Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria.

AIMS: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. METHOD AND RESULTS: A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml(-1) for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml(-1) and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. CONCLUSIONS: The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.

Recombination events in Neurospora crassa may cross a translocation breakpoint by a template-switching mechanism.

To assist investigation of the effect of sequence heterology on recombination in Neurospora crassa, we inserted the Herpes simplex thymidine kinase gene (TK) as an unselected marker on linkage group I, giving a gene order of Cen-his-3-TK-cog-lpl. We show here that in crosses heterozygous for TK, conversion of a his-3 allele on one homolog is accompanied by transfer of the heterologous sequence between cog and his-3 from the other homolog, indicating that recombination is initiated centromere-distal of TK. We have identified a 10-nucleotide motif in the cog region that, although unlikely to be sufficient for hotspot activity, is required for high-frequency recombination and, because conversion of silent sequence markers declines on either side, may be the recombination initiation site. Additionally, we have mapped conversion tracts in His(+) progeny of a translocation heterozygote, in which the translocation breakpoint separates cog from the 5' end of his-3. We present molecular evidence of recombination on both sides of the breakpoint. Because recombination is initiated close to cog and the event must therefore cross the translocation breakpoint, we suggest that template switching occurs in some recombination events, with repair synthesis alternating between use of the homolog and the initiating chromatid as template.

Recombination at his-3 in Neurospora declines exponentially with distance from the initiator, cog.

By deletion of 1.8 kb of sequence between cog(L) and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog(L) and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog(L), a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His(+) recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log(10) (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog(L) in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.

Endotoxemia and enhanced generation of oxygen radicals by neutrophils from patients undergoing cardiopulmonary bypass.

Plasma endotoxin concentrations and oxidative burst response of peripheral blood polymorphonuclear leukocytes were examined in 12 patients undergoing coronary artery bypass. The measurements were made just before the operation, 5 minutes after removal of the aortic crossclamp, and 24 hours after the operation. Endotoxin was quantitated by a combination of a sensitive Limulus amebocyte lysate assay and rocket immunoelectrophoresis measuring picogram amounts of endotoxin. Peripheral blood neutrophils were purified by a two-step dextran sedimentation and metrizoate sodium Ficoll (Lymphoprep., Nyegaard, Oslo, Norway) centrifugation. The oxidative burst response of these cells was measured for their ability to generate superoxide anion and was determined by a cytochrome c reduction assay. Preoperatively, all the plasma samples except one were free of endotoxin. The endotoxin levels reached 100 pg/ml 5 minutes after removal of the aortic crossclamp, and except in one sample they had decreased 24 hours after the operation. Studies on the generation of superoxide by neutrophils showed a decline in the response 5 minutes after removal of the aortic crossclamp and an enhancement of the response to f-Met-Leu-Phe by cells obtained from 11 of 12 patients 24 hours postoperatively. In vitro addition of bacterial lipopolysaccharide to blood from healthy individuals also enhanced the superoxide response of the neutrophils. We conclude that during cardiopulmonary bypass the circulating blood is contaminated by endotoxin and the neutrophils are primed for enhanced generation of oxygen radicals. The released oxygen radicals may be involved in the tissue damage observed in these patients.

Presence of circulating endotoxins during cardiac operations.

Ten patients having coronary artery bypass grafting were intraoperatively and postoperatively analyzed for endotoxins with the Limulus amoebocyte lysate test. A new highly sensitive rocket immunoelectrophoretic assay for reading the reactions of endotoxins with Limulus amoebocyte lysate was used. Preoperatively, all blood samples from the patients had negative Limulus amoebocyte lysate tests, negative blood cultures, normal total white cell counts, and were clinically without signs of infection. Intraoperatively, a substantial amount of endotoxins were found in samples from the extracorporeal circuit, the pulmonary artery, and the cardiac suction lines, which persisted during the cardiopulmonary bypass. The endotoxin content decreased significantly (p less than 0.05) 6 hours after cardiopulmonary bypass and further decreased within the seventh postoperative day (p less than 0.01). A positive Limulus amoebocyte lysate test was also found in some of the fluids administered during the operation, that is, the cardioplegic fluids, the priming fluids for the extracorporeal circuit, the blood transfusions, and the ice for local cooling. Postoperatively, all patients had rectal temperatures below 38.5 degrees C, but no correlation was found between the magnitude of endotoxin content and the degree of fever. Only one of the patients had positive blood cultures. Despite the measured endotoxin content, no intraoperative or postoperative complications were found.

