In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

GIS based Watershed Classification in Lao PDR

Every inclined land surface has a potential for soil and water degradation, the seriousness depends on a multitude of parameters such as slope, soil type, geomorphology, rainfall, land use and natural vegetation cover. In Laos this intensified land use leads to reduced vegetation cover, to increased soil erosion, decreasing yield, and finally is likely to influence the hydrological regime. Against this background the Mekong River Commission (MRC) elaborated a spatial explicit Watershed Classification (WSC) for the Lower Mekong Basin. Based on topographic factors derived from a high-resolution Digital Terrain Model, five watershed classes are calculated, giving indication about the sensitivity to resource degradation by soil erosion. The WSC allows spatial priority setting for watershed management and generally supports informed decision making on reconnaissance level. In the conclusions focus is laid on general considerations when GIS techniques are used for spatial decision support in a development context.

The Mediterranean climate: An overview of the main characteristics and issues

The Mediterranean Region has many morphologic, geographical, historical, and societal characteristics, which make its climate scientifically interesting. The concept of Mediterranean climate is characterized by mild wet winters and warm to hot, dry summers and occur on the west side of continents between about 30° and 40° latitude. However, the presence of a relatively large mass of water is unique to the actual Mediterranean region. The Mediterranean Sea is a marginal and semi-enclosed sea; it is located on the western side of a large continental area and is surrounded by Europe to the North, Africa to the South, and Asia to the East. The chapter discusses that the climate of the Mediterranean region is to a large extent forced by planetary scale patterns. The time and space behavior of the regional features associated with such large-scale forcing is complex. Orography and land–sea distribution play an important role establishing the climate at basin scale and its teleconnections with global patterns. Different levels of services of readiness to emergencies, technological, and economic resources are likely to result in very different adaptation capabilities to environmental changes and new problems. The different economic situations and demographic trends are likely to produce contrasts and conflicts in a condition of limited available resources and environmental stress.

New aspects of airway mechanics in pre-term infants

High-frequency respiratory impedance data measured noninvasively by the high-speed interrupter technique (HIT), particularly the first antiresonance frequency (f(ar,1)), is related to airway wall mechanics. The aim of this study was to evaluate the feasibility and repeatability of HIT in unsedated pre-term infants, and to compare values of f(ar,1) from 18 pre-term (post-conceptional age 32-37 weeks, weight 1,730-2,910 g) and 18 full-term infants (42-47 weeks, 3,920-5,340 g). Among the pre-term infants, there was good short-term repeatability of f(ar,1) within a single sleep epoch (mean (sd) coefficient of variance: 8 (1.7)%), but 95% limits of agreement for repeated measures of f(ar,1) after 3-8 h were relatively wide (-41 Hz; 37 Hz). f(ar,1) was significantly lower in pre-term infants (199 versus 257 Hz), indicating that wave propagation characteristics in pre-term airways are different from those of full-term infants. The present authors suggest that this is consistent with developmental differences in airway wall structure and compliance, including the influence of the surrounding tissue. Since flow limitation is determined by wave propagation velocity and airway cross-sectional area, it was hypothesised that the physical ability of the airways to carry large flows is fundamentally different in pre-term than in full-term infants.