Quantifying climate and management effects on regional crop yield and nitrogen leaching in the north china plain.

Better water and nitrogen (N) management requires better understanding of soil water and N balances and their effects on crop yield under various climate and soil conditions. In this study, the calibrated Root Zone Water Quality Model (RZWQM2) was used to assess crop yield and N leaching under current and alternative management practices in a double-cropped wheat ( L.) and maize ( L.) system under long-term weather conditions (1970-2009) for dominant soil types at 15 locations in the North China Plain. The results provided quantitative long-term variation of deep seepage and N leaching at these locations, which strengthened the existing qualitative knowledge for site-specific management of water and N. In general, the current management practices showed high residual soil N and N leaching in the region, with the amounts varying between crops and from location to location and from year to year. Seasonal rainfall explained 39 to 84% of the variability in N leaching (1970-2009) in maize across locations, while for wheat, its relationship with N leaching was significant ( < 0.01) only at five locations. When N and/or irrigation inputs were reduced to 40 to 80% of their current levels, N leaching generally responded more to N rate than to irrigation, while the reverse was true for crop yield at most locations. Matching N input with crop requirements under limited water conditions helped achieve lower N leaching without considerable soil N accumulation. Based on the long-term simulation results and water resources availability in the region, it is recommended to irrigate at 60 to 80% of the current water levels and fertilize only at 40 to 60% of the current N rate to minimizing N leaching without compromising crop yield.

Soil-test N recommendations augmented with PEST-optimized RZWQM simulations.

Improved understanding of year-to-year late-spring soil nitrate test (LSNT) variability could help make it more attractive to producers. We test the ability of the Root Zone Water Quality Model (RZWQM) to simulate watershed-scale variability due to the LSNT, and we use the optimized model to simulate long-term field N dynamics under related conditions. Autoregressive techniques and the automatic parameter calibration program PEST were used to show that RZWQM simulates significantly lower nitrate concentration in discharge from LSNT treatments compared with areas receiving fall N fertilizer applications within the tile-drained Walnut Creek, Iowa, watershed (>5 mg NL(-1) difference for the third year of the treatment, 1999). This result is similar to field-measured data from a paired watershed experiment. A statistical model we developed using RZWQM simulations from 1970 to 2005 shows that early-season precipitation and early-season temperature account for 90% of the interannual variation in LSNT-based fertilizer N rates. Long-term simulations with similar average N application rates for corn (Zea mays L.) (151 kg N ha(-1)) show annual average N loss in tile flow of 20.4, 22.2, and 27.3 kg N ha(-1) for LSNT, single spring, and single fall N applications. These results suggest that (i) RZWQM is a promising tool to accurately estimate the water quality effects of LSNT; (ii) the majority of N loss difference between LSNT and fall applications is because more N remains in the root zone for crop uptake; and (iii) year-to-year LSNT-based N rate differences are mainly due to variation in early-season precipitation and temperature.

Direct gene transfer into mouse muscle in vivo.

RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Corn stover harvest N and energy budgets in central Iowa.

Harvesting corn stover removes N from the fields, but its effect on subsurface drainage and other N losses is uncertain. We used the Root Zone Water Quality Model (RZWQM) to examine N losses with 0 (NRR) or 50% (RR) corn residue removal within a corn and soybean rotation over a 10-yr period. In general, all simulations used the same pre-plant or post-emergence N fertilizer rate (200 kg ha-1 yr-1). Simulated annual corn yields averaged 10.7 Mg ha-1 for the post emergence applications (NRRpost and RRpost), and 9.5 and 9.4 Mg ha-1 yr-1 for NRRpre and RRpre. Average total N input during corn years was 19.3 kg N ha-1 greater for NRRpre compared to RRpre due to additional N in surface residues, but drainage N loss was only 1.1 kg N ha-1 yr-1 greater for NRRpre. Post-emergence N application with no residue removal (NRRpost) reduced average drainage N loss by 16.5 kg ha-1 yr-1 compared to pre-plant N fertilization (NRRpre). The farm-gate net energy ratio was greatest for RRpost and lowest for NRRpre (14.1 and 10.4 MJ output per MJ input) while greenhouse gas intensity was lowest for RRpost and highest for NRRpre (11.7 and 17.3 g CO2-eq. MJ-1 output). Similar to published studies, the simulations showed little difference in N2O emissions between scenarios, decreased microbial immobilization for RR compared to NRR, and small soil carbon changes over the 10-yr simulation. In contrast to several previous modeling studies, the crop yield and N lost to drain flow were nearly the same between NRR and RR without supplemental N applied to replace N removed with corn stover. These results are important to optimizing the energy and nitrogen budgets associated with corn stover harvest and for developing a sustainable bioenergy industry.

