Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

Predicting organic acid concentration from UV/vis spectrometry measurements - A comparison of machine learning techniques

The concentration of organic acids in anaerobic digesters is one of the most critical parameters for monitoring and advanced control of anaerobic digestion processes. Thus, a reliable online-measurement system is absolutely necessary. A novel approach to obtaining these measurements indirectly and online using UV/vis spectroscopic probes, in conjunction with powerful pattern recognition methods, is presented in this paper. An UV/vis spectroscopic probe from S::CAN is used in combination with a custom-built dilution system to monitor the absorption of fully fermented sludge at a spectrum from 200 to 750 nm. Advanced pattern recognition methods are then used to map the non-linear relationship between measured absorption spectra to laboratory measurements of organic acid concentrations. Linear discriminant analysis, generalized discriminant analysis (GerDA), support vector machines (SVM), relevance vector machines, random forest and neural networks are investigated for this purpose and their performance compared. To validate the approach, online measurements have been taken at a full-scale 1.3-MW industrial biogas plant. Results show that whereas some of the methods considered do not yield satisfactory results, accurate prediction of organic acid concentration ranges can be obtained with both GerDA and SVM-based classifiers, with classification rates in excess of 87% achieved on test data.

A comparison of nitogen fate and transport in catchments underlain by high and low permeability aquifers using chemical and isotopic tools.

Variability in nitrogen fate and transport in different catchments types is often not considered. This research considers the importance of such nitrogen processes within groundwater pathways in two agricultural catchments in Ireland; a well drained catchment, underlain by karstified Carboniferous limestone, and a poorly drained catchment, underlain by Silurian greywacke.
Depth specific low-flow groundwater sampling was used to evaluate the hydrochemical stratification in groundwater. Groundwater samples, as well as surface water samples, along river courses were analysed for nitrogen species (NO3, NH4 and NO2) and nitrate isotopes (d15N and d18O) as well as field parameters and major ions
.
The dominant nitrate (NO3) groundwater pathway in the poorly drained greywacke catchment is through the shallow weathered bedrock, as indicated by transmissivity values and the ionic and isotopic signatures, and a clear reduction in NO3 concentration is observed with depth. A similar chloride trend would suggest dilution is a major factor, however d15N and d18O isotopic values producing an enrichment ratio of 1.8 indicate that denitrification is also an important process involved in the fate of the NO3 within the groundwater flow system. This consistent trend with depth is in contrast to the stratification pattern observed in the karstified catchment. NO3 was not detected in the shallow groundwater pathway; the dominant groundwater pathway is in the deeper groundwater where there is little change in the nitrate isotope values with depth (d15N values range between 4.1 and 4.6 ‰). This deeper groundwater contributes the dominant proportion of the river flow through a number of springs. As a result, the deeper groundwater, springs and river have a similar ionic signature and NO3 concentration range (23 ± 3 mg/l). Despite this pattern, the NO3 isotopes show a distinct difference in isotopic values between the deeper groundwater in the diffuse karst and the springs indicating some denitrification is occurring during groundwater discharge into the river. Furthermore the isotopes give an indication of the variability of the spatial extent of the springs and the complexities of the fissures through which they are fed. The results of this study clearly show the importance of the geology in the fate and transport of NO3 in agricultural catchments.

Associations between maternal characteristics and pregnancy-related stress among low-risk mothers: An observational cross-sectional study

Background: Pregnancy is viewed as a major life event and, while the majority of healthy, low-risk women adapt well to pregnancy, there are those whose levels of stress are heightened by the experience.

Objectives: To determine the level of pregnancy-related stress experienced by a group of healthy, low-risk pregnant women and to relate the level of stress with a number of maternal characteristics.

Design: An observational cross-sectional study.

Setting: A large, urban maternity centre in Northern Ireland.

Participants: Of the 306 pregnant women who were invited to participate, 278 provided informed consent and were administered one self-complete questionnaire. Due to the withdrawal criteria, 15 questionnaires were removed from the analysis, resulting in a final sample of 263 healthy, low-risk pregnant women.

