First Real-Life Data on the Diving Response in Healthy Children.

Swimming and diving are popular recreational activities, representing an effective option in maintaining and improving cardiovascular fitness in healthy people. To date, only little is known about the cardiovascular adaption to submersion in children. This study was conducted to improve an understanding thereof. We used a stepwise apnea protocol with apnea at rest, apnea with facial immersion, and at last apnea during whole body submersion. Continuous measurement of heart rate, oxygen saturation, and peripheral resistance index was done. Physiologic data and analysis of influencing factors on heart rate, oxygen saturation, and peripheral vascular tone response are reported. The current study presents the first data of physiologic diving response in children. Data showed that facial or whole body submersion leads to a major drop in heart rate, and increase of peripheral resistance, while the oxygen saturation seems to be unaffected by static apnea in most children, with apnea times of up to 75 s without change in oxygen saturation.

The Brassicaceae Family Displays Divergent, Shoot-Skewed NLR Resistance Gene Expression.

Nucleotide-binding site leucine-rich repeat resistance genes (NLRs) allow plants to detect microbial effectors. We hypothesized that NLR expression patterns could reflect organ-specific differences in effector challenge and tested this by carrying out a meta-analysis of expression data for 1,235 NLRs from nine plant species. We found stable NLR root/shoot expression ratios within species, suggesting organ-specific hardwiring of NLR expression patterns in anticipation of distinct challenges. Most monocot and dicot plant species preferentially expressed NLRs in roots. In contrast, Brassicaceae species, including oilseed rape (Brassica napus) and the model plant Arabidopsis (Arabidopsis thaliana), were unique in showing NLR expression skewed toward the shoot across multiple phylogenetically distinct groups of NLRs. The Brassicaceae are also outliers in the sense that they have lost the common symbiosis signaling pathway, which enables intracellular infection by root symbionts. While it is unclear if these two events are related, the NLR expression shift identified here suggests that the Brassicaceae may have evolved unique pattern-recognition receptors and antimicrobial root metabolites to substitute for NLR protection. Such innovations in root protection could potentially be exploited in crop rotation schemes or for enhancing root defense systems of non-Brassicaceae crops.

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

Retromer contributes to immunity-associated cell death in Arabidopsis.

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.

Autophagy deficiency leads to accumulation of ubiquitinated proteins, ER stress, and cell death in Arabidopsis.

Autophagy is a homeostatic degradation and recycling process that is also involved in defense against microbial pathogens and in certain forms of cellular suicide. Autophagy has been proposed to negatively regulate plant immunity-associated cell death related to the hypersensitive response (HR), as older autophagy-deficient mutants are unable to contain this type of cell death 5 to 10 d after infection. Such spreading cell death was found to require NPR1 (nonexpressor of PR genes 1), but surprisingly did not occur in younger atg mutants. In contrast, we find that npr1 mutants are not impaired in rapid programmed cell death activation upon pathogen recognition. Furthermore, our molecular evidence suggests that the NPR1-dependent spreading cell death in older atg mutants may originate from an inability to cope with excessive accumulation of ubiquitinated proteins and ER stress which derive from salicylic acid (SA)-dependent signaling (e.g., systemic acquired resistance). We also demonstrate that both senescence and immunity-related cell death seen in older atg mutants can be recapitulated in younger atg mutants primed with ER stress. We therefore propose that the reduction in SA signaling caused by npr1 loss-of-function is sufficient to alleviate the stress levels accumulated during aging in autophagy deficient cells which would otherwise become insurmountable and lead to uncontrolled cell death.

Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1-phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, "sandwich" topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily.

Preservation and analytical procedures for the analysis of chloro-s-triazines and their chlorodegradate products in drinking waters using direct injection liquid chromatography tandem mass spectrometry.

A direct injection, liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the analysis of the chloro-s-triazine herbicides and their degradates in finished drinking water. The target compounds in the method were selected based on their inclusion in a common mechanism group (CMG) because of their ability to induce a similar toxic effect through a common mechanism of toxicity. The target list includes the chloro-s-triazines (atrazine, simazine, cyanazine, and propazine) and their dealkylated degradates (desethylatrazine, desisopropylatrazine, and diaminochlorotriazine). Potential matrix effects are minimized by the use of individual isotopically enriched internal standards. Analyte stability in finished chlorinated drinking water samples is ensured through careful selection of proper dechlorinating and antimicrobial reagents and through buffering sample pH. In the absence of proper dechlorination, the target analytes were found to degrade over a short period of time, even under refrigerated storage conditions. The final method has adequate sensitivity to accurately detect all target analytes at or below 0.1 microg/L and displays sufficient precision and robustness to warrant publication as EPA Method 536.

