A near-deterministic mutational hotspot in Pseudomonas fluorescens is constructed by multiple interacting genomic features.

Mutation - whilst stochastic - is frequently biased toward certain loci. When combined with selection this results in highly repeatable and predictable evolutionary outcomes. Immotile variants of the bacterium Pseudomonas fluorescens (SBW25) possess a 'mutational hotspot' that facilitates repeated occurrences of an identical de novo single nucleotide polymorphism when re-evolving motility, where ≥95% independent lines fix the mutation ntrB A289C. Identifying hotspots of similar potency in other genes and genomic backgrounds would prove valuable for predictive evolutionary models, but to do so we must understand the genomic features that enable such a hotspot to form. Here we reveal that genomic location, local nucleotide sequence, gene strandedness and presence of mismatch repair proteins operate in combination to facilitate the formation of this mutational hotspot. Our study therefore provides a framework for utilising genomic features to predict and identify hotspot positions capable of enforcing near-deterministic evolution.

Pseudomonas aeruginosa adapts to octenidine in the laboratory and a simulated clinical setting, leading to increased tolerance to chlorhexidine and other biocides.

BACKGROUND: Octenidine is frequently used for infection prevention in neonatal and burn intensive care units, where Pseudomonas aeruginosa has caused nosocomial outbreaks. AIM: To investigate the efficacy and impact of using octenidine against P. aeruginosa. METHODS: Seven clinical isolates of P. aeruginosa were exposed to increasing concentrations of octenidine over several days. Fitness, minimum bactericidal concentrations after 1 min, 5 min and 24 h, and minimum inhibitory concentrations (MICs) of a variety of antimicrobials were measured for the parental and octenidine-adapted P. aeruginosa strains. Octenidine and chlorhexidine MICs of a population of P. aeruginosa isolated from a hospital drain trap, exposed to a diluted octenidine formulation four times daily for three months, were also tested. FINDINGS: Some planktonic cultures of P. aeruginosa survived >50% of the working concentration of an in-use octenidine formulation at the recommended exposure time. Seven strains of P. aeruginosa stably adapted following continuous exposure to increasing concentrations of octenidine. Adaptation increased tolerance to octenidine formulations and chlorhexidine up to 32-fold. In one strain, it also led to increased MICs of antipseudomonal drugs. Subsequent to continuous octenidine exposure of a multi-species community in a simulated clinical setting, up to eight-fold increased tolerance to octenidine and chlorhexidine of P. aeruginosa was also found, which was lost upon removal of octenidine. CONCLUSION: Incorrect use of octenidine formulations may lead to inadequate decontamination, and even increased tolerance of P. aeruginosa to octenidine, with resulting cross-resistance to other biocides.

Seasonal variation in copper-mediated low-density lipoprotein oxidation in vitro is related to varying plasma concentration of oxidised lipids in summer and winter.

The seasonal variation of CuCl2-mediated low density lipoprotein (LDL) oxidation (10 microM Cu2+, lag phase, rate of oxidation and maximum absorbance at 234 nm) were measured in 43 men and women on 4-6 occasions (mean 5.7 +/- 0.5) over a 12-month period. The lag phase averaged 52.7 +/- 0.6 min and did not differ by gender. Lag phase and rate of the rapid propagation phase of LDL oxidation showed a sinusoidal pattern over the year (increased and reduced oxidative susceptibility during January and June-July, respectively; both p < 0.001). Changes in plasma alpha-tocopherol, ascorbic acid, lycopene or beta-carotene concentrations did not explain seasonal differences in oxidative susceptibility of LDL in vitro. Nor did plasma lipid content of linoleic acid, the main substrate of lipid peroxidation, vary. However, the amount of hydroperoxy- plus hydroxy-fatty acids in plasma lipids varied according to season (p < 0.024) and was related to the lag phase (r = -0.26, p < 0.001). Seasonal variation in oxidative susceptibility was not significant after adjusting for hydroperoxy- plus hydroxy-fatty acids (p = 0.506). Isolated LDL is more vulnerable to Cu2+-induced lipid peroxidation during the winter and this may be due to the higher amount of oxidised lipids during that period.

Incorporation of (15)N from spiked cattle dung pats into soil under two moorland plant communities.