Methods: Levels of stress were measured using a self-report measure designed to assess specific worries and concerns relating to pregnancy. Maternal characteristics collected included age, marital status, social status, parity, obstetric history, perceived health status and 'wantedness' for the pregnancy. Regression analysis was undertaken using an ordinary linear regression model.

Results: The mean prenatal distress score in the sample was 15.1 (SD = 7.4; range 0-46). The regression model showed that women who had had previous pregnancies, with or without complications, had significantly lower mean prenatal distress scores than primiparous women (p < 0.01). Women reporting poorer physical health had higher mean prenatal distress scores than those who reported at least average health, while women aged 16-20 experienced a mean increase in the reported prenatal distress score (p < 0.05) in comparison to the reference group of 36 years and over.

Conclusions: This study brings to light the prevalence of pregnancy-related stress within a sample representative of healthy, low-risk women. Current antenatal care is ill-equipped to identify women suffering from high levels of stress; yet a growing body of research evidence links stress with adverse pregnancy outcomes. This study emphasises that healthy, low-risk women experience a range of pregnancy-related stress and identification of stress levels, either through the use of a simple stress measurement tool or through the associated factors identified within this research study, provides valuable data on maternal well-being. (C) 2010 Elsevier Ltd. All rights reserved.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Comparison of the acute renal and peripheral vascular responses to frusemide and bumetanide at low and high dose

1. The effects of equipotent doses of frusemide (10 mg and 100 mg) and bumetanide (250 micrograms and 2.5 mg) upon renal and peripheral vascular responses, urinary prostaglandin excretion, plasma renin activity, angiotensin II and noradrenaline were compared in nine healthy volunteers. 2. Frusemide (10 mg and 100 mg) and bumetanide (2.5 mg) increased renal blood flow acutely compared with placebo but bumetanide (250 micrograms) had no effect. The changes in peripheral vascular responses were not significantly different from placebo. 3. Urinary prostaglandin metabolite excretion was acutely increased by all treatments, with no inter-treatment difference. Plasma renin activity was increased acutely by both doses of frusemide and by bumetanide (2.5 mg) compared with placebo and to bumetanide (250 micrograms). There were no differences between the latter two treatments. Angiotensin II was increased significantly 30 min after frusemide 100 mg and bumetanide 2.5 mg, and by all four treatments at 50 min when compared with placebo. There were no significant differences between either of the low doses or the higher doses. Plasma noradrenaline was unchanged by all treatments. 4. Frusemide 100 mg and bumetanide 2.5 mg have the same effects on the renal vasculature and the renin-angiotensin-prostaglandin system. Under the conditions of this study, frusemide 10 mg had different effects on plasma renin activity than bumetanide 250 micrograms.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

A comparison of real catching with catching using stereoscopic visual displays

To date, the usefulness of stereoscopic visual displays in research on manual interceptive actions has never been examined. In this study, we compared the catching movements of 8 right-handed participants (6 men, 2 women) in a real environment (with suspended balls swinging past the participant, requiring lateral hand movements for interception) with those in a situation in which similar virtual ball trajectories were displayed stereoscopically in a virtual reality system (Cave Automated Virtual Environment [CAVE]; Cruz-Neira, Sandin, DeFranti, Kenyon, & Hart, 1992) with the head fixated. Catching the virtual ball involved grasping a lightweight ball attached to the palm of the hand. The results showed that, compared to real catching, hand movements in the CAVE were (a) initiated later, (b) less accurate, (c) smoother, and (d) aimed more directly at the interception point. Although the latter 3 observations might be attributable to the delayed movement initiation observed in the CAVE, this delayed initiation might have resulted from the use of visual displays. This suggests that stereoscopic visual displays such as present in many virtual reality systems should be used circumspectly in the experimental study of catching and should be used only to address research questions requiring no detailed analysis of the information-based online control of the catching movements.

