Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependent drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 degrees C or 30% at 37 degrees C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6?mol/l cryoprotectant (4.65, 4.65, and 3.31?mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at ?80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4?mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P?<?0.05). After 3 and 6?months in vivo valve function was determined by two-dimensional echo-Doppler and at 7?months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P?=?0.0403, P?=?0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P?<?0.05) and infiltrating CD3+ T-cells (P?<?0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at ?80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.     

Above- and belowground fluxes of methane from boreal dwarf shrubs and Pinus sylvestris seedlings

Plant and Soil - Legume break crops provide a series of agronomic benefits to the following wheat crop in a rotation. Phosphorus-efficient break-crop plants can mobilise P from non-labile pools in...     

Responses of aerobic microbial communities and soil respiration to water-level drawdown in a northern boreal fen

On a global basis, peatlands are a major reserve of carbon (C). Hydrological changes can affect the decomposition processes in peatlands and in turn can alter their C balance. Since 1959, a groundwater extraction plant has generated a water-level gradient at our study site that has gradually changed part of the wet fen into a dry peatland forest. The average water-level drawdown of the gradient (from a pristine 9 cm to 26 cm in the dry end) is close to an estimate predicted by an increase in mean global temperature of 3°C. We studied the total microbial community of the aerobic surface peat in four locations along the gradient through phospholipid fatty acid and PCR-DGGE methods. Additionally, field measurements of soil respiration showed a threefold increase in the C-emission rate at the driest location compared with the wettest one, indicating enhanced decomposition. Also, both fungal and bacterial biomass increased in the drier locations. At the species level, the fungal community changed due to water-level drawdown whereas actinobacteria were less sensitive to drying. The majority of fungal sequences were similar to ectomycorrhizal (ECM) fungi, which dominated throughout the gradient. Our results indicate that ECM fungi might act as important facultative decomposers in organic-rich environments such as peatlands.     

Tricholoma matsutake dominates diverse microbial communities in different forest soils

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.     

Carbon flux from decomposing wood and its dependency on temperature,wood N2 fixation rate,moisture and fungal composition in a Norway spruce forest

Globally 40–70 Pg of carbon (C) are stored in coarse woody debris on the forest floor. Climate change may reduce the function of this stock as a C sink in the future due to increasing temperature. However, current knowledge on the drivers of wood decomposition is inadequate for detailed predictions. To define the factors that control wood respiration rate of Norway spruce and to produce a model that adequately describes the decomposition process of this species as a function of time, we used an unprecedentedly diverse analytical approach, which included measurements of respiration, fungal community sequencing, N₂ fixation rate, nifH copy number, ¹⁴C‐dating as well as N%, δ¹³C and C% values of wood. Our results suggest that climate change will accelerate C flux from deadwood in boreal conditions, due to the observed strong temperature dependency of deadwood respiration. At the research site, the annual C flux from deadwood would increase by 27% from the current 117 g C/kg wood with the projected climate warming (RCP4.5). The second most important control on respiration rate was the stage of wood decomposition; at early stages of decomposition low nitrogen content and low wood moisture limited fungal activity while reduced wood resource quality decreased the respiration rate at the final stages of decomposition. Wood decomposition process was best described by a Sigmoidal model, where after 116 years of wood decomposition mass loss of 95% was reached. Our results on deadwood decomposition are important for C budget calculations in ecosystem and climate change models. We observed for the first time that the temperature dependency of N₂ fixation, which has a major role at providing N for wood‐inhabiting fungi, was not constant but varied between wood density classes due to source supply and wood quality. This has significant consequences on projecting N₂ fixation rates for deadwood in changing climate.     

Taxonomy of alkaliphilic Bacillus strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.     

Charcoal as a habitat for microbes and its effect on the microbial community of the underlying humus

Wildfires produce a charcoal layer, which has an adsorbing capacity resembling activated carbon. After the fire a new litter layer starts to accumulate on top of the charcoal layer, which liberates water‐soluble compounds that percolate through the charcoal and the unburned humus layer. We first hypothesized that since charcoal has the capacity to adsorb organic compounds it may form a new habitat for microbes, which decompose the adsorbed compounds. Secondly, we hypothesized that the charcoal may cause depletion of decomposable organic carbon in the underlying humus and thus reduce the microbial biomass. To test our hypotheses we prepared microcosms, where we placed non‐heated humus and on top one of the adsorbents: non‐adsorptive pumice (Pum), charcoal from Empetrum nigrum (EmpCh), charcoal from humus (HuCh) or activated carbon (ActC). We watered them with birch leaf litter extract. The adsorbing capacity increased in the order Pumorg in the litter extract, respectively. After one month, all adsorbents harboured microbes, but their amount and basal respiration was largest in EmpCh and HuCh, and smallest in Pum. In addition, different kinds of microbial communities with respect to their phospholipid fatty acid and substrate utilization patterns were formed in the adsorbents. The amount of microbial biomass and number of bacteria did not differ between humus under different adsorbents, although different microbial communities developed in humus under EmpCh compared with Pum, which is obviously related to the increased pH of the humus under EmpCh, and also ActC. We suggest that charcoal from burning can support microbial communities, which are small in size but have a higher specific growth rate than those of the humus. Although the charcoal layer induces changes in the microbial community of the humus, it does not reduce the amount of humus microbes.