An improved colorimetric method for chlorine dioxide and chlorite ion in drinking water using lissamine green B and horseradish peroxidase.

Lissamine Green B (LGB) was carefully selected as a potential candidate for the development of a new U.S. Environmental Protection Agency (EPA) method that is intended for use at water utilities to determine chlorine dioxide (ClO2) in drinking water. Chlorine dioxide reacts with LGB in aqueous solution to decrease the absorbance of LGB in direct proportion to the ClO2 concentration. LGB was confirmed to have adequate sensitivity, and to suffer less interference than other dyes reported in the literature. The stoichiometry for the reaction between LGB and ClO2 was found not to be 1:1 and is dependent on the LGB concentration. This required calibration of each LGB stock solution and prompted the investigation of alternate means of calibration, which utilized a horseradish peroxidase (HRP)-catalyzed conversion of chlorite ion (ClO2(-)) to ClO2. This approach allowed the simultaneous determination of ClO2(-) concentration, which is also required each day at water plants that use ClO2. Studies were conducted to characterize and carefully optimize the HRP-conversion of ClO2(-) to ClO2 in order to yield reaction conditions that could be accomplished in less than 30 min at modest cost, yet meet EPA's sensitivity and robustness requirements for routine monitoring. An assessment of method detection limit, linearity and slope (or sensitivity), precision, and accuracy in finished drinking water matrices indicated that this approach was suitable for publication as EPA Method 327.0.

Statistical procedures for determination and verification of minimum reporting levels for drinking water methods.

The United States Environmental Protection Agency's Office of Ground Water and Drinking Water has developed a single-laboratory quantitation procedure: the lowest concentration minimum reporting level (LCMRL). The LCMRL is the lowest true concentration for which future recovery is predicted to fall, with high confidence (99%), between 50% and 150%. The procedure takes into account precision and accuracy. Multiple concentration replicates are processed through the entire analytical method and the data are plotted as measured sample concentration (y-axis) versus true concentration (x-axis). If the data support an assumption of constant variance over the concentration range, an ordinary least-squares regression line is drawn; otherwise, a variance-weighted least-squares regression is used. Prediction interval lines of 99% confidence are drawn about the regression. At the points where the prediction interval lines intersect with data quality objective lines of 50% and 150% recovery, lines are dropped to the x-axis. The higher of the two values is the LCMRL. The LCMRL procedure is flexible because the data quality objectives (50-150%) and the prediction interval confidence (99%) can be varied to suit program needs. The LCMRL determination is performed during method development only. A simpler procedure for verification of data quality objectives at a given minimum reporting level (MRL) is also presented. The verification procedure requires a single set of seven samples taken through the entire method procedure. If the calculated prediction interval is contained within data quality recovery limits (50-150%), the laboratory performance at the MRL is verified.

Impact of methanol and acetonitrile on separations based on pi-pi interactions with a reversed-phase phenyl column.

Studies were performed to investigate the roles of methanol and acetonitrile on the retention mechanism of an active pharmaceutical ingredient (API) and related compounds with a reversed phase phenyl column. Different retention orders were observed depending upon whether acetonitrile or methanol was used as the organic modifier. We propose that acetonitrile impedes the selective pi-pi interactions between the analyte molecules and the phenyl groups in the stationary phase. Further study with 1-naphthoic acid and 1-naphthol as test compounds in the HPLC separation provides additional support for the influence of acetonitrile on pi-pi interactions between analyte molecules and a phenyl stationary phase. This study suggests that methanol be used as the preferred organic modifier with phenyl columns to achieve selectivity based upon pi-pi interactions.

Development of U.S. EPA method 527 for the analysis of selected pesticides and flame retardants in the UCMR survey.

Method 527 was developed to address the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) under its second unregulated contaminant monitoring rule (UCMR 2). This method includes a wide range of semivolatile organic contaminants, including pesticides that were deferred during the first UCMR, flame retardants, and pyrethroid pesticides. This paper discusses the rationale for selection and inclusion of the various contaminants included in Method 527 and describes the challenges associated with developing analytical methods that will be used for the occurrence monitoring of such a diverse group of organic molecules. Method 527 employs solid-phase extraction with analysis by gas chromatography/ mass spectrometry (GC/MS). The final method preservation scheme requires the storage of samples in amber bottles buffered at pH 3.8 using citric acid to prevent degradation from acid-catalyzed hydrolysis and from UV light. Citric acid is also an effective antimicrobial reagent, preventing this mode of loss during storage. Ethylenediaminetetraacetic acid (EDTA) is added to remove transition metals such as copper, which was determined to degrade target analytes upon storage. Finally, free available chlorine (FAC), which is present in many finished waters and found to degrade a number of the targets, is removed using ascorbic acid. The final method meets all of the EPA UCMR survey requirements for sample storage, precision, accuracy, and sensitivity and will be proposed for use under the UCMR 2.