Can we trust mass spectrometry for determination of arsenic peptides in plants:comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.

TMAX – a Database System Integrating Tissue Biomarker & Genomic Data

Background: Popular approaches in human tissue-based biomarker discovery include tissue microarrays (TMAs) and DNA Microarrays (DMAs) for protein and gene expression profiling respectively. The data generated by these analytic platforms, together with associated image, clinical and pathological data currently reside on widely different information platforms, making searching and cross-platform analysis difficult. Consequently, there is a strong need to develop a single coherent database capable of correlating all available data types.

Method: This study presents TMAX, a database system to facilitate biomarker discovery tasks. TMAX organises a variety of biomarker discovery-related data into the database. Both TMA and DMA experimental data are integrated in TMAX and connected through common DNA/protein biomarkers. Patient clinical data (including tissue pathological data), computer assisted tissue image and associated analytic data are also included in TMAX to enable the truly high throughput processing of ultra-large digital slides for both TMAs and whole slide tissue digital slides. A comprehensive web front-end was built with embedded XML parser software and predefined SQL queries to enable rapid data exchange in the form of standard XML files.

Results & Conclusion: TMAX represents one of the first attempts to integrate TMA data with public gene expression experiment data. Experiments suggest that TMAX is robust in managing large quantities of data from different sources (clinical, TMA, DMA and image analysis). Its web front-end is user friendly, easy to use, and most importantly allows the rapid and easy data exchange of biomarker discovery related data. In conclusion, TMAX is a robust biomarker discovery data repository and research tool, which opens up the opportunities for biomarker discovery and further integromics research.

The institutionalisation of the European Internal Cross-Border Co-operation Policy: A first appraisal

In the space of just 20 years, internal cross-border co-operation (CBC) has transformed from a marginal issue for European integration into an important strand of the third objective of European Union's (EU's) regional policy. How might this process of transformation be explained? This study intends to reconstruct the chronology of its development through interviews and use of archival material. The emergence of the current CBC policy was not, we argue, an inevitable solution to the problem of border management but, rather, the result of a struggle between the actors of that policy sub-system. The dramatic rise of CBC is the result of a series of factors that originated with the signing of the Single European Act in 1986. The construction of CBC as a set of problems and solutions by a network of policy actors at the margins of the EU through a series of technical reports, together with the policy window opened by the appointment of the Delors Commission, allowed the launching of an innovative CBC policy which has consolidated over time.

A comparison of two measures of facial activity during pain in the newborn child

Facial activity is strikingly visible in infants reacting to noxious events. Two measures that reduce this activity to composite events, the Neonatal Facial Coding System (NFCS) and the Facial Action Coding System (FACS), were used to examine facial expressions of 56 neonates responding to routine heel lancing for blood sampling purposes. The NFCS focuses upon a limited subset of all possible facial actions that had been identified previously as responsive to painful events, whereas the FACS is a comprehensive system that is inclusive of all facial actions. Descriptions of the facial expressions obtained from the two measurement systems were very similar, supporting the convergent validity of the shorter, more readily applied system. As well, the cluster of facial activity associated with pain in this sample, using either measure, was similar to the cluster of facial activity associated with pain in adults and other newborns, both full-term and preterm, providing construct validity for the position that the face encodes painful distress in infants and adults.

Organization specific predictors of job satisfaction: findings from a Canadian multi-site quality of work life cross-sectional survey

Background: Organizational features can affect how staff view their quality of work life. Determining staff perceptions about quality of work life is an important consideration for employers interested in improving employee job satisfaction. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent health care organizations.

Methods: 5,486 full, part and causal time (non-physician) staff on active payroll within six organizations (2 community hospitals, 1 community hospital/long-term care facility, 1 long-term care facility, 1 tertiary care/community health centre, and 1 visiting nursing agency) located in five communities in Central West Ontario, Canada were asked to complete a 65-item quality of work life survey. The self-administered questionnaires collected staff perceptions of: co-worker and supervisor support; teamwork and communication; job demands and decision authority; organization characteristics; patient/resident care; compensation and benefits; staff training and development; and impressions of the organization. Socio-demographic data were also collected.