Effects of glutamine on the immune system: influence of muscular exercise and HIV infection.

Glutamine increased the proliferative response and the lymphokine-activated killer cell activity of blood mononuclear cells isolated from normal healthy subjects (n = 6) in a dose-dependent manner, with optimum at 0.3-1.0 mM. The relative fraction of CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells was not changed by glutamine at a concentration of 0.6 mM, except in the phytohemagglutinin-stimulated proliferation experiment where the fraction of CD4+, and therefore CD3+ cells, increased. The natural killer cell activity was not influenced by glutamine. Human immunodeficiency virus (HIV)-seropositive subjects (n = 8) who performed concentric bicycle exercise for 1 h at 75% of maximal O2 consumption had an overall lower phytohemagglutinin-stimulated proliferative response, compared with the HIV-seronegative control group (n = 7). The proliferation during exercise was lower in both the HIV-seropositive and the HIV-seronegative group. Addition of glutamine in vitro did not normalize the lower proliferation in the HIV-seropositive group or the attenuated proliferation seen during exercise in both groups.

Cardiac function and hypercarbia.

In 12 patients with heart disease, hypercarbia was induced for carotid endarterectomy. Anesthesia was maintained with nitrous oxide in oxygen and methoxyflurane. In addition to intra-arterial measurements of blood pressure, cardiac output, systolic time intervals (STI), and pressure time indices (PTI) were determined in order to assess cardiovascular responses in these patients. Internal carotid stump blood pressure was measured in five patients before and after induction of hypercarbia. Mild elevation of the Paco2 level affected systolic time intervals but not heart rate and blood pressure. When Paco2 levels reached 56 to 65 torr, systolic but not diastolic blood pressure rose significantly, heart rate and cardiac output increased, while the shortening in the preejection period (PEP), left ventricular ejection time (LVET), and the decrease in the PEP/LVET ratio signified increased mechanical cardiac activity. Hypercarbia caused intense sympathetic stimulation as demonstrated by twofold to threefold increases in plasma catecholamine levels. Stump blood pressure was elevated. Cardiac oxygen demand was significantly increased, while coronary filling time was shortened, as indicated by the increase in the tension time index and shortening in the diastolic time. This signified a relative myocardial underperfusion. Thus, while hypercarbia to levels of 66 to 70 torr increased internal carotid artery stump pressure, it also caused increased cardiac mechanical activity and concomitant unfavorable balance between myocardial oxygen consumption and supply. The measurement of STI and the computation of PTI provided early detection of alterations in cardiac function.

Ventilation in ARDS and asthma: the optimal blood gas values.

Artificial ventilation of patients with acute respiratory diseases, i.e. ARDS and severe asthma, may involve the risk of pulmonary oxygen toxicity as well as volutrauma. The relationship between ventilator treatment and volutrauma suggests that only in patients with normal lungs the aim of ventilator treatment should be an arterial carbon dioxide tension and pH within the normal ranges. In patients suffering from a lung disease the clinical target must be based not only upon the arterial blood gases but also upon airway pressure and respiratory tidal volume. Thus during artificial ventilation of a patient with an acute pulmonary disease the following arterial pH and pCO2 optima are proposed: pH 7.35, with a range from 7.1 to 7.4; pCO2 is related to pH but an acceptable range is 5-12 kPa. The lowest acceptable fraction of inspired oxygen and thereby the safe lower level of arterial pO2 for an individual patient depends on many factors. The lower limit may be about 3 kPa, but the arterial pO2 should not be evaluated as an isolated parameter. It is related to the general oxygen transport capability of arterial blood, extractable oxygen, cardiac output and the microcirculation.