Measurement of perchlorate in water by use of an 18O-enriched isotopic standard and ion chromatography with mass spectrometric detection.

Currently, the most promising analytical methodology for low-level determination and confirmation of perchlorate (ClO4-) in drinking water is ion chromatography followed by electrospray ionization mass spectrometric detection (IC-ESI-MS). However, there are still potentially limiting situations that must be considered when analyzing real world samples by IC-ESI-MS. They are: (1) co-elution of contaminants with fragment ions having the same m/z as ClO4-, (2) high background counts at the m/z of interest leading to a subsequent decrease in signal-to-noise, (3) gradual loss of sensitivity occurring over time as the sampling cone and/or ion optics of the mass spectrometer are fouled, and (4) suppression of gas phase ion formation (ionization suppression) that can occur if high concentrations of contaminants co-elute with ClO4-. An internal standard whose response, on the column and in the electrospray, is similar to that of ClO4- would give the highest degree of accuracy possible in situations 3 and 4 listed above. Commercially available NaClO4 that was enriched with oxygen-18 was evaluated for its potential as an internal standard. The level of oxygen-18 (18O) enrichment was verified, the stability of the enriched ClO4- in aqueous solutions was determined, and method performance parameters such as method detection limits, accuracy, precision and ruggedness using the enriched ClO4 as an internal standard were determined. Internal and external calibration yielded similar results with respect to calibration, detection limits, and short-term precision. However, the enriched isotopic internal standard showed superior results with respect to ruggedness and percent recovery of spikes in ground water and synthetic water samples.

Challenges encountered in extending the sensitivity of US Environmental Protection Agency Method 314.0 for perchlorate in drinking water.

Concerns about the potential adverse health effects of perchlorate at concentrations below the minimum reporting level (MRL) of US Environmental Protection Agency (EPA) Method 314.0 (generally recognized as 4.0 microg/l) have led to an interest in increasing the sensitivity of the method. This work describes the use of 2 mm columns with a large-loop direct injection method, a column concentration technique and this concentration technique with a background reduction step, to increase the sensitivity for the analysis of trace levels of perchlorate in high ionic strength matrices. The concentrator columns studied were the Dionex TAC LP-1 and a new Dionex high capacity Cryptand concentrator column. The use of a surrogate to monitor trapping efficiency for the concentration technique and the use of confirmational columns to minimize the potential for false positives are also discussed. The large-loop direct injection method and the column concentration methods provided acceptable data when the samples were pre-treated with solid phase pretreatment cartridges. The background reduction technique did not provide acceptable data with either of the concentrator columns evaluated.

Optimizing the determination of haloacetic acids in drinking waters.

Three methods are currently approved by the US Environmental Protection Agency for the compliance monitoring of haloacetic acids in drinking waters. Each derivatizes the acids to their corresponding esters using either acidic methanol or diazomethane. This study was undertaken to characterize the extent of methylation of these analytes by these methods, and to fully optimize methylation chemistries to improve analytical sensitivity, precision and accuracy. The approved methods were shown to have little to no esterification efficiencies for the brominated trihaloacetic acids (HAA3). Methylation with acidic methanol was determined to be more efficient and rugged than methylation with diazomethane. A new higher boiling solvent, tertiary-amyl methyl ether, is reported which has significantly improved methylation efficiencies for HAA3. Additional modifications to the method have been made that improve method ruggedness. The revised method, EPA Method 552.3, outperforms the currently approved methods, especially for HAA3.

Validating sample preservation techniques and holding times for the approved compliance monitoring methods for haloacetic acids under the US EPA's stage 1 D/DBP rule.

Haloacetic acids (HAAs), which are formed during the disinfection of drinking waters with chlorine, are regulated by the US Environmental Protection Agency (EPA) under the Stage 1 Disinfectant/Disinfection Byproducts (D/DBP) Rule. Recently, three studies have been reported indicating that low concentrations of HAAs can also be formed during disinfection with chloramines. Methods currently approved for compliance monitoring under the Stage 1 Rule arrest the chlorine-mediated formation of HAAs by adding ammonium chloride, which forms chloramines. Studies were undertaken using an in-process water that favored the formation of HAAs with moderate total organic carbon concentration and high levels of chlorine to investigate the potential formation of HAAs under sample storage conditions. The ammonium chloride-quenched sample did form a small amount of HAAs, but total formation over a period equal to the 14-day sample storage time was less than 2 microg/l, whereas the unquenched samples increased 41 microg/l during the same period. Pour plate studies indicated that chlorinated drinking waters quenched with ammonium chloride are protected from microbial growth, which is an important additional advantage to this preservation scheme. The presence of a combined chlorine residual should prevent microbial degradation of HAAs in samples. These studies support the preservation protocols and the sample storage times promulgated for compliance monitoring under the Stage 1 D/DBP Rule.