Results: Depending on the organization, between 15 and 30 (of the 40 potential predictor) variables were found to be statistically associated with job satisfaction (univariate analyses). Logistic regression analyses identified the best predictors of job satisfaction and these are presented for each of the six organizations and for all organizations combined.

Conclusions: The findings indicate that job satisfaction is a multidimensional construct and although there appear to be some commonalities across organizations, some predictors of job satisfaction appear to be organization and context specific.

Nurses' perceptions of and satisfaction with the medication administration system in long-term-care homes

The purpose of this study was to explore nurses' perceptions of and level of satisfaction with the medication administration system in long-term care (LTC). The cross-sectional survey design included both quantitative and open-ended questions. Data were collected from licensed registered nurses (RNs) and registered practical nurses (RPNs) at 9 LTC residences in southwestern Ontario, Canada. Using independent sample f tests, the researchers found that RNs were significantly less satisfied than RPNs with their medication administration system, particularly with respect to safety issues. RNs identified a number of related barriers, including time constraints, poor packaging, insufficient drug information, prescription changes, lack of staff competency, and unwieldy medication carts. Implications for practice and policy are discussed, including recommendations for improving medication administration practices and for addressing the workload demands of LTC nurses. © McGill University School of Nursing.

Self-reported use of natural health products:A cross-sectional telephone survey in older Ontarians

Background: The self-reported use of natural health products (NHPs) (herbal products and vitamin and mineral supplements) has increased over the past decade in Canada. Because the elderly population might have comorbidities and concurrently administered medications, there is a need to explore the perceptions and behaviors associated with NHPs in this age group. Objective: The goal of this study was to assess the use of NHPs in a cohort of older Canadian residents and the characteristics, perceptions, and behaviors associated with NHP use. Methods: Survey participants aged =60 years were randomly selected from telephone listings in the area of greater Hamilton, Ontario, Canada. Data were collected using a standardized computer-assisted telephone interview system. Self-reported data covering 7 domains were collected: (1) demographics; (2) self-reported 12-month NHP use; (3) reasons for NHP use; (4) self-reported 12-month prescription medication use; (5) expenditures on NHPs; (6) patient-reported adverse events and drug-NHP interactions; and (7) perceptions of physicians' attitudes regarding NHPs. Descriptive statistics were used to compare the characteristics of NHP users with those of nonusers and to assess the characteristics of NHP users across these 7 domains. Multivariate regression analysis was conducted to determine the demographic variables that might be associated with NHP user status. Results: Of 2528 persons identified as age =60 years, 1206 (48%) completed the telephone interview. Six hundred sixteen of these respondents (51%) reported the use of =1 NHP during the previous 12 months. On the initial univariate analysis, younger age and higher income were significantly associated with reporting NHP use (mean age, users vs nonusers, 71.1 vs 72.7 years, respectively; 95% CI, 1.02-1.06; P <0.001; income more than Can $26,000 was 28% and 22% in users and nonusers, respectively; P = 0.028). One hundred seventy of 616 users (28%) used an NHP to treat the same condition for which they were concurrently receiving a prescription medication, and 43 (25%) had not informed their physicians about their NHP use. Patients' characteristics such as sex, education, smoking status, and self-reported health status did not differ significantly between users and nonusers. In individuals who regularly spent money to purchase NHPs (n = 394), the mean cost was $20.38/mo. NHP expenditure was not significantly associated with age, sex, or income. Conclusion: Based on these findings, a substantial proportion of those Ontarians aged =60 years reported NHP use, and there is a need for greater communication with physicians to avoid potential drug-NHP interactions. © 2009 Excerpta Medica Inc. All rights reserved.