The rate and depth of cattle dung incorporation into moorland soil may be an important factor influencing plant community dynamics through its effects on soil nutrient availability. This study traces the incorporation of (15)N-labelled dung into a moorland soil under two vegetation types in Dartmoor National Park, UK. Cores of treated and control soil 10 cm deep were collected at 2, 4, 8 and 16 week intervals and divided into 2 cm depth increments. Soil samples were freeze-dried, ground and analysed for atom% (15)N and %N content using continuous-flow isotope-ratio mass spectrometry. The contribution of dung N to the soil N pool was estimated by changes in atom% (15)N of the soil. The incorporation of dung dry matter into the soil was also calculated. The labile component of the dung N was incorporated deeper and more rapidly into soil under grass than under heather vegetation. The implications of these processes for the dynamics of upland plant communities are considered in relation to the ability of plants to compete for nutrients.

Rye bran and soy protein delay growth and increase apoptosis of human LNCaP prostate adenocarcinoma in nude mice.

BACKGROUND: In this study, we investigated whether dietary intervention could inhibit tumor growth of an androgen-sensitive human prostatic cancer. METHODS: LNCaP cells were transplanted subcutaneously in nude-mice. The animals were then put on different diets and tumor take, tumor growth and prostate specific antigen (PSA) secretion were studied during 9 weeks. RESULTS: Palpable tumors developed in 75% of the tumor-cell injected sites in animals fed a control diet (corn starch, sucrose, etc.) whereas, for animals given rye bran (RB), ethyl acetate extraction from rye bran supplemented cellulose based diets (CCEE), palpable tumors were seen in only 30% and for soy protein based diets (SCC) 50% of the transplantation sites, respectively. The tumors that grew to palpable size in the rye (RB) and soy (SCC) groups were smaller and secreted less PSA than those in the control group. In the rye and soy groups tumor cell apoptosis was increased, but cell proliferation was unaffected. Addition of fat to the rye diet reduced its effect on prostate cancer growth. CONCLUSIONS: Factors in rye bran and soy protein may inhibit prostate cancer growth. The effect is more apparent for rye than for soy. Further studies are needed to identify the effective substances and to explore the mechanism of action.

Ochratoxin A in dried vine fruit: method development and survey.

A method is described for the determination of concentrations of the mycotoxin ochratoxin A in dried vine fruits (currants, raisins and sultanas) using acidic methanolic extraction, immunoaffinity chromatography clean-up and HPLC determination. The limit of detection was estimated as 0.2 microgram/kg, and recoveries of 63-77% were achieved at 5 micrograms/kg. HPLC-mass spectrometric confirmation of the identity of ochratoxin was obtained. Ochratoxin A and aflatoxins were determined in 60 samples of retail dried vine fruits purchased in the United Kingdom. Ochratoxin A was found in excess of 0.2 microgram/kg in 19 of 20 currant, 17 of 20 sultana and 17 of 20 raisin samples examined, an overall incidence of 88%. The maximum level found was 53.6 micrograms/kg. No aflatoxin was found in any sample analysed, using a method with a detection limit of 0.2 microgram/kg for each of aflatoxin B1, B2, G1 and G2.

Determination of 19-nortestosterone and trenbolone in animal tissues by high-performance liquid chromatography with immunoaffinity clean-up.

A method for the simultaneous determination of residues of 17 beta-trenbolone and 17 beta, 19-nortestosterone and their epimers in animal tissues is described, involving immunoaffinity chromatography clean-up and high-performance liquid chromatography with dual-wavelength UV detection. The method has been validated at 2 micrograms/kg in pig and cattle liver and corned beef with recoveries of 41% upwards. The method has been applied to the determination of incurred residues of 19-nortestosterone and trenbolone. Various alternative extraction steps for incurred trenbolone have been investigated, including direct extraction, protease digestion, heating and ultrasonic probe treatment. Glucuronidase digestion has been shown to be the most effective method for this analyte.

A microbore high performance liquid chromatography/electrospray ionization mass spectrometry method for the determination of the phytoestrogens genistein and daidzein in comminuted baby foods and soya flour.

