Transdermal enhancement through rat skin of luciferase plasmid DNA loaded in elastic nanovesicles

Transdermal absorption of luciferase plasmid (pLuc) was enhanced by loading in elastic cationic liposomes and niosomes and the application of iontophoresis or the stratum corneum (SC) stripping method. Cationic liposomes (DPPC/Chol/DDAB at a 1:1:1 molar ratio) and niosomes (Tween61/Chol/DDAB at a 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes method. The elastic vesicles were prepared by hydrating the lipid or surfactant film by 25% of ethanol instead of distilled water. Gel electrophoresis of all nanovesicles showed the 100% pLuc entrapment efficiency. All nanovesicles loaded with pLuc showed larger vesicular sizes than the nonloaded vesicles of about 1.4 times for liposomes and 1.7 times for niosomes. The nanovesicles loaded with pLuc demonstrated less positive zeta potential than the nonloaded vesicles. The pLuc loaded in elastic vesicles kept at 4 ± 2 and 27 ± 2°C for 8 weeks gave the remaining pLuc of about 70 and 60% for liposomes and 85 and 73% for niosomes, respectively. For nonelastic vesicles kept at 4 ± 2°C, 56 and 61% of the remaining pLuc were observed for liposomes and niosomes, respectively, while at 27 ± 2°C, all pLuc were degraded. The deformability indices of the elastic liposomes and niosomes loaded with the pLuc were 16.64 ± 2.92 and 20.72 ± 0.82, whereas the nonelastic vesicles gave 9.35 ± 0.09 and 10.08 ± 0.12, respectively. Transdermal absorption through rat skin pretreated with SC stripping or treated with iontophoresis of pLuc loaded in nanovesicles by vertical Franz diffusion cells was investigated at 37°C. The cells were stopped and the skin and the receiving solution were withdrawn at 1, 3, and 6 hours and the pLuc contents in the stripped SC, whole skin (viable epidermis and dermis; VED), and the receiving solution were assayed by the modified gel electrophoresis and gel documentation. Without the SC stripping technique or iontophoresis, the pLuc loaded and nonloaded in nonelastic cationic liposomes or niosomes were not found in SC, VED, and receiving solution. The fluxes in the whole skin of pLuc loaded in nonelastic liposomes and niosomes with SC stripping and iontophoresis at 6 hours gave 2.73 ± 0.46 and 3.83 ± 0.73, and 7.01 ± 1.22 and 9.60 ± 1.31 g/cm²/h, respectively, while pLuc loaded in elastic liposomes and niosomes without the SC stripping and iontophoresis at 6 hours showed 2.79 ± 0.09 and 2.84 ± 0.04 g/cm²/h, respectively. The pLuc loaded in elastic niosomes or in nonelastic niosomes with iontophoresis was found in the receiving solution with a higher amount than that loaded in elastic liposomes or nonelastic liposomes with iontophoresis. The fluxes in the receiving solution of pLuc loaded in nonelastic liposomes and niosomes with iontophoresis at 6 hours were 6.71 ± 0.31 and 8.82 ± 0.28 g/cm²/h, respectively. For elastic liposomes and niosomes, the fluxes of the loaded pLuc in the receiving solution were the same, at about 1.9 g/cm²/h. Although pLuc loaded in nonelastic niosomes with iontophoresis gave the highest delivery of the plasmid in VED and receiving solution, a more promising applicable approach for gene delivery has been suggested to be the elastic niosomal systems, since no equipment is required.     

Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing

Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater. Received: 19 May 1998; Accepted: 29 October 1998     

A new semi-automated instrument to improve the fine needle aspiration procedure during breast lesion cell sampling

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for some type of breast lesion. Fine Needle Aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. However, the frequency of conclusive samples using this method varies and is often too low, especially when performed by unexperienced operators. In this study we have developed and tested a new semi-automated instrument (“CytoTest”) designed for FNA which is intended to improve the efficacy of the technique by increasing the percentage of conclusive samples. A total of 443 consecutive aspiration procedures on palpable breast lesions were performed to compare this new “CytoTest” equipment with the standard protocol using the same type of needles. We conclude that by increasing the extent and frequency of the reciprocatory motions used by an experienced sampling operator as well as enhancing the ejection pressure, the cellular yield can be increased almost three folded compared to the standard protocol. For cases with high amounts of non-diagnostic material (such as blood or cystic fluid) which were discarded, up to four times more sample could be obtained. Furthermore, the frequency of sparse samples under 1 mg was halved with use of the “CytoTest”.     

Four-year growth dynamics of beech-spruce model ecosystems under CO2 enrichment on two different forest soils

To elucidate how atmospheric CO₂ enrichment, enhanced nutrient supply and soil quality interact to affect regrowth of temperate forests, young Fagus sylvatica and Picea abies trees were grown together in large model ecosystems. Identical communities were established on a nutrient-poor acidic and on a more fertile calcareous soil and tree growth, leaf area index, fine root density and soil respiration monitored over four complete growing seasons. Biomass responses to CO₂ enrichment and enhanced N supply at the end of the experiment reflected compound interest effects of growth stimulation during the first two to three seasons rather than persistent stimulation over the whole duration of the experiment. Whereas biomass of Picea was enhanced in elevated CO₂ on both soils, Fagus responded negatively to CO₂ on acidic but positively on calcareous soil. Biomass of both species profited from enhanced N supply on the poor acidic soil only. Leaf area index on both soils was greater in high N supply as a consequence of a stimulation early in the experiment, but was unaffected by CO₂ enrichment. Fine root density on acidic soil was increased in high N supply, but this did not stimulate soil respiration rate. In contrast, elevated CO₂ stimulated both fine root density and soil CO₂ efflux on calcareous soil, especially towards the end of the experiment. Our experiment suggests that future species dominance in beech-spruce forests is likely to change in response to CO₂ enrichment, but this response is subject to complex interactions with environmental factors other than CO₂, particularly soil type.     

Environment versus dispersal in the assembly of western Amazonian palm communities

Aim It is a central issue in ecology and biogeography to understand what governs community assembly and the maintenance of biodiversity in tropical rain forest ecosystems. A key question is the relative importance of environmental species sorting (niche assembly) and dispersal limitation (dispersal assembly), which we investigate using a large dataset from diverse palm communities. Location Lowland rain forest, western Amazon River Basin, Peru. Methods We inventoried palm communities, registering all palm individuals and recording environmental conditions in 149 transects of 5 m × 500 m. We used ordination, Mantel tests and indicator species analysis (ISA) to assess compositional patterns, species responses to geographical location and environmental factors. Mantel tests were used to assess the relative importance of geographical distance (as a proxy for dispersal limitation) and environmental differences as possible drivers of dissimilarity in palm species composition. We repeated the Mantel tests for subsets of species that differ in traits of likely importance for habitat specialization and dispersal (height and range size). Results We found a strong relationship between compositional dissimilarity and environmental distance and a weaker but also significant relationship between compositional dissimilarity and geographical distance. Consistent with expectations, relationships with environmental and geographical distance were stronger for understorey species than for canopy species. Geographical distance had a higher correlation with compositional dissimilarity for small‐ranged species compared with large‐ranged species, whereas the opposite was true for environmental distance. The main environmental correlates were inundation and soil nutrient levels. Main conclusions The assembly of palm communities in the western Amazon appears to be driven primarily by species sorting according to hydrology and soil, but with dispersal limitation also playing an important role. The importance of environmental characteristics and geographical distance varies depending on plant height and geographical range size in agreement with functional predictions, increasing our confidence in the inferred assembly mechanisms.