Small-scale agricultural grassland management can affect soil fungal community structure as much as continental scale geographic patterns.

A European transect was established, ranging from Sweden to the Azores, to determine the relative influence of geographic factors and agricultural small-scale management on the grassland soil microbiome. Within each of five countries (factor 'Country'), which maximized a range of geographic factors, two differing growth condition regions (factor 'GCR') were selected: a favorable region with conditions allowing for high plant biomass production and a contrasting less favorable region with a markedly lower potential. Within each region, grasslands of contrasting management intensities (factor 'MI') were defined: intensive and extensive, from which soil samples were collected. Across the transect, 'MI' was a strong differentiator of fungal community structure, having a comparable effect to continental scale geographic factors ('Country'). 'MI' was also a highly significant driver of bacterial community structure, but 'Country' was clearly the stronger driver. For both, 'GCR' was the weakest driver. Also at the regional level, strong effects of MI occurred on various measures of the soil microbiome (i.e. OTU richness, management-associated indicator OTUs), though the effects were largely regional-specific. Our results illustrate the decisive influence of grassland MI on soil microbial community structure, over both regional and continental scales, and, thus, highlight the importance of preserving rare extensive grasslands.

Quality control for handling of accidental blood exposures.

A regional counselling service was established to handle all accidental blood exposures using a standardized protocol. Levels of risk were assessed using an algorithm. Accidents that posed a risk for the transmission of hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) were classified as 'high risk', whereas accidents that posed a risk for HBV alone were classified as 'low risk'. Medical interventions were implemented according to the level of risk. During a one-year period, all accidents were registered and analysed for adherence to the standard protocol. In 2003, the centre handled 454 incidents. Of these, 36 (7.9%) incidents were assessed as no risk, 329 (72.5%) were assessed as low risk, and 67 (14.8%) were assessed as high risk. Due to incomplete registration, 22 (4.8%) incidents could not be analysed further. In total, 36% of the incidents with risk for HBV transmission and 40% of the incidents with risk for HCV and HIV transmission were not handled according to the proposed protocol. Breaches consisted of over-reaction (25/396) as well as insufficient response (123/396). Potentially inadequate treatment occurred for HIV postexposure prophylaxis in 12 of 63 incidents. Incomplete follow-up for HCV occurred in 11 of 63 incidents, and lack of HBV immunoglobulin administration occurred in five of 396 incidents, including three high-risk incidents. In 21 of 396 low-risk exposures, the breaches in protocol resulted from late reporting. It remains difficult to achieve an acceptable level of standardized care when using standard operational procedures. Documentation and evaluation of flaws are essential to improve the system.

Differences between hospital- and community-acquired blood exposure incidents revealed by a regional expert counseling center.

OBJECTIVE: One year (2003) regional analysis of all blood exposure incidents from hospitals as well as from the community. DESIGN: Establishment of an easily accessible regional expert counseling center, operating 24 h a day, for all accidental blood exposures. Tasks of the center were to register incoming calls, to inform and counsel the victim, to assess the risk of the incident, and to provide a plan of further actions, including prophylactic measures. SETTING: A Dutch region (Northeast Brabant) with 500,000 inhabitants and two major hospitals (1,786 beds). RESULTS: A total of 454 incidents (1.2 per day) were recorded. Only half of the incidents occurred in the hospital setting (n = 234), whereas the others (n = 220) took place in the community setting. Nearly all (95%, n = 432) incidents occurred during work, and most of them (84%, n = 385) were related to health care activities. In the hospital setting injuries occurred with physicians (13%), nursing staff (45%), operating room (OR) staff (13%), ancillary (18%), others (10%). In the community setting, incidents took place among healthcare workers (48%), detention and police officers (10%), civilians (10%), general practitioners/dentists and their staff (8%), cleaning staff (4%) and work-related incidents not falling into any of the above categories (7%). More low risk incidents took place outside the hospital (87% vs. 68% in hospital), while high-risk incidents predominantly occurred within the hospital setting (23% vs. 6%). The hepatitis-B immunization rate was significantly lower in victims from the community than in those working in hospitals (38% vs. 96%). Reports from incidents in the community setting were delayed. CONCLUSIONS: Incidents that expose individuals to blood-borne pathogens occur equally frequent in the hospital and non-hospital (community) setting. Therefore, a regional expert counseling center, accessible around-the-clock, for all types of blood-exposure incidents is needed. Blood-exposure prevention programs should aim at a reduction of high-risk incidents within hospitals, and at increasing the awareness for vaccination and early reporting within the community setting.

