Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses.

The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP

Free energy landscape of siRNA-polycation complexation: Elucidating the effect of molecular geometry, polymer flexibility, and charge neutralization

The success of medical threatments with DNA and silencing interference RNA is strongly related to the design of efficient delivery technologies. Cationic polymers represent an attractive strategy to serve as nucleic-acid carriers with the envisioned advantages of efficient complexation, low cost, ease of production, well-defined size, and low polydispersity index. However, the balance between efficacy and toxicity (safety) of these polymers is a challenge and in need of improvement. With the aim of designing more effective polycationic-based gene carriers, many parameters such as carrier morphology, size, molecular weight, surface chemistry, and flexibility/rigidity ratio need to be taken into consideration. In the present work, the binding mechanism of three cationic polymers (polyarginine, polylysine and polyethyleneimine) to a model siRNA target is computationally investigated at the atomistic level. In order to better understand the polycationic carrier-siRNA interactions, replica exchange molecular dynamic simulations were carried out to provide an exhaustive exploration of all the possible binding sites, taking fully into account the siRNA flexibility together with the presence of explicit solvent and ions. Moreover, well-tempered metadynamics simulations were employed to elucidate how molecular geometry, polycation flexibility, and charge neutralization affect the siRNA-polycations free energy landscape in term of low-energy binding modes and unbinding free energy barriers. Significant differences among polymer binding modes have been detected, revealing the advantageous binding properties of polyarginine and polylysine compared to polyethyleneimine

CO2 increases 14C-primary production in an Arctic plankton community

Responses to ocean acidification in plankton communities were studied during a CO2-enrichment experiment in the Arctic Ocean, accomplished from June to July 2010 in Kongsfjorden, Svalbard (78°56′ 2′′ N, 11°53′ 6′′ E). Enclosed in 9 mesocosms (volume: 43.9–47.6 m3), plankton was exposed to CO2 concentrations, ranging from glacial to projected mid-next-century levels. Fertilization with inorganic nutrients at day 13 of the experiment supported the accumulation of phytoplankton biomass, as indicated by two periods of high chl a concentration. This study tested for CO2 sensitivities in primary production (PP) of particulate organic carbon (PPPOC) and of dissolved organic carbon (PPDOC). Therefore, 14C-bottle incubations (24 h) of mesocosm samples were performed at 1 m depth receiving about 60% of incoming radiation. PP for all mesocosms averaged 8.06 ± 3.64 μmol C L−1 d−1 and was slightly higher than in the outside fjord system. Comparison between mesocosms revealed significantly higher PPPOC at elevated compared to low pCO2 after nutrient addition. PPDOC was significantly higher in CO2-enriched mesocosms before as well as after nutrient addition, suggesting that CO2 had a direct influence on DOC production. DOC concentrations inside the mesocosms increased before nutrient addition and more in high CO2 mesocosms. After addition of nutrients, however, further DOC accumulation was negligible and not significantly different between treatments, indicating rapid utilization of freshly produced DOC. Bacterial biomass production (BP) was coupled to PP in all treatments, indicating that 3.5 ± 1.9% of PP or 21.6 ± 12.5% of PPDOC provided on average sufficient carbon for synthesis of bacterial biomass. During the later course of the bloom, the response of 14C-based PP rates to CO2 enrichment differed from net community production (NCP) rates that were also determined during this mesocosm campaign. We conclude that the enhanced release of labile DOC during autotrophic production at high CO2 exceedingly stimulated activities of heterotrophic microorganisms. As a consequence, increased PP induced less NCP, as suggested earlier for carbon-limited microbial systems in the Arctic

Response of bacterioplankton activity in an Arctic fjord system to elevated pCO2: results from a mesocosm perturbation study