Modeling nitrogen and water management effects in a wheat-maize double-cropping system.

Excessive N and water use in agriculture causes environmental degradation and can potentially jeopardize the sustainability of the system. A field study was conducted from 2000 to 2002 to study the effects of four N treatments (0, 100, 200, and 300 kg N ha(-1) per crop) on a wheat (Triticum aestivum L.) and maize (Zea mays L.) double cropping system under 70 +/- 15% field capacity in the North China Plain (NCP). The root zone water quality model (RZWQM), with the crop estimation through resource and environment synthesis (CERES) plant growth modules incorporated, was evaluated for its simulation of crop production, soil water, and N leaching in the double cropping system. Soil water content, biomass, and grain yield were better simulated with normalized root mean square errors (NRMSE, RMSE divided by mean observed value) from 0.11 to 0.15 than soil NO(3)-N and plant N uptake that had NRMSE from 0.19 to 0.43 across these treatments. The long-term simulation with historical weather data showed that, at 200 kg N ha(-1) per crop application rate, auto-irrigation triggered at 50% of the field capacity and recharged to 60% field capacity in the 0- to 50-cm soil profile were adequate for obtaining acceptable yield levels in this intensified double cropping system. Results also showed potential savings of more than 30% of the current N application rates per crop from 300 to 200 kg N ha(-1), which could reduce about 60% of the N leaching without compromising crop yields.

N loss to drain flow and N2O emissions from a corn-soybean rotation with winter rye.

Anthropogenic perturbation of the global nitrogen cycle and its effects on the environment such as hypoxia in coastal regions and increased N2O emissions is of increasing, multi-disciplinary, worldwide concern, and agricultural production is a major contributor. Only limited studies, however, have simultaneously investigated NO3- losses to subsurface drain flow and N2O emissions under corn-soybean production. We used the Root Zone Water Quality Model (RZWQM) to evaluate NO3- losses to drain flow and N2O emissions in a corn-soybean system with a winter rye cover crop (CC) in central Iowa over a nine year period. The observed and simulated average drain flow N concentration reductions from CC were 60% and 54% compared to the no cover crop system (NCC). Average annual April through October cumulative observed and simulated N2O emissions (2004-2010) were 6.7 and 6.0kgN2O-Nha-1yr-1 for NCC, and 6.2 and 7.2kgNha-1 for CC. In contrast to previous research, monthly N2O emissions were generally greatest when N loss to leaching were greatest, mostly because relatively high rainfall occurred during the months fertilizer was applied. N2O emission factors of 0.032 and 0.041 were estimated for NCC and CC using the tested model, which are similar to field results in the region. A local sensitivity analysis suggests that lower soil field capacity affects RZWQM simulations, which includes increased drain flow nitrate concentrations, increased N mineralization, and reduced soil water content. The results suggest that 1) RZWQM is a promising tool to estimate N2O emissions from subsurface drained corn-soybean rotations and to estimate the relative effects of a winter rye cover crop over a nine year period on nitrate loss to drain flow and 2) soil field capacity is an important parameter to model N mineralization and N loss to drain flow.

Hyperplastic and neoplastic changes in the mammary glands of feral mice free of endogenous mouse mammary tumor virus provirus.

Laboratory colonies of feral mice (Mus musculus domesticus) have been established with specific mouse mammary tumor virus (MuMTV) genotype, including colonies lacking any proviral DNA (ev-) or carrying only a single copy of MuMTV DNA (ev+). No evidence of a decline in reproductive capacity has been observed in the first 8 generations. Both the ev- and ev+ mice showed normal mammary gland development and the development of hyperplastic lesions in the older females. The mice were very resistant to spontaneous or chemically induced mammary tumors. However, the occurrence of 1 mammary tumor in an ev- mouse indicates that mammary neoplasias can occur in the absence of MuMTV DNA. The few tumors that do occur in the ev- mice provide a unique opportunity to study the neoplastic process in the absence of proviral DNA.