Improving the performance of US Environmental Protection Agency Method 300.1 for monitoring drinking water compliance.

In 1998, the United States Environmental Protection Agency (EPA) promulgated the maximum contaminant level (MCL) for bromate in drinking water at 10 microg/l, and the method for compliance monitoring of bromate in drinking water was established under Stage 1 of the Disinfectants/Disinfection By-Products Rule (D/DBP) as EPA Method 300.1. In January 2002, the United States Food and Drug Administration (FDA) regulated the bromate concentration in bottled waters at 10 microg/l. EPA anticipates proposing additional methods, which have improved performance for bromate monitoring, in addition to EPA Method 300.1, in the Stage 2 DBP Rule. Until the Stage 2 Rule is promulgated, EPA Method 300.1 will continue to be the only method approved for compliance monitoring of bromate. This manuscript describes the work completed at EPA's Technical Support Center (TSC) to assess the performance of recently developed suppressor technologies toward improving the trace level performance of EPA Method 300.1, specifically for the analysis of trace levels of bromate in high ionic matrices. Three different types of Dionex suppressors were evaluated. The baseline noise, return to baseline after the water dip, detection limits, precision and accuracy, and advantages/disadvantages of each suppressor are discussed. Performance data for the three different suppressors indicates that chemical suppression of the eluent, using the AMMS III suppressor, is the most effective means to reduce baseline noise, resulting in the best resolution and the lowest bromate detection limits, even when a high ionic matrix is analyzed. Incorporation of the AMMS III suppressor improves the performance of EPA Method 300.1 at and below 5.0 microg/l and is a quick way for laboratories to improve their bromate compliance monitoring.

Improvements to EPA method 531.1 for the analysis of carbamates that resulted in the development of U.S. EPA Method 531.2.

This project is undertaken to fully optimize the U.S. Environmental Protection Agency Method 531.1 post-column chemistries and to incorporate recent advances in liquid chromatographic separation, post-column derivatization, and detection techniques. Sample preservation and storage stability studies establish citric acid as a suitable replacement for the caustic monochloroacetic acid in the current method and confirm its antimicrobial effectiveness. Performance of an alternate set of commercially available post-column reagents is also investigated. This research has resulted in the publication of Method 531.2, a high-performance liquid chromatographic direct injection method for the analysis of N-methylcarbamoyloximes and N-methylcarbamates using post-column derivatization and fluorescence detection.

US Environmental Protection Agency Method 326.0, a new method for monitoring inorganic oxyhalides and optimization of the postcolumn derivatization for the selective determination of trace levels of bromate.

The development of US Environmental Protection Agency (EPA) Method 317.0 provided a more sensitive, acceptable alternative to EPA Method 300.1 to be proposed as one of the recommended compliance monitoring methods for Stage II of the Disinfectants/Disinfection By-Products (DBP) Rule. This work was initiated to evaluate other postcolumn reagents (PCRs) that might be utilized to provide an additional, alternative method in order to augment compliance monitoring flexibility for inorganic oxyhalide DBP anions. Modifications of the method reported by Salhi and von Gunten, which included adjustment and optimization of flow-rates, reaction temperature, and delivery of the PCR, improved the method performance. Method 326.0 incorporates an acidic solution of potassium iodide containing catalytic amounts of molybdenum(VI) as the PCR and provides acceptable precision and accuracy for all analytes and a postcolumn bromate detection limit in reagent water of 0.17 microg/l.

The application of tris buffer and copper sulfate for the preservation of phenylurea pesticides analyzed using U.S. EPA method 532 in the UCMR Survey.

A high performance liquid chromatographic method was developed to meetthe U.S. Environmental Protection Agency's (EPA) Unregulated Contaminant Monitoring Rule (UCMR) Survey need for the analysis of phenylurea pesticides in drinking waters. Many of these phenylurea compounds were demonstrated to degrade rapidly in the presence of the residual chlorine disinfectant in drinking waters. This degradation was halted by the addition of a tris buffer, which was initially chosen to optimize the sample pH prior to extraction. Copper sulfate was found to prevent the regrowth of microorganisms in surface waters, which was observed upon dechlorination. Tris buffer provided the additional benefit of keeping the copper sulfate preservative in solution even in groundwater matrices that caused precipitation of copper in its absence. A C18 solid phase, in cartridge or disk form, was used to efficiently extract target compounds from the preserved drinking water matrices. A 21-day storage stability study, together with precision and accuracy studies, demonstrated thatthis method had suitable sensitivity, selectivity, accuracy, precision, and ruggedness for use in the EPA's UCMR drinking water occurrence survey.