Predictive factors for sacral neuromodulation in chronic lower urinary tract dysfunction.

OBJECTIVES: To investigate data from 211 patients who underwent a trial stimulation (percutaneous nerve evaluation [PNE]) to determine the clinical parameters that can enhance the prediction of PNE success. The advantageous effect of sacral neuromodulation depends on the accurate identification of suitable candidates during the preimplantation PNE. METHODS: A total of 211 patients (161 women and 50 men), with refractory urge incontinence, urgency-frequency syndrome, and urinary retention, underwent a PNE. Patient data (demographics, medical history, urologic investigations, and diagnosis) were collected. The PNE results were evaluated from a voiding diary and patient history. More than 50% improvement of voiding parameters was considered a successful PNE, and those patients were selected for implantation. Logistic regression analysis was performed. The factors tested for predicting the test result were sex, patient age, diagnosis, previous surgery, neurogenic bladder dysfunction, duration of complaints, and previous treatments. RESULTS: The PNEs were positive in 85 patients (40.3%) and negative in 105 patients (49.8%). In 18 patients (8.5%), the test electrode had migrated; 3 more patients were not assessable and were also excluded. Missing data on the variable "duration of complaints" reduced the number of patients in the analyses from 190 to 174 patients. CONCLUSIONS: Intervertebral disk prolapse, duration of complaints, neurogenic bladder dysfunction, and urge incontinence were found to be significant predictive factors. However, a PNE remains necessary to evaluate a patient's chance of implant success objectively.

Effects of elevated carbon dioxide and arbuscular mycorrhizal infection on Trifolium repens.

Trifolium repens L. cv. aran was grown for 58 d at ambient (350 µol mol(-1) ) and elevated (700 µol mol(-1) ) atmospheric CO2 , wish and without the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe cv. YV. Plant biomass, mycorrhizal infection, non-structural carbohydrates, C, N and P content were examined. Elevated CO2 (a) significantly increased above- and below-ground biomass, (b) decreased specific leaf area and specific root length, (c) decreased tissue %N and increased the C:N ratio, and (d) significantly increased total non-structural carbohydrates. Inoculating T. ripens with Glomus mosseae (a) significantly increased above- and below-ground biomass. (b) increased the total root length and total leaf area, and (c) significantly decreased tissue of Evidence of an increased influence of mycorrhiza on the P nutrition of T. repens at elevated CO2 was found in the 22%, increase in leaf total P (P < 005) of mycorrhizal plants grown at elevated CO2 compared with non-mycorrhizal plants. No significant interactions were found between CO2 and mycorrhiza treatments. The proportion of T. repens root length colonized by Glomus mosseae was not affected by CO2 concentration. The percentage mycorrhizal infection was 29% at ambient CO2 and 35%, et al elevated CO2 . However, exposure to elevated CO2 significantly increased the total mycorrhizal foot length from 3.4 to 6.1 m per plant. The results show little evidence that the role of arbuscular mycorrhiza in the growth and nutrition of T. repens would increase if atmospheric CO2 were to increase as predicted.

Determination of methyl 5-hydroxy-2-benzimidazole carbamate in urine by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic (HPLC) assay for methyl 5-hydroxy-2-benzimidazole carbamate (5-HBC) in urine was developed in order to assess the exposure of workers to the pesticide carbendazim. 5-HBC is measured in urine after hydrolysis, sample clean-up through a strong cation-exchange (SCX) column and extraction with ethyl acetate. HPLC with electrochemical detection provides selective and sensitive determination of 5-HBC with a detection limit of 5 micrograms/l. A C18 reversed-phase column was used with 0.06 M ammonium acetate solution (pH 8)-methanol (73:27) as mobile phase. The method was validated with respect to hydrolysis of urine samples, analytical recovery of spiked 5-HBC, stability of 5-HBC conjugates, limit of detection, background and precision. The overall analytical recovery from urine was better than 60%. 5-HBC, excreted in urine as a conjugate, was stable for at least one year when stored at -20 degrees C. A background of ca. 5 micrograms/l was detected in urine from some non-occupationally exposed persons. Between-day coefficients of variation as calculated from the results of the stability test were 7, 4 and 4% for concentrations of 61, 244 and 295 micrograms/l 5-HBC, respectively (n = 16).

Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection.

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic-mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 micrograms/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at -20 degrees C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 micrograms/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.