A microbore high performance liquid chromatographic/electrospray/mass spectrometric (HPLC/ESI-MS) method has been developed for the determination of the phytoestrogens daidzein and genistein in soya flours and baby foods. The samples were hydrolysed and extracted with acetonitrile-water prior to analysis. LC was performed on a microbore Primesphere 5C8 column using a water/acetonitrile/acetic acid mobile phase at a flow rate of 60 microliters/min. Atmospheric pressure ionization in the form of pneumatically assisted electrospray mass spectrometry (ESI-MS) was used as the method of detection. The limit of detection was 0.2 mg/ kg for daidzein and 0.7 mg/kg for genistein in the flour and food samples. The method proved both robust and reliable when operated over a long time period (10 days, 463 injections) generating precision data with a coefficient of variation of 4-15%.

Quantitative gas chromatography-mass spectrometry isomer-specific measurement of hydroxy fatty acids in biological samples and food as a marker of lipid peroxidation.

We have developed a capillary gas chromatography-mass spectrometry method for the quantitative analysis of individual positional isomers of monohydroxy fatty acids derived from linoleic, arachidonic, eicosapentaenoic, or docosahexaenoic acid. Peroxidation of a particular polyunsaturated fatty acid results already in a complex mixture of positional isomers of hydroperoxy and hydroxy fatty acids. Catalytic hydrogenation of lipid extracts produces stable saturated hydroxy lipids from the complex mixtures typical of oxidized biological samples, simultaneously simplifying the analytical problem and eliminating oxidation artifacts. After saponification and methylation, monohydroxy fatty acid methyl esters are purified by solid-phase extraction and partially resolved using a CP Sil 19 column following on-column derivatization of the hydroxy groups with tetramethylammonium hydroxide. The resulting methoxy fatty acid methyl esters are subjected to electron impact mass spectroscopy. Two characteristic ions are produced for each positional isomer. Quantitative measurements were achieved by using odd chain C17 and C19 monohydroxy fatty acids as internal standards. The limit of detection of individual hydroxy fatty acid isomers is dependent on the total number of ions monitored. Monitoring 11 pairs of ions simultaneously gives limits of detection of 10 ng. Sensitivity is much higher by monitoring fewer ions and as little as 0.2 ng of a single isomer can be detected. The method has been applied for the quantitative analysis of hydroxy (plus hydroperoxy) fatty acids in plasma, adipose tissue, oils, and foods. To date over 1000 samples have been analyzed using the method described in this paper.

On-line high-performance liquid chromatographic/gas chromatographic/tandem ion trap mass spectrometric determination of levamisole in milk.

On-line high-performance liquid chromatography/high-resolution gas chromatography/tandem mass spectrometry, performed on a quadrupole ion trap, has been used as a rapid method for the quantification of the anthelmintic drug levamisole in raw milk extracts following a simple extraction procedure. Detection at the 0.5 p.p.b. level and a linear response in the 10-0.5 p.p.b. range is demonstrated. Multiple-scan monitoring techniques have been used to acquire chemical ionization tandem mass spectra and electron impact mass spectra in a single chromatographic run.

Modified on-column interface for coupled high-performance liquid chromatography-gas chromatography and its application to the determination of levamisole in milk.

A modified on-column interface is reported for the coupling of high-performance liquid chromatography with gas chromatography, incorporating an adapted, commercially available multidimensional gas chromatography switching system. Novel features include cryogenic analyte focusing, total solvent exclusion from the analytical column and independent carrier gas supplies to the analytical GC column and uncoated pre-column. The instrumentation was used for the determination of the veterinary anthelmintic drug levamisole in milk with analyte detection by both flame ionisation and nitrogen-phosphorus detectors. Detection limits for the assay were 2.2 micrograms l-1 and 0.4 micrograms l-1 by flame ionisation and nitrogen-phosphorus detectors, respectively. The assay was applied to a survey of fourteen milk samples from different dairy outlets.

A comparison between amoxycillin/clavulanate and mezlocillin in abdominal surgical prophylaxis.