Fiber-optic chemical sensors (Gas-Stat) for blood gas monitoring during hypothermic extracorporeal circulation.

Measurements of pO2, pCO2 and pH by optical fluorescence microsensing technology has recently become available for monitoring blood gases during extracorporeal circulation ECC). We have compared simultaneous measurements with fiber-optic sensors (Gas-Stat, Bentley) and electrochemical sensors (ABL-4, Radiometer) on discrete samples. In 10 patients undergoing coronary artery bypass grafting during hypothermic (25 degrees C) ECC and hemodilution (hemoglobin concentration 4 mmol.l-1) arterial and venous pO2, pCO2 and pH were measured in-line in the extracorporeal circuit at the actual blood temperature. Simultaneous and anaerobically collected blood samples in glass syringes were analyzed within five minutes at 37 degrees C in the ABL-4. Linear regression analysis of the values at actual temperature shows the following equations: Gas-Stat = Y, ABL-4 = X: pO2 (kPa): Y = 1.04 X + 0.5 r = 0.95 n = 136; pCO2 (kPa): Y = 0.71 X + 1.5 r = 0.79 n = 136; pH: Y = 0.788 X + 1.590 r = 0.76 n = 136. The advantage of the Gas-Stat is continuous monitoring of blood gas parameters during ECC. The present study shows that measurements of pO2, pCO2 and pH with fiber-optic chemical sensors may be reliable. The differences between the two principles of measurement may be due to unknown factors interfering with the in-line measurements or to variations in sensitivity and stability of the individual sensor.

Rapid preparation of cyanobacterial DNA for real-time PCR analysis.

AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.

Cardiac output--pulse contour analysis vs. pulmonary artery thermodilution.

BACKGROUND: The aims of this study were to determine the agreement between pulmonary artery thermodilution (PA-TD), transpulmonary thermodilution (TP-TD) and the pulse contour method, and to test the ability of the pulse contour method to track changes in cardiac output. METHODS: Cardiac output was determined twice before cardiac surgery with both PA-TD and TP-TD. The precision (two standard deviations of the difference between repeated measurements) and agreement of the two methods were calculated. Post-operatively, cardiac output was determined with the PA-TD and pulse contour methods, and the bias and limits of agreement were again calculated. Finally, in patients with heart rates below 60 beats/min or a cardiac index of less than 2.5 l/min/m2, atrial pacing was started and the haemodynamic consequences were monitored with the PA-TD and pulse contour methods. RESULTS: Twenty-five patients were included. The precisions of PA-TD and TP-TD were 0.41 l/min [95% confidence interval (CI), +/- 0.07] and 0.48 l/min (95% CI, +/- 0.08), respectively. The bias and limits of agreement between PA-TD and TP-TD were - 0.46 l/min (95% CI, +/- 0.11) and +/- 1.10 l/min (95% CI, +/- 0.19), respectively. Post-operatively, the bias and limits of agreement between the PA-TD and pulse contour methods were 0.07 l/min and +/- 2.20 l/min, respectively. The changes in cardiac output with atrial pacing were in the same direction and of the same magnitude in 15 of the 16 patients. CONCLUSION: The precision of cardiac output measurements with PA-TD and TP-TD was very similar. The transpulmonary method, however, overestimated the cardiac output by 0.46 l/min. Post-operatively, cardiac output measurements with the PA-TD and pulse contour methods did not agree, but the pulse contour method reliably tracked pacing-induced changes in cardiac output.

Precision of bolus thermodilution cardiac output measurements in patients with atrial fibrillation.