The effect of elevated seawater carbon dioxide (CO2) on the activity of a natural bacterioplankton community in an Arctic fjord system was investigated by a mesocosm perturbation study in the frame of the European Project on Ocean Acidification (EPOCA). A pCO2 range of 175–1085 μatm was set up in nine mesocosms deployed in the Kongsfjorden (Svalbard). The bacterioplankton communities responded to rising chlorophyll a concentrations after a lag phase of only a few days with increasing protein production and extracellular enzyme activity and revealed a close coupling of heterotrophic bacterial activity to phytoplankton productivity in this experiment. The natural extracellular enzyme assemblages showed increased activity in response to moderate acidification. A decrease in seawater pH of 0.5 units roughly doubled rates of β-glucosidase and leucine-aminopeptidase. Activities of extracellular enzymes in the mesocosms were directly related to both seawater pH and primary production. Also primary production and bacterial protein production in the mesocosms at different pCO2 were positively correlated. Therefore, it can be suggested that the efficient heterotrophic carbon utilization in this Arctic microbial food web had the potential to counteract increased phytoplankton production that was achieved under elevated pCO2 in this study. However, our results also show that the transfer of beneficial pCO2-related effects on the cellular bacterial metabolism to the scale of community activity and organic matter degradation can be mitigated by the top-down control of bacterial abundances in natural microbial communities

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Faculty authors explore Spanish vanguard poetry, fatherhood, multi-modal learning, and more

A comparison of drug transport in pulmonary absorption models: isolated perfused rat lungs, respiratory epithelial cell lines and primary cell culture

Purpose: To evaluate the ability of human airway epithelial cell layers and a simple rat isolated perfused lung (IPL) model to predict pulmonary drug absorption in rats in vivo. Method: The permeability of seven compounds selected to possess a range of lipophilicity was measured in two airway cell lines (Calu-3 and 16HBE14o-), in normal human bronchial epithelial (NHBE) cells and using a simple isolated perfused lungs (IPL) technique. Data from the cell layers and ex vivo lungs were compared to published absorption rates from rat lungs measured in vivo. Results: A strong relationship was observed between the logarithm of the in vivo absorption half-life and the absorption half-life in the IPL (r = 0.97; excluding formoterol). Good log-linear relationships were also found between the apparent first-order absorption rate in vivo and cell layer permeability with correlation coefficients of 0.92, 0.93, 0.91 in Calu-3, 16HBE14o- and NHBE cells, respectively. Conclusion: The simple IPL technique provided a good prediction of drug absorption from the lungs, making it a useful method for empirical screening of drug absorption in the lungs. Permeability measurements were similar in all the respiratory epithelial cell models evaluated, with Calu-3 having the advantage for routine permeability screening purposes of being readily availability, robust and easy to culture

Expansion of Agriculture in Northern Cold-Climate Regions: A Cross-Sectoral Perspective on Opportunities and Challenges

Agriculture in the boreal and Arctic regions is perceived as marginal, low intensity and inadequate to satisfy the needs of local communities, but another perspective is that northern agriculture has untapped potential to increase the local supply of food and even contribute to the global food system. Policies across northern jurisdictions target the expansion and intensification of agriculture, contextualized for the diverse social settings and market foci in the north. However, the rapid pace of climate change means that traditional methods of adapting cropping systems and developing infrastructure and regulations for this region cannot keep up with climate change impacts. Moreover, the anticipated conversion of northern cold-climate natural lands to agriculture risks a loss of up to 76% of the carbon stored in vegetation and soils, leading to further environmental impacts. The sustainable development of northern agriculture requires local solutions supported by locally relevant policies. There is an obvious need for the rapid development of a transdisciplinary, cross-jurisdictional, long-term knowledge development, and dissemination program to best serve food needs and an agricultural economy in the boreal and Arctic regions while minimizing the risks to global climate, northern ecosystems and communities

Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum

This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression

Solid-State Dynamic Nuclear Polarization at 263 GHz: Spectrometer Design and Experimental Results

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz 1H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water–glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.National Institutes of Health (U.S.) (NIH grant EB-002804)National Institutes of Health (U.S.) (NIH grant EB-002026