Gas chromatographic determination of the pesticide dodemorph for assessment of occupational exposure.

As part of a health hazard survey of occupational exposure to pesticides in greenhouse growing of ornamentals, analytical methods are developed and validated for measurement of exposure of workers to the fungicide dodemorph. A gas chromatographic method is developed using on-column injection and nitrogen-phosphorus detection for quantification. Methods for the determination of (potential) dermal exposure by the analysis of foliar dislodgeable residues and cotton gloves are validated with respect to background, analytical recovery, stability, limit of detection, and between-day coefficients of variation. Analytical recovery from 'foliar dislodgeable residue solutions' and cotton gloves is more than 95%. Dodemorph in 'foliar dislodgeable residue solutions' and on cotton gloves is stable for at least five and six months, respectively, when stored in the refrigerator. Between-day coefficients of variation are 6% for both matrices. The limit of detection is 3 micrograms per leaf sample and 150 micrograms per pair of gloves. Institute of Occupational Medicine (IOM) samplers, designed for the collection of a defined inspirable fraction of aerosols, are tested for sampling air-borne dodemorph. IOM samplers equipped with glass-fiber or cellulose filters appear unsuitable for reliable sampling of the fungicide because of breakthrough or breakdown during sampling.

Assessment of dermal exposure of greenhouse workers to the pesticide bupirimate.

An HPLC method was developed for estimation of dermal exposure of greenhouse workers to the pesticide bupirimate. Chromatography was performed on a cyano-modified silica column with methanol-water (6:4 by volume) containing 5 g/L ammonium sulfate as eluent. UV detection at 310 nm was used for quantitation. Dermal exposure was assessed by letting the workers wear cotton gloves and by measuring foliar dislodgable residues in the greenhouses as potential exposure. The analytical procedure was validated for measurement of bupirimate on cotton gloves and in solutions used for the estimation of foliar dislodgable residues. Gloves were extracted with methanol. Recovery of bupirimate from fortified gloves was complete. Methanol extracts with one volume of water added and solutions containing dislodgable residues were injected directly onto the HPLC system. The limit of detection was 30 micrograms/L. Between-day coefficients of variation were 7 and 4% at concentrations of 0.6 and 28 mg/L, respectively.

An automated method for the determination of acetyl and pseudo cholinesterase in hemolyzed whole blood.

The aim of the present study was to develop a method which allows determination of pseudo (PsChE) and acetyl cholinesterase (AChE) activities in single hemolyzed blood samples of workers exposed to cholinesterase-inhibiting compounds, avoiding the time-consuming and laborious separation of plasma and erythrocytes. Two methods based on Ellman's colorimetric determination of cholinesterase activity were compared, and three different substrates were tested. The best results were obtained with the substrates butyrylthiocholine and acetyl(beta-methyl)thiocholine, both showing a substrate specificity of more than 97% with respect to PsChE and AChE, respectively. The method showed sensitivity to detect low levels of inhibition of AChE and PsChE in blood. The between-day precision was less than 4% for both cholinesterase activities. It was demonstrated with this method that hemolyzed blood can be stored at -20 degrees C at least 18 months without loss of cholinesterase activity. The method has been used for 18 months in a monitoring program for laboratory employees working with various cholinesterase-inhibiting compounds. The average co-efficients of intraindividual variation amounted to 6.8% (range 2.2-9.6%; 90 percentile, 8%) and 6.6% (range 2.9-9.9%; 90 percentile, 7.9%) for PsChE and AChE, respectively. In a group of non-exposed workers the average intraindividual variations were 4.0% (range 1.5-7.7%; 90 percentile, 7.6%) and 3.6% (range 0.6-6.6%; 90 percentile, 5.3%), respectively. Using the value of 4.0%, it appears possible to detect an individual decrease in cholinesterase activity of more than 8% below a baseline based on three determinations. The method can thus be used to detect relatively low levels of cholinesterase inhibition.

Ethanol stimulates phospholipid turnover and inositol 1,4,5-trisphosphate production in Chlamydomonas eugametos gametes.