BACKGROUND: The precision of bolus thermodilution cardiac output measurements in patients with atrial fibrillation (AF) has not previously been determined. A priori we suspected that the precision would be lower in patients with AF than in patients with sinus rhythm (SR). Consequently, we also determined if the precision could be improved by injecting the thermal indicator into the right ventricle instead of the right atrium. METHODS: Cardiac output was determined as the average result of four injections of 10 ml of iced saline. Replicate measurements were performed with thermal indicator injections into the right atrium and ventricle. The coefficients of variation and the precisions were calculated. RESULTS: In the 25 patients with AF, mean cardiac output was 3.96 l min(-1) (range 2.4-7.4), the coefficient of variation 0.073 (95% CI +/- 0.011), and the precision 0.38 l min(-1) (95% CI +/- 0.14) with injection into the right atrium. In the 25 patients with SR, mean cardiac output was 4.73 l min(-1) (range 2.4-7.3), the coefficient of variation 0.047(95% CI +/- 0.006), and the precision 0.38 l min(-1) (95% CI +/- 0.14). In both groups, an agreement analysis demonstrated that the injection of indicator into the right ventricle resulted in a significantly higher cardiac output [AF+0.25 (95% CI +/- 0.15) l min(-1), SR+0.29 ( +/- 0.20) l min(-1)]. CONCLUSION: The coefficient of variation for cardiac output determinations is 55% higher in patients with AF. Two measurements, separated by time or intervention, must differ by 15% in AF patients and 9% in SR patients before one can be 95% confident that a real change has taken place.

Cardiac function during halothane anf fluroxene anesthesia expressed by systolic time intervals.

A non-invasive method of measuring the systolic time intervals (STI) during anesthesia is described. Using the ratio between the pre-ejection period and the left ventricular ejection time (PEP/LVET-ratio) as an expression of cardiac function, it is shown in 39 patients that thiopentone exerted a marked depression on the heart (increase in PEP/LVET-ratio). This depression was further aggravated by halothane, while fluroxene caused an improvement of PEP/LVET-ratio. In the immediate post-anesthetic period PEP/LVET-ratio almost reached control value after discontinuation of fluroxene in contrast to halothane, where an increased PEP/LVET-ratio (25%) still persisted. The changes were mainly due to a prolongation of PEP without any specific change in LVET. During the study, PEP/LVET-ratio showed a close correlation to the reciprocal value of the square of the pre-ejection period (1/PEP-2). STI form 27 volunteers were measured and compared to the values from the patients before anesthesia was induced. PEP and PEP/LVET-ratio were significantly shorter in the patient group.

Transcutaneous oxygen measurement during thoracic anaesthesia.

The value of transcutaneous oxygen tension (tcPO2) as an oxygen parameter during uncomplicated thoracic anaesthesia was examined in ten patients anaesthetized with oxygen-nitrous oxide and enflurane or flunitrazepam/fentanyl. tcPO2 was measured with the Radiometer TCM-I monitor at 45 degrees C. Measuring interference due to the anaesthetic agents was not observed. tcPO2 was found to be lower than the arterial tension (PaO2) at any inspiratory oxygen fraction (FIO2). When the peroperative readings were related to the preoperative values, no statistically significant difference was found between PaO2 and tcPO2 at FIO2 - 0.5, 0.4 and 0.3 (P greater than 0.3). Linear regression between PaO2 and tcPO2 shows disparity in pre- and peroperative regression. tcPO2 (preoperative) = -2.2 + 1.03 X PaO2 (4 = 0.89) tcPO2 (preoperative) = +3.1 + 0.56 X PaO2 (r = 0.87). This disparity indicates a decrease in the tcPO2/PaO2 ratio with increasing PaO2. It is concluded that tcPO2 cannot substitute for PaO2, but tcPO2 and PaO2 proved to be equally useful as oxygen parameters in the examined patients. Interpretation of tcPO2 during anaesthesia, however, necessitates a preoperative measurement as reference.

Continuous measurement of the oxygen tension in blood. A comparative study in the dog.

An equipment constructed by the International Biophysics Corporation, the Differential Oxygen Analyzer, which was designed for continuous recording of the oxygen tension in the blood, particularly during extracorporeal circulation, was tested. It proved to react rather slowly to changes in the PO2 and the magnitude of the error of the single measurements is far too high at the present stage of development.