Genetic mitigation strategies to tackle agricultural GHG emissions: The case for biological nitrification inhibition technology.

Accelerated soil-nitrifier activity and rapid nitrification are the cause of declining nitrogen-use efficiency (NUE) and enhanced nitrous oxide (N2O) emissions from farming. Biological nitrification inhibition (BNI) is the ability of certain plant roots to suppress soil-nitrifier activity, through production and release of nitrification inhibitors. The power of phytochemicals with BNI-function needs to be harnessed to control soil-nitrifier activity and improve nitrogen-cycling in agricultural systems. Transformative biological technologies designed for genetic mitigation are needed, so that BNI-enabled crop-livestock and cropping systems can rein in soil-nitrifier activity, to help reduce greenhouse gas (GHG) emissions and globally make farming nitrogen efficient and less harmful to environment. This will reinforce the adaptation or mitigation impact of other climate-smart agriculture technologies.

Novel, Precise, Accurate Ion-Pairing Method to Determine the Related Substances of the Fondaparinux Sodium Drug Substance: Low-Molecular-Weight Heparin.

Fondaparinux sodium is a synthetic low-molecular-weight heparin (LMWH). This medication is an anticoagulant or a blood thinner, prescribed for the treatment of pulmonary embolism and prevention and treatment of deep vein thrombosis. Its determination in the presence of related impurities was studied and validated by a novel ion-pair HPLC method. The separation of the drug and its degradation products was achieved with the polymer-based PLRPs column (250 mm × 4.6 mm; 5 µm) in gradient elution mode. The mixture of 100 mM n-hexylamine and 100 mM acetic acid in water was used as buffer solution. Mobile phase A and mobile phase B were prepared by mixing the buffer and acetonitrile in the ratio of 90:10 (v/v) and 20:80 (v/v), respectively. Mobile phases were delivered in isocratic mode (2% B for 0-5 min) followed by gradient mode (2-85% B in 5-60 min). An Evaporative Light Scattering Detector (ELSD) was connected to the LC system to detect the responses of chromatographic separation. Further, the drug was subjected to stress studies for acidic, basic, oxidative, photolytic, and thermal degradations as per ICH guidelines and the drug was found to be labile in acid, base hydrolysis, and oxidation, while stable in neutral, thermal, and photolytic degradation conditions. The method provided linear responses over the concentration range of the LOQ to 0.30% for each impurity with respect to the analyte concentration of 12.5 mg/mL, and regression analysis showed a correlation coefficient value (r(2)) of more than 0.99 for all the impurities. The LOD and LOQ were found to be 1.4 µg/mL and 4.1 µg/mL, respectively, for fondaparinux. The developed ion-pair method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

LivestockPlus: Forages, sustainable intensification, and food security in the tropics.

The increased use of grain-based feed for livestock during the last two decades has contributed, along with other factors, to a rise in grain prices that has reduced human food security. This circumstance argues for feeding more forages to livestock, particularly in the tropics where many livestock are reared on small farms. Efforts to accomplish this end, referred to as the 'LivestockPlus' approach, intensify in sustainable ways the management of grasses, shrubs, trees, and animals. By decoupling the human food and livestock feed systems, these efforts would increase the resilience of the global food system. Effective LivestockPlus approaches take one of two forms: (1) simple improvements such as new forage varieties and animal management practices that spread from farmer to farmer by word of mouth, or (2) complex sets of new practices that integrate forage production more closely into farms' other agricultural activities and agro-ecologies.

Potential for biological nitrification inhibition to reduce nitrification and N2O emissions in pasture crop-livestock systems.