Alcohols induce mating-structure activation in Chlamydomonas eugametos gametes. From the effect of ethanol on the (32)P-labelling of polyphosphoinositides, we conclude that the synthesis of these lipids is stimulated. Biologically inactive concentrations of ethanol (<6%) had no effect on synthesis, but 6-8% ethanol stimulated synthesis for upto 60 min. The (32)P incorporated into polyphosphoinositides and phosphatidic acid during ethanol treatment was readily chased out when 1 mM unlabelled Na3PO4 was added. Using a binding assay for inositol 1,4,5-trisphosphate, we show that the production of this phospholipid constituent is dramatically increased after ethanol treatment. This effect, coupled to a rise in intracellular calcium concentration, could explain gamete activation. The significance of these results in explaining other ethanol-induced phenomena in algae is discussed.

High-performance liquid chromatographic method for the determination of occupational exposure to the pesticide abamectin.

As part of a survey of occupational exposure to pesticides in greenhouses for growing ornamentals, analytical methods were developed and validated for the measurement of exposure of workers to the pesticide abamectin. Abamectin consists of a mixture of avermectin-B1a and avermectin-B1b, which are members of a class of fermentation products of the soil microorganism Streptomyces Avermitilis. Because of the high molecular weight of the avermectins (greater than 800 daltons), high-performance liquid chromatography (HPLC) was the analytical method of choice. Previously described HPLC methods that used fluorescence detection were adapted and validated for the determination of dermal exposure by the analysis of cotton gloves and foliar dislodgeable residue. IOM samplers (developed at the Institute of Occupational Medicine, Edinburgh, U.K.) for collecting the inspirable fraction of dust or aerosols were tested for the determination of airborne abamectin concentrations in greenhouses. An analytical procedure considerably simpler than published methods appeared suitable for the determination of abamectin residues on cotton gloves and on greenhouse foliage. Analytical recovery from cotton gloves, solutions of foliar dislodgeable residues, and air-sampling filters was essentially complete. However, air concentrations of abamectin could not be reliably measured by using the IOM sampling device because of breakdown during sampling. Between-day coefficients of variation for solutions of dislodgeable residue and cotton glove extracts were between 3% and 6% for abamectin concentrations between 5 and 140 micrograms/L.

Determination of the pesticide chlorothalonil by HPLC and UV detection for occupational exposure assessment in greenhouse carnation culture.

An HPLC method was developed for application in the measurement of occupational exposure to the pesticide chlorothalonil. In addition, sampling methods were validated for the determination of exposure to chlorothalonil in the greenhouse culturing of carnations. Procedures for sampling of the inspirable fraction of aerosols, for the determination of hand contamination by hand rinse and the use of cotton gloves, and for the determination of dislodgable chlorothalonil residues on carnation leaves were validated. Normal phase HPLC with hexane-dioxane as the eluent and UV detection at either 254 or 325 nm appeared suitable for the determination of chlorothalonil in the described matrices. The limit of detection, after concentration on SepPak C18 cartridges was approximately 0.5 micrograms/L. Linear calibration curves were obtained in concentration ranges from 0.5 microgram/L to 100 mg/L. In general, no interferences were noticed in the analysis of the matrices. However, cotton gloves for determination of hand contamination had to be washed before use because they contained interfering material, and in the case of air sampling, glass fiber filters for air sampling appeared to degrade chlorothalonil very rapidly. Therefore, cellulose filters were used for collection of the inspirable fraction of aerosols containing chlorothalonil.

Pharmacokinetics of 24,25-dihydroxyvitamin D3 in humans.

Pharmacokinetic properties of pharmacological doses of 24,25-dihydroxyvitamin-D3 [24,25(OH)2D3] were determined in healthy volunteers. Four male subjects received 25 micrograms of 24,25(OH)2D3 as an intravenous bolus injection. Plasma concentrations of 24,25(OH)2D3, 25-hydroxyvitamin D and 1,25-dihydroxy-vitamin D were monitored during 14 days. In addition, serum ionized calcium, total calcium, inorganic phosphate, albumin, creatinine and intact hPTH(1-84) were measured during 14 days. The concentration-time curve of 24,25(OH)2D3 could be described by a two-exponential curve with half-lives of 3.0 +/- 0.9 hrs and 8.2 +/- 2.9 days (mean +/- SD). The volume of distribution was 0.19 +/- 0.02 liters/kg. None of the mentioned biochemical parameters, except serum 24,25(OH)2D3, changed markedly. In 18 subjects suffering from primary hyperparathyroidism, taking 25 micrograms of 24,25(OH)2D3 daily during three months, an average plateau level of 39 +/- 12 nmol/l of serum was observed. Bioavailability as estimated from this plateau level was approximately 70%.

The effect of vitamin D supplementation on vitamin D status and parathyroid function in elderly subjects.