A randomized comparative study was conducted to evaluate the clinical efficacy of amoxycillin/clavulanate (Augmentin) compared with mezlocillin for the prevention of wound infection in patients undergoing abdominal surgery. There was no difference in overall wound infection rates between the amoxycillin/clavulanate treated group and the mezlocillin group. When sub-groups were examined for total infections no significant difference was seen between antibiotic groups in patients undergoing clean/potentially contaminated operations or contaminated operations, although more deep infections were encountered in the amoxycillin/clavulanate group in comparison with the mezlocillin group, in contaminated operations. The type of operation performed also failed to show any difference in those patients undergoing upper gastro-intestinal, appendiceal, colonic, or biliary operations. The infections in those receiving amoxycillin/clavulanate were largely of bowel origin and predominantly sensitive to amoxycillin/clavulanate. Those in the mezlocillin group were predominantly staphylococcal in origin. Amoxycillin/clavulanate appears to be an effective antibiotic for use as a single agent in surgical prophylaxis.

Non-aqueous size-exclusion chromatography coupled on-line to reversed-phase high-performance liquid chromatography. Interface development and applications to the analysis of low-molecular-weight contaminants and additives in foods.

An interface has been developed which permits the on-line coupling of size-exclusion chromatography in tetrahydrofuran with aqueous reversed-phase high-performance liquid chromatography. The interface isolates the required size exclusion chromatography fraction and dilutes it with water to ensure reconcentration of analytes on the reversed-phase column prior to gradient elution. Operational parameters and the influence of analyte polarity have been examined in detail. A predictive system is presented for determining the applicability of the system to any analyte, based on solute retention times on an ODS phase eluted with a methanol-water gradient. The method is illustrated with examples of direct analyses of crude lipid extracts from a snack product for 2,6-di-tert.-4-methylphenol and from chocolate for dibutyl phthalate. Detection limits of ca. 0.5 mg/kg have been achieved.

The role of gut micro-organisms in the metabolism of deoxynivalenol administered to rats.

1. Oral administration of deoxynivalenol (DON) to control rats resulted in the appearance of a de-epoxy metabolite in urine and faeces. 2. When DON was administered to rats treated with antibiotics to deplete their gut microflora there was very little excretion of radioactivity as the de-epoxy metabolite in faeces or urine. 3. Incubation of DON with a strictly anaerobic preparation of gut contents resulted in the progressive appearance of de-epoxy DON during a 24 h incubation period. 4. Incubation of DON with liver homogenate did not result in the appearance of the de-epoxy DON metabolite. 5. These results indicate that the presence of de-epoxy DON in rat excreta, following the oral administration of DON, is the result of metabolism by micro-organisms in the gut.

Enzyme-linked immunosorbent (ELISA) determination of aflatoxin B1 in peanut butter: collaborative trial.

Fourteen laboratories in the United Kingdom participated in a collaborative trial of a commercially available ELISA test kit for the determination of aflatoxin B1 in peanut butter. Each laboratory carried out six replicate analyses of each of six individual samples. Collaborators received a control, uncontaminated sample, together with samples prepared by blending naturally-contaminated and control material to give target levels of 8 micrograms/kg, 25 micrograms/kg and 75 micrograms/kg. Two of these samples (8 micrograms/kg and 25 micrograms/kg) were supplied as undisclosed duplicates. The repeatabilities of the assay ranged from 6.2 to 16.7 micrograms/kg. The reproducibilities for aflatoxin B1 concentrations in naturally-contaminated samples ranged from 3.6 to 18.7 micrograms/kg using uncontaminated peanut butter as a reference blank. Modifications to the format of the commercial kit were recommended as a result of the collaborative trial.

A survey of the occurrence of aflatoxin B1 in peanut butters by enzyme-linked immunosorbent assay.

A survey was carried out in 1986 for the occurrence of aflatoxin B1 in peanut butters (129 samples) obtained from specialist Health Food outlets. The results showed that 6.2% of the samples exceeded 10 micrograms/kg of aflatoxin, 8% contained between 2.5 and 10 micrograms/kg, and in the remainder (86%) aflatoxin could not be detected at a limit of 2.5 micrograms/kg. These results show a lower contamination by aflatoxin than found in these products in previous surveys (1982-1984). An aflatoxin B1-specific enzyme-linked immunosorbent assay (ELISA) was employed for the first time in these analyses; and to make an assessment of its performance positive aflatoxin results, together with a random selection of those below the ELISA limit of detection, were additionally analysed by conventional extraction and clean-up followed by HPLC. The ELISA technique offered a significant improvement in speed of analysis over conventional approaches, enabling a six-fold increase in sample throughput compared to that required for conventional analysis, together with other advantages.