Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

Background

Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.

Results

Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.

Conclusions

Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.     

Identification of a novel archaebacterial thioredoxin: determination of function through structure

As part of a high-throughput, structural proteomic project we have used NMR spectroscopy to determine the solution structure and ascertain the function of a previously unknown, conserved protein (MtH895) from the thermophilic archeon Methanobacterium thermoautotrophicum. Our findings indicate that MtH895 contains a central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other. It has an overall fold superficially similar to that of a glutaredoxin. However, detailed analysis of its three-dimensional structure along with molecular docking simulations of its interaction with T7 DNA polymerase (a thioredoxin-specific substrate) and comparisons with other known members of the thioredoxin/glutaredoxin family of proteins strongly suggest that MtH895 is more akin to a thioredoxin. Furthermore, measurement of the pK(a) values of its active site thiols along with direct measurements of the thioredoxin/glutaredoxin activity has confirmed that MtH895 is, indeed, a thioredoxin and exhibits no glutaredoxin activity. We have also identified a group of previously unknown proteins from several other archaebacteria that have significant (34-44%) sequence identity with MtH895. These proteins have unusual active site -CXXC- motifs not found in any known thioredoxin or glutaredoxin. On the basis of the results presented here, we predict that these small proteins are all members of a new class of truncated thioredoxins.     

Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation

Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4-mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1 alpha, MIP-1 beta, IL-8) and cytokines (TNF-alpha, IL-1 beta, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.     

Machine learning of large‐scale spatial distributions of wild turkeys with high‐dimensional environmental data

Species distribution modeling often involves high‐dimensional environmental data. Large amounts of data and multicollinearity among covariates impose challenges to statistical models in variable selection for reliable inferences of the effects of environmental factors on the spatial distribution of species. Few studies have evaluated and compared the performance of multiple machine learning (ML) models in handling multicollinearity. Here, we assessed the effectiveness of removal of correlated covariates and regularization to cope with multicollinearity in ML models for habitat suitability. Three machine learning algorithms maximum entropy (MaxEnt), random forests (RFs), and support vector machines (SVMs) were applied to the original data (OD) of 27 landscape variables, reduced data (RD) with 14 highly correlated covariates being removed, and 15 principal components (PC) of the OD accounting for 90% of the original variability. The performance of the three ML models was measured with the area under the curve and continuous Boyce index. We collected 663 nonduplicated presence locations of Eastern wild turkeys (Meleagris gallopavo silvestris) across the state of Mississippi, United States. Of the total locations, 453 locations separated by a distance of ≥2 km were used to train the three ML algorithms on the OD, RD, and PC data, respectively. The remaining 210 locations were used to validate the trained ML models to measure ML performance. Three ML models had excellent performance on the RD and PC data. MaxEnt and SVMs had good performance on the OD data, indicating the adequacy of regularization of the default setting for multicollinearity. Weak learning of RFs through bagging appeared to alleviate multicollinearity and resulted in excellent performance on the OD data. Regularization of ML algorithms may help exploratory studies of the effects of environmental factors on the spatial distribution and habitat suitability of wildlife.     

Dietary protein deficiency in pregnant mice and offspring

Previous studies suggest an association between dermal contact hypersensitivity and preterm delivery. We hypothesized that dietary protein deficiency produces cell-mediated immune hypersensitivity in pregnant animals and their offspring akin to those known to produce tissue damage. We compared the effects of feeding a 20% protein diet (controls) to those of feeding a 10% protein (deficient) diet ad libitum to pregnant BALB/c mice. We measured dermal contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) by the increment in ear skin thickness (swelling) 72 h after immunization and parity by the number of viable pups delivered. Dams fed the protein-deficient diet ingested less food, gained less weight and delivered fewer viable pups than the dams fed the control diet. Greater DNFB-stimulated increment in ear skin thickness was found in the protein-deficient mothers and in their offspring than in the control mothers and their offspring. We conclude that dietary protein deficiency limits parity and induces immune hypersensitivity. These findings suggest the potential for dietary protein deficiency to activate a T-cell-mediated branch of the immune response that may put pregnant animals at risk for preterm delivery.     