Agriculture and livestock production systems are two major emitters of greenhouse gases. Methane with a GWP (global warming potential) of 21, and nitrous oxide (N2O) with a GWP of 300, are largely emitted from animal production agriculture, where livestock production is based on pasture and feed grains. The principal biological processes involved in N2O emissions are nitrification and denitrification. Biological nitrification inhibition (BNI) is the natural ability of certain plant species to release nitrification inhibitors from their roots that suppress nitrifier activity, thus reducing soil nitrification and N2O emission. Recent methodological developments (e.g. bioluminescence assay to detect BNIs in plant root systems) have led to significant advances in our ability to quantify and characterize the BNI function. Synthesis and release of BNIs from plants is a highly regulated process triggered by the presence of NH4 + in the rhizosphere, which results in the inhibitor being released precisely where the majority of the soil-nitrifier population resides. Among the tropical pasture grasses, the BNI function is strongest (i.e. BNI capacity) in Brachiaria sp. Some feed-grain crops such as sorghum also have significant BNI capacity present in their root systems. The chemical identity of some of these BNIs has now been established, and their mode of inhibitory action on Nitrosomonas has been characterized. The ability of the BNI function in Brachiaria pastures to suppress N2O emissions and soil nitrification potential has been demonstrated; however, its potential role in controlling N2O emissions in agro-pastoral systems is under investigation. Here we present the current status of our understanding on how the BNI functions in Brachiaria pastures and feed-grain crops such as sorghum can be exploited both genetically and, from a production system's perspective, to develop low-nitrifying and low N2O-emitting production systems that would be economically profitable and ecologically sustainable.

A paradigm shift towards low-nitrifying production systems: the role of biological nitrification inhibition (BNI).

BACKGROUND: Agriculture is the single largest geo-engineering initiative that humans have initiated on planet Earth, largely through the introduction of unprecedented amounts of reactive nitrogen (N) into ecosystems. A major portion of this reactive N applied as fertilizer leaks into the environment in massive amounts, with cascading negative effects on ecosystem health and function. Natural ecosystems utilize many of the multiple pathways in the N cycle to regulate N flow. In contrast, the massive amounts of N currently applied to agricultural systems cycle primarily through the nitrification pathway, a single inefficient route that channels much of this reactive N into the environment. This is largely due to the rapid nitrifying soil environment of present-day agricultural systems. SCOPE: In this Viewpoint paper, the importance of regulating nitrification as a strategy to minimize N leakage and to improve N-use efficiency (NUE) in agricultural systems is highlighted. The ability to suppress soil nitrification by the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI), an active plant-mediated natural function that can limit the amount of N cycling via the nitrification pathway. The development of a bioassay using luminescent Nitrosomonas to quantify nitrification inhibitory activity from roots has facilitated the characterization of BNI function. Release of BNIs from roots is a tightly regulated physiological process, with extensive genetic variability found in selected crops and pasture grasses. Here, the current status of understanding of the BNI function is reviewed using Brachiaria forage grasses, wheat and sorghum to illustrate how BNI function can be utilized for achieving low-nitrifying agricultural systems. A fundamental shift towards ammonium (NH4(+))-dominated agricultural systems could be achieved by using crops and pastures with high BNI capacities. When viewed from an agricultural and environmental perspective, the BNI function in plants could potentially have a large influence on biogeochemical cycling and closure of the N loop in crop-livestock systems.

Evidence for biological nitrification inhibition in Brachiaria pastures.

Nitrification, a key process in the global nitrogen cycle that generates nitrate through microbial activity, may enhance losses of fertilizer nitrogen by leaching and denitrification. Certain plants can suppress soil-nitrification by releasing inhibitors from roots, a phenomenon termed biological nitrification inhibition (BNI). Here, we report the discovery of an effective nitrification inhibitor in the root-exudates of the tropical forage grass Brachiaria humidicola (Rendle) Schweick. Named "brachialactone," this inhibitor is a recently discovered cyclic diterpene with a unique 5-8-5-membered ring system and a gamma-lactone ring. It contributed 60-90% of the inhibitory activity released from the roots of this tropical grass. Unlike nitrapyrin (a synthetic nitrification inhibitor), which affects only the ammonia monooxygenase (AMO) pathway, brachialactone appears to block both AMO and hydroxylamine oxidoreductase enzymatic pathways in Nitrosomonas. Release of this inhibitor is a regulated plant function, triggered and sustained by the availability of ammonium (NH(4)(+)) in the root environment. Brachialactone release is restricted to those roots that are directly exposed to NH(4)(+). Within 3 years of establishment, Brachiaria pastures have suppressed soil nitrifier populations (determined as amoA genes; ammonia-oxidizing bacteria and ammonia-oxidizing archaea), along with nitrification and nitrous oxide emissions. These findings provide direct evidence for the existence and active regulation of a nitrification inhibitor (or inhibitors) release from tropical pasture root systems. Exploiting the BNI function could become a powerful strategy toward the development of low-nitrifying agronomic systems, benefiting both agriculture and the environment.