The counterion influence on cationic lipid-mediated transfection of plasmid DNA.

A panel of DOTAP analogs was prepared by altering the anionic counterion that accompanies the trimethylammonium polar domain. The transfection of plasmid DNA into NIH3T3 cells and mouse lung was examined using the counterion analogs. The in vitro transfection activity decreased as follows: DOTAP.bisulfate > trifluoromethanesulfonate approximately equal to iodide approximately equal to bromide > dihydrogenphosphate approximately equal to chloride approximately equal to acetate > sulfate. A similar activity trend was observed in vivo.

Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid.

Simple, nontoxic, and pharmaceutically defined methods for genetic modification of respiratory tissues may enable development of a variety of molecular medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmonary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccination. "Free," "direct," or "naked" plasmid administration is a simple, apparently safe, and pharmaceutically defined gene delivery method. Murine, macaque, and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transduction, and specifically to provide a theoretical framework for developing a category of adjuvants, nuclease inhibitors, that augment the transfection activity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose escalation studies were performed using lung homogenate assays to characterize transfection. Potential toxicity was assessed histologically. The data indicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expression. The effective dose for ATA enhancement of direct lung transfection was 0.5 microg/g mouse weight, and the LD50 was approximately 6 microg/g. These findings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory transfection technology.

Gene expression following direct injection of DNA into liver.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Cationic lipid-mediated gene delivery to murine lung: correlation of lipid hydration with in vivo transfection activity.

A panel of lipidic tetraalkylammonium chlorides has been prepared and screened in studies of both lipid hydration and in vivo mouse transfection. The effect of cationic lipid structure on liposome surface hydration was determined using differential scanning calorimetry. Increases in headgroup steric bulk and the inclusion of cis-unsaturation in the hydrophobic domain led to greater lipid hydration, indicative of a decrease in lipid polar domain associations. Cationic lipids containing hydrogen-bonding functionality in the polar domain exhibited a corresponding decrease in observed lipid hydration, indicative of an increase in lipid polar domain associations. To explore a potential correlation of the hydration data with transfection activity, we examined the in vivo transfection activity of the lipid panel by direct intratracheal instillation of cationic liposome-DNA complexes into BALB/c mice. The more active transfection agents were the lipids that featured headgroup structures promoting close polar domain association in combination with fatty acyl cis-unsaturation. The hydration data suggest that the more effective transfection lipids for mouse lung delivery are those possessing the greatest imbalance between the cross-sectional areas occupied by the polar and hydrophobic domains.

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Improved method for the removal of endotoxin from DNA.

Contaminating endotoxin in solutions used in gene therapy and genetic immunization can result in various deleterious effects both in vitro and in vivo. In order to avoid such complications, attempts were made to characterize the extent of the problem of endotoxin contamination and develop a solution to this problem. After screening for endotoxin in plasmid DNA preparations using the Limulus Amoebocyte Lysate (LAL) assay, nearly half of all samples displayed high endotoxin levels. Therefore, a simple one-step procedure was developed for the removal of endotoxin using a polymyxin B resin.

Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy.

Immunotherapy has been successfully used to treat some human malignancies, principally melanoma and renal cell carcinoma. Genetic-based cancer immunotherapies were proposed which prime T lymphocyte recognition of unique neo-antigens arising from specific mutations. Genetic immunization (polynucleotide vaccination, DNA vaccines) is a process whereby gene therapy methods are used to create vaccines and immunotherapies. Recent findings indicate that genetic immunization works indirectly via a bone marrow derived cell, probably a type of dendritic antigen presenting cell (APC). Direct targeting of genetic vaccines to these cells may provide an efficient method for stimulating cellular and humoral immune responses to infectious agents and tumor antigens. Initial studies have provided monocytic-derived dendritic cell (DC) isolation and culture techniques, simple methods for delivering genes into these cells, and have also uncovered potential obstacles to effective cancer immunotherapy which may restrict the utility of this paradigm to a subset of patients.

