an analogue dust from Salten Skov in Denmark allows simulation of the Martian aerosol. Here experiments can be tested in preparation for a planned mission to the planet (Mars Exploration Rovers to be launched in 2003). Observations of adhesion and cohesion effects have been made in the wind tunnel, which are relevant to particle transport and of significance for validating the performance of specific experiments on Mars. Preliminary studies have been made, at Mars atmospheric pressure, of dust capture on magnet arrays similar to those flown on the Mars Pathfinder mission.

A Facility for Long-Term Mars Simulation Experiments: The Mars Environmental Simulation Chamber (MESCH)

We describe the design, construction, and pilot operation of a Mars simulation facility comprised of a cryogenic environmental chamber, an atmospheric gas analyzer, and a xenon/mercury discharge source for UV generation. The Mars Environmental Simulation Chamber (MESCH) consists of a double-walled cylindrical chamber. The double wall provides a cooling mantle through which liquid N(2) can be circulated. A load-lock system that consists of a small pressure-exchange chamber, which can be evacuated, allows for the exchange of samples without changing the chamber environment. Fitted within the MESCH is a carousel, which holds up to 10 steel sample tubes. Rotation of the carousel is controlled by an external motor. Each sample in the carousel can be placed at any desired position. Environmental data, such as temperature, pressure, and UV exposure time, are computer logged and used in automated feedback mechanisms, enabling a wide variety of experiments that include time series. Tests of the simulation facility have successfully demonstrated its ability to produce temperature cycles and maintain low temperature (down to -140 degrees C), low atmospheric pressure (5-10 mbar), and a gas composition like that of Mars during long-term experiments.

Activity and stability of a complex bacterial soil community under simulated Martian conditions

A simulation experiment with a complex bacterial soil community in a Mars simulation chamber was performed to determine the effect of Martian conditions on community activity, stability and survival. At three different depths in the soil core short-term effects of Martian conditions with and without ultraviolet (UV) exposure corresponding to 8 Martian Sol were compared. Community metabolic activities and functional diversity, measured as glucose respiration and versatility in substrate utilization, respectively, decreased after UV exposure, whereas they remained unaffected by Martian conditions without UV exposure. In contrast, the numbers of culturable bacteria and the genetic diversity were unaffected by the simulated Martian conditions both with and without UV exposure. The genetic diversity of the soil community and of the colonies grown on agar plates were evaluated by denaturant gradient gel electrophoresis (DGGE) on DNA extracts. Desiccation of the soil prior to experimentation affected the functional diversity by decreasing the versatility in substrate utilization. The natural dominance of endospores and Gram-positive bacteria in the investigated Mars-analogue soil may explain the limited effect of the Mars incubations on the survival and community structure. Our results suggest that UV radiation and desiccation are major selecting factors on bacterial functional diversity in terrestrial bacterial communities incubated under simulated Martian conditions. Furthermore, these results suggest that forward contamination of Mars is a matter of great concern in future space missions.

Comparison of the mineralogical effects of an experimental forest fire on a goethite/ferrihydrite soil with a topsoil that contains hematite, maghemite and goethite

Abstract A long-standing unresolved puzzle related to the Danish temperate humid climate is the presence of extended areas with large Fe contents, where goethite and ferrihydrite are present in the topsoil along with hematite and maghemite. Hematite and, particularly, maghemite would normally be interpreted as the result of high temperature as found after forest fires. However, a body of evidence argues against these sites having been exposed to fire. In an attempt to get closer to an explanation of this Fe mineralogy, an experimental forest fire was produced. The results showed a clear mineralogical zonation down to 10 cm depth. This was not observed at the natural sites, which contained a mixture of goethite/ferrihydrite, hematite and maghemite down to 20 cm depth. The experimental forest fire left charcoal and ashes at the topsoil, produced high pH and decreased organic matter content, all of which is in contrast to the natural sites. The conclusion from this work is that the mineralogy of these sites is not consistent with exposure to forest fire, but may instead result from long-term transformation in a reducing environment, possibly involving microbiology.

Effects of Long-Term Simulated Martian Conditions on a Freeze-Dried and Homogenized Bacterial Permafrost Community

Indigenous bacteria and biomolecules (DNA and proteins) in a freeze-dried and homogenized Arctic permafrost were exposed to simulated martian conditions that correspond to about 80 days on the surface of Mars with respect to the accumulated UV dose. The simulation conditions included UV radiation, freeze-thaw cycles, the atmospheric gas composition, and pressure. The homogenized permafrost cores were subjected to repeated cycles of UV radiation for 3 h followed by 27 h without irradiation. The effects of the simulation conditions on the concentrations of biomolecules; numbers of viable, dead, and cultured bacteria; as well as the community structure were determined. Simulated martian conditions resulted in a significant reduction of the concentrations of DNA and amino acids in the uppermost 1.5 mm of the soil core. The total number of bacterial cells was reduced in the upper 9 mm of the soil core, while the number of viable cells was reduced in the upper 15 mm. The number of cultured aerobic bacteria was reduced in the upper 6 mm of the soil core, whereas the community structure of cultured anaerobic bacteria was relatively unaffected by the exposure conditions. As explanations for the observed changes, we propose three causes that might have been working on the biological material either individually or synergistically: (i) UV radiation, (ii) UV-generated reactive oxygen species, and (iii) freeze-thaw cycles. Currently, the production and action of reactive gases is only hypothetical and will be a central subject in future investigations. Overall, we conclude that in a stable environment (no wind-/pressure-induced mixing) biological material is efficiently shielded by a 2 cm thick layer of dust, while it is relatively rapidly destroyed in the surface layer, and that biomolecules like proteins and polynucleotides are more resistant to destruction than living biota.

Non-dissociative and dissociative ionization of CO, CO2 and CH4 by positron impact

Cross sections for direct and total non-dissociative ionization of CO, CO2 and CH4 by positron impact have been measured for projectile energies ranging from threshold to 2000 eV. For CO and CO2 direct and total dissociative cross sections for several different fragments have been measured. The non-dissociative cross sections are observed to behave similarly to the single ionization cross sections of the light noble gases. The dissociative cross sections resemble that seen recently for N2, and can be related to models for double ionization of the light noble gases.

Antihydrogen production and precision experiments

The study of CPT invariance with the highest achievable precision in all particle sectors is of fundamental importance for physics. Equally important is the question of the gravitational acceleration of antimatter. In recent years, impressive progress has been achieved in capturing antiprotons in specially designed Penning traps, in cooling them to energies of a few milli-electron volts, and in storing them for hours in a small volume of space. Positrons have been accumulated in large numbers in similar traps, and low energy positron or positronium beams have been generated. Finally, steady progress has been made in trapping and cooling neutral atoms. Thus the ingredients to form antihydrogen at rest are at hand. Once antihydrogen atoms have been captured at low energy, spectroscopic methods can be applied to interrogate their atomic structure with extremely high precision and compare it to its normal matter counterpart, the hydrogen atom. Especially the 1S-2S transition, with a lifetime of the excited state of 122 msec and thereby a natural linewidth of 5 parts in 10{sup 16}, offers in principle the possibility to directly compare matter and antimatter properties at a level of 1 part in 10{sup 16}.

Ionization of argon and krypton by positron impact

Total single and double ionization cross sections for argon and krypton by positron impact have been measured for projectile energies ranging from threshold to ~1 keV. The single ionization cross sections are seen to behave as expected, showing large contributions from positronium formation at low energies. In the double ionization cross sections there seems to be a lack of positronium formation at threshold, while a clear contribution from the process is seen at intermediate energies.