A global assessment using PCR techniques of mycorrhizal fungal populations colonising Tithonia diversifolia.

Tithonia diversifolia (Mexican sunflower) is a shrub commonly used as a green manure crop in Central and South America, Asia and Africa as it accumulates high levels of phosphorus and other nutrients, even in depleted soils. In root samples collected from the global distribution of Tithonia, we examined the degree of mycorrhizal colonisation and estimated the families of associated arbuscular mycorrhizal (AM) fungi. No colonisation by ectomycorrhizas was found. The degree of colonisation by AM fungi was on average 40%, but ranged between 0 and 80%. No mycorrhizal colonisation was found in the samples collected from the Philippines or in one each of the Rwandan and Venezuelan samples. Throughout its global distribution (Costa Rica, Nicaragua, Indonesia, Honduras, Mexico, Kenya and Rwanda), Tithonia forms mainly associations with Glomaceae. Only in one location in Nicaragua were associations with another family ( Acaulosporaceae) found.

The high level of aluminum resistance in signalgrass is not associated with known mechanisms of external aluminum detoxification in root apices.

Al resistance of signalgrass (Brachiaria decumbens Stapf cv Basilisk), a widely sown tropical forage grass, is outstanding compared with the closely related ruzigrass (Brachiaria ruziziensis Germain and Evrard cv Common) and Al-resistant genotypes of graminaceous crops such as wheat, triticale, and maize. Secretion of organic acids and phosphate by root apices and alkalinization of the apical rhizosphere are commonly believed to be important mechanisms of Al resistance. However, root apices of signalgrass secreted only moderately larger quantities of organic acids than did those of ruzigrass, and efflux from signalgrass apices was three to 30 times smaller than from apices of Al-resistant genotypes of buckwheat, maize, and wheat (all much more sensitive to Al than signalgrass). In the presence, but not absence, of Al, root apices of signalgrass alkalinized the rhizosphere more than did those of ruzigrass. The latter was associated with a shortening of the alkalinizing zone in Al-intoxicated apices of ruzigrass, indicating that differences in alkalinizing power were a consequence, not a cause of, differential Al resistance. These data indicate that the main mechanism of Al resistance in signalgrass does not involve external detoxification of Al. Therefore, highly effective resistance mechanisms based on different physiological strategies appear to operate in this species.

Roots of nutrient-deprived Brachiaria species accumulate 1,3-di-O-trans-feruloylquinic acid.

A novel di-hydroxycinnamoylquinic acid ester, 1,3-di-O-trans-feruloylquinic acid (DFQA), was isolated from roots of nutrient-deprived Brachiaria species--the most widely sown tropical forage grasses in South America. In contrast to other so far characterized quinic-acid esters, DFQA exists in a chair conformation with the carboxylic group in the axial orientation. It accumulates in older parts of the root system, but not in root apices or shoots. Higher levels were found in B. ruziziensis, which is poorly adapted to infertile acid soils, than in well adapted B. decumbens. DFQA was also found in the soil, most likely as a result of root decay, because it was not detected in root exudates of plants cultivated in solution culture. Nitrogen and phosphorus deficiency--but not aluminum toxicity or deprivation of other nutrients--stimulated its synthesis in roots. Its accumulation was correlated with a shift in biomass partitioning toward the root system.

Univentricular repair. Early and midterm results.

A total of 202 patients (62 with tricuspid atresia and 140 without tricuspid atresia) underwent univentricular repair at our unit from January 1990 to September 1994. Of these patients, 182 had nonfenestrated and 20 had fenestrated interatrial baffles. Early mortality was 15.9% (29/182) in the group with nonfenestrated baffles and 5% (1/20) in the group with fenestrated baffles. The follow-up period ranged from 2 to 58 months. Seven late deaths occurred, and five patients were lost to follow-up. Of 160 patients who have been evaluated in the outpatient department in the past 3 months, 142 (88.75%) required no cardiac medicines and were in functional class I. Risk factors analyzed for early mortality and significant effusion were age, preoperative diagnosis, type of Fontan modification, cardiopulmonary bypass time, aortic crossclamp time, pulmonary artery size, associated pulmonary arterioplasty, takedown of systemic-pulmonary artery shunt, and pulmonary artery debanding, along with the Fontan operation. Bypass time exceeding 120 minutes was associated with a higher early mortality (12/47 vs 18/155; p = 0.0187). Bypass time exceeding 120 minutes (p = 0.0456) and aortic crossclamp time exceeding 60 minutes (p = 0.0278) were associated with significant postoperative effusion. Other factors were not associated with any significantly increased risk for early mortality or postoperative effusions. Fenestration of the interatrial baffle appeared to decrease early mortality, although the numbers are too small to be statistically significant. The prevalence of effusions did not differ significantly between the group with fenestrated baffles and the group without fenestrated baffles.

Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet (IV. Changes with Time Following Increased Supply of Phosphate to Low-Phosphate Plants).

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).

Strategies for repair of congenital heart defects in infants without the use of blood.

Eleven infants and children with a body weight of less than 10 kg (median weight, 6.8 kg) whose parents were Jehovah's Witnesses underwent repair (n = 10) or palliation (n = 1) of congenital heart defects without the use of blood products and with (n = 9) or without (n = 2) cardiopulmonary bypass (CPB). In 1 neonate (weight, 3.2 kg) with critical aortic stenosis, moderate hypothermia and a 3.5-minute period of inflow occlusion and circulatory arrest allowed an aortic valvotomy; in another patient (weight, 7.0 kg) with tricuspid and pulmonary atresia, transposition of the great arteries, and persistent left superior vena cava, a bilateral bidirectional cavopulmonary shunt procedure was performed without CPB. Use of heparin-bonded tubing allowed reduction of the initial dose of heparin sodium to 1 mg/kg. Tissue perfusion and oxygenation on bypass were adequate, as evidenced by a mean lowest pH of 7.38 +/- 0.09 and a mean lowest venous oxygen tension of 65.0 +/- 36.2 mm Hg. Although the mean postoperative hematocrit (Hct) was lower than the mean preoperative Hct (p < 0.05, analysis of variance and Scheffe's F test), the Hct within 2 hours after CPB was restored to a value (mean Hct, 27.5% +/- 1.0%) between the preoperative Hct (mean value, 42.7% +/- 3.5%) and the lowest Hct on CPB (mean value, 18.4% +/- 1.4%). The Hct at discharge was 31.8% +/- 1.1%. The median postoperative blood loss was 9 mL/kg. There was no perioperative mortality. The median stay in the intensive care unit and the hospital was 2 days and 6 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Anterior pulmonary translocation for repair of truncus arteriosus with interrupted arch.

A newborn was found to have truncus arteriosus and an interrupted aortic arch, and underwent primary repair. The patient did well initially, but, by 8 weeks postoperatively, showed evidence of severe compression of the right pulmonary artery. At reoperation, the pulmonary artery was found to be compressed by a large aortic root (truncal root) and the retroaortic area was narrowed. The pulmonary artery bifurcation was therefore translocated anteriorly to alleviate the compression.

Characterization of progesterone biosynthesis in a transformed granulosa cell line.

The study of regulation of steroidogenesis in primary cultures of rat granulosa cells is difficult because the cells do not undergo more than one cell doubling in culture. Furthermore, there is size and steroidogenic heterogeneity in granulosa cells and it is difficult to obtain pure, functionally defined populations. Hence, it is advantageous to develop a homogeneous population of granulosa cells. In this report we describe the characterization of one such cell line (Rao-gcl-29) developed from diethylstilbestrol treated immature rat granulosa cells by transformation with SV40 T antigen. In this cell line cyclic AMP analogs induce high levels of progesterone biosynthesis, though there was no effect on estradiol biosynthesis. Also, FSH and hCG have no effect on progesterone biosynthesis. In the presence of FBS medium (20% fetal bovine serum in DMEM/F-12) and enriched medium (10% fetal bovine serum, 10% horse serum and 2% UltraSer G in DMEM/F-12 medium), 1 mM cAMP analogs induce high levels of progesterone biosynthesis up to 96 h. Ultrastructural features of the cell line resemble those of primary granulosa cells, in addition to forming gap junctions. Cyclic AMP analogs also induced cytochrome P450scc mRNA in this cell line by 48 h, and this effect is apparent by 24 h. Thus, this cell line could be useful in understanding the molecular mechanisms of regulation of cytochrome P450scc gene regulation.