Aquatic heterotrophic bacteria have highly flexible phosphorus content and biomass stoichiometry

Bacteria are central to the cycling of carbon (C), nitrogen (N) and phosphorus (P) in every ecosystem, yet our understanding of how tightly these cycles are coupled to bacterial biomass composition is based upon data from only a few species. Bacteria are commonly assumed to have high P content, low biomass C:P and N:P ratios, and inflexible stoichiometry. Here, we show that bacterial assemblages from lakes exhibit unprecedented flexibility in their P content (3% to less than 0.01% of dry mass) and stoichiometry (C:N:P of 28: 7: 1 to more than 8500: 1200: 1). The flexibility in C:P and N:P stoichiometry was greater than any species or assemblage, including terrestrial and aquatic autotrophs, and suggests a highly dynamic role for bacteria in coupling multiple element cycles.Terrestrial ecosystems are an important source of nutrients and organic carbon (C) to freshwater rivers and lakes as well as the coastal ocean. Past work has shown that heterotrophic bacteria, a group of organisms that process terrestrial inputs of organic carbon, nitrogen (N) and phosphorus (P) (Biddanda et al., 2001), are C-poor and P-rich (Makino et al., 2003) relative to terrestrial inputs characterized by high C:P ratios. As a result, bacterial assemblages in freshwater ecosystems should experience elemental imbalance and act as efficient exporters of organic carbon to downstream ecosystems. However, freshwater ecosystems metabolize most of the organic C they receive from terrestrial ecosystems (Cole et al., 2007) and it has been shown recently that strains (Scott et al., 2012) and assemblages (Godwin and Cotner, 2014) of bacteria from lakes can be P-poor and stoichiometrically flexible. Here, we demonstrate that bacterial assemblages from lakes exhibit unprecedented plasticity in their stoichiometry and discuss the implications of flexible composition to ecosystem processes.To determine the extent of stoichiometric flexibility within assemblages, we performed two experiments in which we cultured the bacteria-sized fraction of plankton from a northern temperate lake under varying C:P supply ratios and measured their biomass composition (Supplementary Methods). We created C:P_supply ratios from 31.6:1 to more than 2 20 000:1 by manipulating the supply of phosphate in a defined medium, with all other nutrients in excess of C and P. At each level of C:P_supply, we enriched the lake assemblages in batch cultures and used these enrichments to inoculate chemostats at the same C:P_supply. The chemostats were maintained at a dilution rate (0.33 d⁻¹) that is low relative to assemblage growth rates measured in lakes (Cotner et al., 2001).The bacterial P content decreased from a mean of 3.55% of dry mass when the assemblage was C-limited to 0.006–0.05% when the assemblage was most P-limited (Figure 1). The range of P content measured in the assemblage cultures was nearly equal to the range of existing data in the literature, particularly for P relative to dry mass (Supplementary Table 6). Single-cell measurements from plankton environments indicated the potential for even lower phosphorus quotas (Norland et al., 1995; Cotner et al., 2010), although many of those cells may not be actively growing, potentially decreasing their demand for P-rich RNA, where much of the P resides in bacterial cells (Makino et al., 2003). The P relative to dry mass values measured here were lower than those reported for a bacterium grown in the absence of added phosphate and high concentrations of arsenate (0.012% of dry mass as P, Wolfe-Simon et al., 2010). The results presented here clearly demonstrate that bacteria can have P content less than 0.01% of dry mass when growing at low levels of P.Open in a separate windowFigure 1Effect of C:P_supply ratio on biomass P/cell (a), P/dry mass (b), C:P_biomass (c) and N:P_biomass (d) ratios in chemostats diluted at 0.33 d⁻¹. Data from Experiment 1 are displayed as solid circles, and open circles denote data from Experiment 2. At each level of C:P_supply, the data from replicate chemostat are staggered to improve clarity. The error bars represent the s.e. of the ratio for each chemostat, following propagation of errors from the numerator and denominator. In Experiment 1, C:P_biomass and N:P_biomass (analysis of variance, P<1 × 10^-5) increased and P/dry mass and P/cell decreased (P<0.005) significantly with increasing C:P_supply. On the basis of changes in C:P_biomass, the assemblage was defined as P-sufficient at C:P_supply of 31.6:1 and P-limited at C:P_supply of 10 000:1 and greater. At C:P_supply of 1 00 000:1, only one chemostat had P content above the analytical detection limit and only two chemostats without added P had N above the detection limit.The C:P_biomass and N:P_biomass of the bacterial assemblages increased from 28:1 and 6:1, respectively, when C-limited to a maximum of >8500:1 and >1200:1 when P-limited (Figure 1). The ranges of C:P_biomass and N:P_biomass observed in this study cover nearly the entire range of measurements recorded in previous studies for bacterial cultures and assemblages (Figure 2; Supplementary Table 6) and nearly match the ranges of C:P_biomass and N:P_biomass observed in vascular plant tissues (Elser et al., 2000; Sterner and Elser, 2002; Reich and Oleksyn, 2004). Furthermore, the bacterial assemblage (of multiple strains) exhibited greater stoichiometric plasticity than has been documented in any other species or assemblage, including terrestrial and aquatic primary producers (Sterner and Elser, 2002; Persson et al., 2010). These experiments demonstrate that previous assumptions of low and invariant C:P_biomass (Tanaka et al., 2009; Fanin et al., 2013) and high relative P content for bacteria (Wolfe-Simon et al., 2010) do not represent the physiological flexibility of bacteria in natural assemblages. Although mean cellular P content decreased under P limitation, much of the flexibility in C:P_biomass was due to a substantial increase in cellular C content (Supplementary Figure 1), likely owing to the accumulation of C-rich storage molecules (Thingstad et al., 2005).Open in a separate windowFigure 2Ranges of C:P_biomass and N:P_biomass for bacterial cultures and other organisms, with separate panels for C:P_biomass (panel a) and N:P_biomass (panel b). Data for heterotrophic bacteria are separated by sources: literature data (Supplementary Table 7), assemblage chemostat cultures (Godwin and Cotner, 2014) and this study. Ranges for other organisms were from ^a (Cross et al., 2005) and ^b (Elser et al., 2000). ^cRanges for E. coli were compiled from multiple studies (Supplementary Table 7). Seston refers to suspended particulate matter (phytoplankton, heterotrophs and detritus). The boxplots display data for individual replicate cultures where data are available, with the centerline representing the median, the edges of the box representing the 25% and 75% quantiles, and the whiskers representing the maximum and minimum values. Dashed lines indicate the Redfield ratio (C:N:P=106:16:1).The range of stoichiometric flexibility present in natural assemblages is critical to understanding homeostasis within ecosystems. Strict homeostasis of assemblage C:N:P_biomass leads to the prediction that the ratio of regenerated C:P increases dramatically with increasing resource C:P (Sterner, 1990), but flexible biomass stoichiometry allows tight coupling and negative feedback between bacterial biomass stoichiometry and resource stoichiometry, facilitating the inherent resilience of ecosystems to nutrient perturbations (Scheffer et al., 2001). It is increasingly recognized that much of the organic matter metabolized in rivers and lakes originates in terrestrial ecosystems where C:P and N:P ratios can be much higher than for organic matter originating in aquatic ecosystems (Lennon and Pfaff, 2005). The observations in this study of extreme flexibility in bacterial biomass stoichiometry are consistent with observations of higher and more variable biomass C:P and N:P in the seston (suspended particulate matter) in freshwaters than in pelagic (offshore) marine systems where terrestrial influences and nutrient gradients are less profound (Cotner et al., 2010).The bacteria in inland waters and the coastal ocean experience stoichiometric imbalance when they process terrestrial inputs of dissolved and particulate organic matter with high C:P ratios. Compared with bacteria with low and invariant C:P_biomass, assemblages that increase their C:P_biomass in response to this imbalance will remineralize less ‘excess'' C through respiration and could decrease the export of organic matter to downstream ecosystems. In ecosystems where internal nutrient cycling processes are dominant and bacteria regenerate a large fraction of available nutrients (for example, offshore marine systems), flexible bacterial stoichiometry likely stabilizes dissolved inorganic nutrient concentrations and inhibits fluctuations.The capacity of heterotrophic bacteria to continue to buffer C and nutrient feedbacks in ecosystems is likely challenged by the use of inorganic fertilizers that decrease the exported C:N and C:P ratios to aquatic systems (Arbuckle and Downing, 2001) and anthropogenic warming that increases both the export of organic carbon and the C:N:P stoichiometry of that material (Freeman et al., 2001; Urban et al., 2011). Additionally, because stoichiometric flexibility decreases with increasing relative growth rates (Makino and Cotner., 2004; Hillebrand et al., 2013), bacterial assemblages in low-temperature environments could become less flexible as the result of anthropogenic warming. By examining the capacity of aquatic bacterial assemblages to respond to C:N:P imbalance, we can evaluate the influence of stoichiometric flexibility on aquatic ecosystem productivity and the extent and periodicity of nutrient fluctuations.