Application of microcosm experiments for quantifying lateral flow and evapotranspiration on recovering bog ecotypes.

The importance of characterizing the ecohydrological interactions in natural, damaged/drained, and restored bogs is underscored by the importance of peatlands to global climate change and the growing need for peatland restoration. An understudied aspect of peatland ecohydrology is how shallow lateral flow impacts local hydrological conditions and water balance, which are critical for peatland restoration success. A novel method is presented using microcosms installed in the field to understand the dynamics of shallow lateral flow. Analysis of the difference in water table fluctuation inside and outside the microcosm experimental areas allowed the water balance to be constrained and the calculation of lateral flow and evapotranspiration. As an initial demonstration of this method, a series of four microcosm experiments were set up in locations with differing ecological quality and land management histories, on a raised bog complex in the midlands of Ireland. The timing and magnitude of the lateral flow differed considerably between locations with differing ecological conditions, indicating that shallow lateral flow is an important determining factor in the ecohydrological trajectory of a recovering bog system. For locations where Sphagnum spp. moss layer was present, a slow continuous net lateral input of water from the upstream catchment area supported the water table during drought periods, which was not observed in locations lacking Sphagnum. Consistent with other studies, evapotranspiration was greater in locations with a Spaghnum moss layer than in locations with a surface of peat soil.

Pubertal probiotic blocks LPS-induced anxiety and the associated neurochemical and microbial outcomes, in a sex dependent manner.

Puberty is a critical period of neural development, and exposure to stress and inflammation during this period is thought to increase vulnerability to mental illness. The gut microbiome influences brain functioning and behavior and impacts mental health. Yet, the role of the gut microbiome during puberty, a period during which mental health conditions tend to onset, remains largely uninvestigated. We first examined age and sex differences in gut microbial changes among CD-1 mice exposed to an immune challenge (lipopolysaccharide; LPS) at 6 weeks of age (during the pubertal stress-sensitive period) or at 10 weeks of age (in adulthood) (Experiment 1). Compared to their adult counterparts, pubertal males and females showed more significant changes in gut microbial composition following LPS treatment, including the depletion of numerous bacterial genera such as Lactobacillus. Given the beneficial effects of Lactobacillus strains on stress and behaviour, we next investigated whether replenishment of the gut with the probiotic Lactobacillus reuteri (L. reuteri) throughout pubertal development would modulate LPS-induced sickness and enduring effects on memory dysfunction, anxiety-like behaviour and stress reactivity in adulthood (Experiment 2). LPS treatment at 6 weeks of age created enduring changes in anxiety-like behaviors among males only. Similarly, only males showed the protective effects of L. reuteri supplementation during puberty in preventing longstanding LPS-induced changes in anxiety-like behavior and stress-induced brain activation. These findings demonstrate that colonizing the gut with L. reuteri during puberty modulates sickness responses and enduring behavioural and neurochemical outcomes in a sex-specific manner. Therefore, colonizing the gut with beneficial microbes may protect against the development of mental illnesses in adulthood.

Structural characterization of dissolved organic matter: a review of current techniques for isolation and analysis.

Natural dissolved organic matter (DOM) in aquatic systems plays many environmental roles: providing building blocks and energy for aquatic biota, acting as a sunscreen in surface water, and interacting with anthropogenic compounds to affect their ultimate fate in the environment. Such interactions are a function of DOM composition, which is difficult to ascertain due to its heterogeneity and the co-occurring matrix effects in most aquatic samples. This review focuses on current approaches to the chemical structural characterization of DOM, ranging from those applicable to bulk samples and in situ analyses (UV-visible spectrophotometry and fluorescence spectroscopy) through the concentration/isolation of DOM followed by the application of one or more analytical techniques, to the detailed separation and analysis of individual compounds or compound classes. Also provided is a brief overview of the main techniques used to characterize isolated DOM: mass spectrometry (MS), nuclear magnetic resonance mass spectrometry (NMR) and Fourier transform infrared spectroscopy (FTIR).

Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation.

An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling ¹D peaks. Transfer, transient storage, and subsequent second dimension (²D) separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Using existing valve technology we find that the approach is repeatable (%RSD of peak area <1.5%), even at very short first dimension sampling times--as low as 1s. We have also systematically studied the critical influence of the volume and composition of fractions transferred from the first to the second dimension of the sLC × LC system with reversed-phase columns in both dimensions, and the second dimension operated isocratically. We find that dilution of the transferred fraction, so that it contains 10-20% less organic solvent than the ²D eluent, generally mitigates the devastating effects of large transfer volumes on ²D performance in this type of system. Several example applications of the sLC × LC approach are described in the second part of this two-part series. We anticipate that future advances in the valve technology used here will significantly widen the scope of possible applications of the sLC × LC approach.

Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: applications.

In this second paper of a two-part series, we demonstrate the utility of an approach to enhancing the resolution of select portions of conventional 1D-LC separations, which we refer to as selective comprehensive two-dimensional HPLC (sLC × LC), in three quite different example applications. In the first paper of the series we described the principles of this approach, which breaks the long-standing link in online multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. In the first example, the power of the sLC × LC approach to significantly reduce the analysis time and method development effort is demonstrated by selectively enhancing the resolution of critical pairs of peaks that are unresolved by a one-dimensional separation (1D-LC) alone. Transfer and subsequent ²D separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peaks in the 2D separation space. The added time dimension of sLC × LC chromatograms also facilitates the application of sophisticated chemometric curve resolution algorithms to further resolve peaks that are otherwise chromatographically unresolved. This is demonstrated in this work by the targeted analysis of phenytoin in urban wastewater effluent using UV diode array detection. Quantitation by both standard addition and external calibration methods yielded results that were not statistically different from 2D-LC/MS/MS analysis of the same samples. Next, we demonstrate the utility of sLC × LC for reducing ion suppression due to matrix effects in electrospray ionization mass spectrometry through the analysis of cocaine in urban wastewater effluent. Finally, we explore the flexibility of the approach in its application to two select regions of a single ¹D separation of triclosan and cocaine. The diversity of these applications demonstrates the power and versatility of the sLC × LC approach, which will benefit tremendously from further optimization and advances in valve technology that specifically address the needs of this new technique.

1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum.

We report the (1)H, (13)C, and (15)N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, isolated from the acidophilic gram-negative facultatively anaerobic metal-reducing alphaproteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c (2) and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on (13)C- and (15)N- labeled substrates.

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices.

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

A timeline for predicting durable medical equipment needs and interventions for amyotrophic lateral sclerosis patients.

ALS is progressive with increasing patient needs for durable medical equipment (DME) and interventions (gastric feeding tube - PEG, and non-invasive ventilation - NIV). We performed a chart review of deceased patients to determine the time-course of needs and their estimated costs. A timeline of needs was based on when clinic personnel felt an item was necessary. The point in time when an item or intervention was needed was expressed as a percentage of a patient's total disease duration. A wide range of DME and interventions was needed irrespective of site of ALS symptom onset (bulbar, upper, lower extremity), beginning at 10% of disease duration of lower extremity onset and increasing thereafter for all sites. The cumulative probability of costs of items and interventions began at 25%-50% of disease duration and increased to between $18,000 and $32,000 (USD), highest for lower extremity onset due to the cost of wheelchairs. We conclude that a high percentage of ALS patients will need a full spectrum of major DME items and interventions during the second half of disease duration. This results in a linear rise in costs over the second half of the disease duration.

Alaska sentinel surveillance for antimicrobial resistance in Helicobacter pylori isolates from Alaska native persons, 1999-2003.

BACKGROUND: Previous studies in Alaska have demonstrated elevated proportions of antimicrobial resistance among Helicobacter pylori isolates. MATERIALS AND METHODS: We analyzed H. pylori data from the Centers for Disease Control and Prevention (CDC)'s sentinel surveillance in Alaska from July 1999 to June 2003 to determine the proportion of culture-positive biopsies from Alaska Native persons undergoing routine upper-endoscopy, and the susceptibility of H. pylori isolates to metronidazole [minimum inhibitory concentration (MIC) of > 8 g metronidazole/mL), clarithromycin (MIC > or = 1), tetracycline (MIC > or = 2) and amoxicillin (MIC > or = 1)] using agar dilution. RESULTS: Nine-hundred sixty-four biopsy specimens were obtained from 687 participants; 352 (51%) patients tested culture positive. Mean age of both culture-positive and culture-negative patients was 51 years. Metronidazole resistance was demonstrated in isolates from 155 (44%) persons, clarithromycin resistance from 108 (31%) persons, amoxicillin resistance from 8 (2%) persons, and 0 for tetracycline resistance. Metronidazole and clarithromycin resistance varied by geographic region. Female patients were more likely than male subjects to show metronidazole resistance (p < .01) and clarithromycin resistance (p = .05). CONCLUSIONS: Resistance to metronidazole and clarithromycin is more common among H. pylori isolates from Alaska Native persons when compared with those from elsewhere in the USA.

Recruitment and community interactions in the GOCADAN study.