Vitamin D deficiency is common in the elderly and may lead to secondary hyperparathyroidism, cortical bone loss, and hip fractures. The effect of vitamin D supplementation for 1 yr was studied in 72 people living in a nursing home and 70 people living in an aged people's home. The subjects were randomized into 3 groups: control, and 400 or 800 IU vitamin D3/day. The initial vitamin D status of each subject was classified as deficient or borderline [serum 25-hydroxyvitamin D (25OHD) less than 30 nmol/L] in 79% and adequate (serum 25OHD greater than or equal to 30 nmol/L) in 21%. Serum 25OHD concentrations increased about 3-fold in both groups receiving vitamin D supplementation. Serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations increased slightly but significantly, and the increase was inversely related to the initial serum 25OHD concentration. Serum intact PTH-(1-84) concentrations decreased about 15% during supplementation in both nursing home and aged people's home residents, whereas serum osteocalcin significantly decreased in the nursing home residents only. We conclude that a vitamin D3 supplement of 400 IU/day adequately improves vitamin D status in elderly people and increases 1,25-(OH)2D concentrations in those with vitamin D deficiency. Supplementation decreases parathyroid function and may depress bone turnover to some degree.

Improvements in the simultaneous determination of calcidiol and calcitriol in human serum or plasma.

Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.

Determinants of vitamin D status in patients with hip fracture and in elderly control subjects.

The factors that influence vitamin D status were investigated in 125 patients with hip fracture and in 74 elderly control subjects. The serum concentrations of 25-hydroxyvitamin D [25(OH)D] varied with sunshine score and were paralleled by serum 1,25-dihydroxyvitamin D [1,25(OH)2D]. The control subjects showed a higher sunshine score and higher serum 24(OH)D levels than the patients with hip fracture. Dietary vitamin D intake was similar in both groups (mean 115 IU/d). A positive correlation between vitamin D intake and serum 25(OH)D was observed in the patients with low sunshine exposure. It appeared from this relation that dietary vitamin D intake should be approximately 300 IU/d to maintain an adequate serum (25(OH)D concentration. Vitamin D status was very poor in patients who were institutionalized before hip fracture. Multiple regression analysis on serum 25(OH)D confirmed the primary role of sunshine exposure as determinant of vitamin D status. The principal determinants of serum 1,25(OH)2D were serum 25(OH)D, serum creatinine, and serum phosphate.

Effect of dietary calcium, phosphate and vitamin D deprivation on the pharmacokinetics of 1,25-dihydroxyvitamin D3 in the rat.

The in vivo regulation of circulating 1,25(OH)2D3 concentrations by vitamin D status and by dietary calcium and phosphate deficiency was studied. Adult rats were cannulated in the jugular vein and the clearance of physiological doses of 1,25(OH)2D3 monitored. In vitamin D-replete rats we investigated the effects of dietary calcium and phosphate deficiency on the elimination half life of 1,25(OH)2D3 The results showed no effect of dietary phosphate deficiency on the elimination half life of 1,25(OH)2D3. Dietary calcium deficiency resulted in a small increase of the 1,25(OH)2D3 elimination half life (P = 0.04) (normal diet: 16.3 +/- 1.8 hrs, n = 6; -Ca diet: 18.6 +/- 1.1 hrs, n = 5; -P diet: 16.0 +/- 1.4 hrs, n = 6; mean +/- SD). The experiments with the vitamin D deficient rats showed a marked increase in the elimination half life of 1,25(OH)2D3 (36.4 +/- 6.8 hrs, n = 7), when compared to the rats on the normal diet (P = 0.001). From the experiments in the vitamin D replete rats one can infer that regulation of circulating 1,25(OH)2D3 concentrations by dietary calcium or phosphate takes place at the production site and not by changes in elimination rate. However, vitamin D status appears to regulate circulating 1,25(OH)2D3 concentrations also through an effect on the elimination rate.

A simplified cannulation procedure for pharmacokinetic experiments in rats.

A convenient, simplified method for cannulating the jugular vein of rats is described. The technique has been developed for use in pharmacokinetic experiments. The construction of the cannula has been simplified compared with previously described methods. In addition, a small and simple outlet device for the cannula is described which allows the connection of tubing for sampling at short time intervals or for short-term infusions (several hours). In order to reduce stress on the animal, the tubing can be disconnected during sampling at long time intervals, allowing free movement of the animal with only a very small external outlet device.