Cholesterol enhances cationic liposome-mediated DNA transfection of human respiratory epithelial cells.

Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphosphatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.

Residual and contact herbicide transport through field lysimeters via preferential flow.

Usage of glyphosate [N-(phosphonomethyl)-glycine] and glufosinate [2-amino-4-(hydroxy-methylphosphinyl)butanoic acid] may reduce the environmental impact of agriculture because they are more strongly sorbed to soil and may be less toxic than many of the residual herbicides they replace. Preferential flow complicates the picture, because due to this process, even strongly sorbed chemicals can move quickly to ground water. Therefore, four monolith lysimeters (8.1 m(2) by 2.4 m deep) were used to investigate leaching of contact and residual herbicides under a worst-case scenario. Glufosinate, atrazine (6-chloro-N(2)-ethyl-N(4)-isopropyl-1,3,5-triazine-2,4-diamine), alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl) acetamide], and linuron (3-3,4-dichlorophenyl-1-methoxy-1-methylurea) were applied in 1999 before corn (Zea mays L.) planting and glyphosate, alachlor, and metribuzin [4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one] were applied in 2000 before soybean [Glycine max (L.) Merr.] planting. A high-intensity rainfall was applied shortly after herbicide application both years. Most alachlor, metribuzin, atrazine, and linuron losses occurred within 1.1 d of rainfall initiation and the peak concentration of the herbicides coincided (within 0.1 d of rainfall initiation in 2000). More of the applied metribuzin leached compared with alachlor during the first 1.1 d after rainfall initiation (2.2% vs. 0.035%, P < 0.05). In 1999, 10 of 24 discrete samples contained atrazine above the maximum contaminant level (atrazine maximum contaminant level [MCL] = 3 mug L(-1)) while only one discrete sample contained glufosinate (19 mug L(-1), estimated MCL = 150 mug L(-1)). The results indicate that because of preferential flow, the breakthrough time of herbicides was independent of their sorptive properties but the transport amount was dependent on the herbicide properties. Even with preferential flow, glyphosate and glufosinate were not transported to 2.4 m at concentrations approaching environmental concern.

Cationic liposome-mediated RNA transfection.

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/lipofectin transfection procedure, we have analyzed the role of capping and beta-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with beta-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.

Specific inhibition of macrophage TNF-alpha expression by in vivo ribozyme treatment.

The overproduction of the cytokine TNF-alpha is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-alpha production with ribozymes directed against TNF-alpha mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-alpha ribozymes were more effective inhibitors of TNF-alpha secretion than catalytically inactive ribozyme controls. Inhibition of TNF-alpha secretion was proportional to the concentration of ribozyme administered, with an IC50 of approximately 10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated approximately 6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-alpha secretion by approximately 50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1alpha. Ribozyme-specific TNF-alpha mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.

Mucosal immune responses associated with polynucleotide vaccination.

A variety of gene delivery technologies can be used to express antigens within somatic tissues, resulting in systemic humoral and cellular immune responses. This observation has led to the development of polynucleotide vaccine preparations for stimulation of systemic immunity. Mucosal immune responses are functionally distinct from systemic immune responses, and are stimulated by antigen presentation within specialised mucosal-associated inductor tissues. We hypothesize that mucosal genetic vaccine will require gene transfer methods which target mucosal-associated inductor tissues such as the oropharyngeal Waldeyer's ring or intestinal Peyer's patches. We have tested this hypothesis by expressing a test antigen using a replication-defective recombinant Semliki Forest Virus (SFV) preparation. Mice treated with recombinant SFV via an intravascular or intratracheal route generated systemic immune responses against the test antigen. In contrast, intranasal inoculation resulted in the production of IgA within pulmonary fluids, one hallmark of a mucosal immune response. These results indicate that transfection of mucosal effector tissues may not be sufficient for the generation of a universal mucosal immune response. Furthermore, the results predict that techniques which target transfection or transduction to mucosal inductor tissues will enable the development of a new class of polynucleotide vaccines which exploit current concepts in mucosal immunology.