OBJECTIVES: To study heart and vascular disease in Alaskan Eskimos. To identify risk factors for CVD in Norton Sound Eskimos. STUDY DESIGN: Participatory research. In this paper, procedures for selection and enrollment and providing feedback and referrals are described. Our working relationships with the Norton Sound Health Corporation (NSHC) Board, the village councils, individuals, and communities are also described. METHODS: This study was conducted in the Norton Sound region of Alaska. The participants were members of Alaskan Eskimo families. RESULTS: Procedures were formed for selecting and enrolling extended families into the study and for working with the NSHC Board, the village councils, and individual participants. The average participation was 82.6% of the age-eligible villagers in seven villages. A four-level referral system was designed. Test results were provided to participants in the form of letters, with duplicates sent to health care providers and medical records. A senior researcher returned to the village to explain the results to the participants. CONCLUSIONS: Principles of participatory research applied and developed in this study led to successful screening of 1214 Eskimos in nine villages between October 2000 and June 2004. This partnership developed into a relationship with the community, in which researchers and the communities mutually participated in the study, from the initiation of the design to the return of the data to the individuals, communities, and health care providers.

Cardiovascular disease and risk factors in three Alaskan Eskimo populations: the Alaska-Siberia project.

OBJECTIVES: To determine the prevalence of CVD and to identify and characterize associated risk factors in three distinct Eskimo populations. STUDY DESIGN: Cross-sectional. METHODS: A slightly modified Strong Heart Study protocol was followed to examine 454 participants, aged 25-91, from four villages. RESULTS: Overall, 6% of the participants under 55 years of age and 26% of those > or = 55 years of age showed evidence of CHD by ECG, or in patient records. The prevalence of "definite coronary heart disease" (CHD) in women with glucose intolerance (GI) was 21.0%, compared to 2.4% in those with normal glucose tolerance (NGT). Men had comparable values of 26.7% and 6.3%. In addition, comparable values for "possible CHD" were 29.7% vs 6.0% for women and 21.4% vs 8.0% for men. GI was associated with relatively higher prevalences of CHD in women than in men (prevalence ratio = 8.5 vs 4.3). CHD was significantly related to age, glucose intolerance and insulin. Hypertension and obesity were significantly associated with CHD only in some ethnic groups. The prevalence of current smokers was 56%. CONCLUSIONS: Recent changes in lifestyle and diet of Alaskan Eskimos, leading to obesity, hypertension, insulin resistance and DM, contribute to an increased risk for cardiovascular disease.

A successful diabetes prevention study in Eskimos: the Alaska Siberia project.

OBJECTIVES: To test the efficacy of a simple intervention method to reduce risk factors for type 2 diabetes (DM) and cardiovascular disease (CVD) in Alaskan Eskimos. STUDY DESIGN: The study consisted of 1) a comprehensive screening for risk factors of 454 individuals in 4 villages, 2) a 4-year intervention and 3) a repetition of the screening in year 5 to test the efficacy of the intervention. METHODS: Personal counseling (1hr/year) stressed the consumption of more traditional foods high in omega-3 fatty acids and less of certain specific store-bought foods high in palmitic acid, which was identified as being associated with glucose intolerance. RESULTS: The intervention resulted in significant reductions in plasma concentrations of total cholesterol (p = 0.0001), LDL cholesterol (p = 0.0001), fasting glucose (p = 0.0001), diastolic blood pressure (p = 0.0007) and improved glucose tolerance (p = 0.0006). This occurred without loss of body weight. Sixty percent of the participants had improved glucose tolerance; only one of the 44 originally identified with impaired glucose tolerance (IGT) developed DM during the study. CONCLUSIONS: Dramatic improvements of risk factors for DM and CVD were achieved in the intervention by primarily stressing the need for changes in the consumption of specific fats. The results suggest that fat consumption is an important risk factor for DM.

A genetic and epidemiologic study of cardiovascular disease in Alaska natives (GOCADAN): design and methods.

This article is a report of the design and methods of the Genetics of Coronary Artery Disease in Alaska Natives (GOCADAN) Study. This longitudinal, population-based study was initiated to investigate the genetic determinants of cardiovascular disease and its risk factors. Between October 2000 and April 2004, this family study enrolled 1,214 Eskimos from several coastal villages in the Norton Sound region of Western Alaska. Examinations included a physical, laboratory determinations, and measures of subclinical disease. This study will generate a genome-wide scan for loci influencing cardiovascular disease-related traits. Relations between subclinical atherosclerosis and markers of inflammation will be examined using historic and newly drawn samples. The study will provide data on CVD prevalence, risk factors and the relative contribution of genetic and environmental determinants in Alaska Native peoples. Data from this study will contribute to the delivery of health-care and prevention of CVD in Alaska Eskimos and other populations.

The electronic record as infrastructure.

An electronic record will be a significant component of a mental health agency's infrastructure in the future. It is a convenient, safe, time-saving way of managing records in larger systems. This paper describes an electronic record system and its advantages and disadvantages at a large, private, non-profit mental health agency.