A novel plasmid for detection of N-acyl homoserine lactones.

Many bacteria utilize acyl-homoserine lactones as cell to cell signals that can regulate the expression of numerous genes. Structural differences in acyl-homoserine lactones produced by different bacteria, such as acyl side chain length and the presence or absence of an oxy group, make many of the commonly used detection bioassays impractical for broad range detection. Here we present a simple, broad range acyl-homoserine lactone detection bioassay that can be used to detect a wide range of these chemical signals. A plasmid (pEAL01) was constructed and transformed into Pseudomonas aeruginosa strain QSC105 to allow for detection of a broad range of acyl-homoserine lactones through induction of a lasB'-lacZ transcriptional fusion. Monitoring beta-galactosidase activity from this bioassay showed that P. aeruginosa strain QSC105 (pEAL01) could detect the presence of eight acyl-homoserine lactones tested at physiological concentrations. This novel strain could also detect acyl-homoserine lactones from the extracts of four different bacteria that produce different acyl-homoserine lactones signals. These data indicate that strain QSC105 (pEAL01) can be used to detect a wide variety of acyl-homoserine lactones by a simple beta-galactosidase assay and this bioassay could be a useful and inexpensive tool to quickly identify the presence of these signal molecules.

Deliberate hypotension in orthopedic surgery reduces blood loss and transfusion requirements: a meta-analysis of randomized controlled trials.

PURPOSE: To determine if deliberate hypotension decreases blood loss and transfusion requirements in patients undergoing orthopedic surgery, a systematic review of all randomized trials addressing this issue was undertaken. METHODS: Electronic databases, citations lists and review articles were searched for potential articles. Relevant articles met the following inclusion criteria: English language, humans undergoing orthopedic surgery, deliberate hypotension used by any method, intraoperative blood loss measured as an outcome, and the trial methodology being randomized and controlled. Four outcomes were analyzed, including estimated blood loss, blood transfused, surgery duration, and quality of the surgical field. For all analyses, the random-effects model was used. RESULTS: Seventeen articles met the inclusion criteria. The surgeries studied included total hip arthroplasty (seven), orthognathic surgery (eight), total knee arthroplasty (one) and spinal fusion (one). A total of 636 patients were randomized across all studies. For blood loss, the overall weighted mean difference favoured treatment, with a savings of about 287 mL of blood [95% confidence interval (CI): -447, -127]. The mean differences also showed a statistically significant benefit for deliberate hypotension in reducing transfusion requirements (-667 mL of blood transfused; 95% CI: -963, -370). Deliberate hypotension was not shown to reduce the duration of surgery (-1.9 min of surgery; 95% CI: -7.2, 3.5) or improve surgical conditions (surgical field quality rating -0.5; 95% CI: -1.1, 0.2). CONCLUSION: This review provides some support for the use of deliberate hypotension in reducing blood loss and transfusion requirements in orthopedic surgery, but these results are tempered by the small sample sizes and poor methodological quality of published studies.

Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

Both the OxyArm and Capnoxygen mask provide clinically useful capnographic monitoring capability in volunteers.

PURPOSE: To compare the capnography monitoring performance of the new OxyArm (OA) with the Capnoxygen mask (CM), a conventional oxygen mask with a carbon dioxide sampling port. METHODS: Eleven healthy volunteer adult subjects underwent capnographic monitoring (in a non-randomized, un-blinded crossover study) at baseline and while receiving oxygen at seven different flow rates (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10 L x min(-1)), applied first with the CM and then with the OA. RESULTS: Both the OA and CM produced acceptable capnographs with consistent waveforms. The measured end-tidal (ET) CO(2) was equivalent for the two devices at all seven oxygen flow rates. On average, the ETCO(2) measured with the OA was about 2 mmHg greater than that of the CM. Regression analysis showed an inverse relationship between oxygen therapy flow rate and measured ETCO(2) whereby the measured value of CO(2) decreased as the oxygen flow rate was increased (P < 0.001). Both the CM and OA produced consistent measurements of ETCO(2) as illustrated by their reliability coefficients, 0.95 and 0.86 respectively. The biggest source of variation in measured CO(2) for both devices was inter-subject differences, followed by variable oxygen flow rates. CONCLUSIONS: This study suggests that the OA and CM can prove useful for respiratory monitoring and oxygen delivery in spontaneously breathing volunteers, and the OA could potentially be used as an alternative to the conventional methods of oxygen delivery and CO(2) sampling in patients.

Agreements between the prothrombin times of blood treated In Vitro with heparinase during cardiopulmonary bypass (CPB) and blood sampled after CPB and systemic protamine.

UNLABELLED: The prothrombin time (PT) is useful for identifying coagulation factor deficits after cardiopulmonary bypass (CPB). However, long processing times and the need for fresh frozen plasma (FFP) to be thawed cause delays in factor replacement. We hypothesized that, by treating with heparinase, blood sampled toward the end of CPB can provide PT results that help to determine the requirement for FFP after CPB. Laboratory delays can be eliminated with point-of-care monitors. We studied 158 adults undergoing nonemergent cardiac surgery. Blood taken before separation from CPB was mixed with heparinase, and PT was measured in the laboratory with a HemoTec timer. Agreements between these results and laboratory measurements of blood taken after systemic protamine were compared by using Bland and Altman plots with the threshold of +/-1.0 s. We found that the laboratory PT measurements during CPB versus after CPB were compara-ble, but the limits of agreement exceeded these thresholds. Similarly, there was unsatisfactory agreement between the HemoTec and laboratory PT results measured before, during, and after CPB. For each PT measured during CPB, the corresponding confidence interval for the postprotamine PT was calculated. During CPB, a laboratory PT of < or =16 s or > or =18 s suggests a > or =83% or > or =93% probability of not requiring or potentially requiring, respectively, FFP after CPB. We conclude that the majority of PT measurements obtained from blood taken before weaning from CPB and treated in vitro with heparinase was associated with a high probability of whether or not FFP would be needed after CPB. IMPLICATIONS: Coagulation dysfunction after cardiopulmonary bypass may contribute to bleeding. Obtaining coagulation tests and fresh frozen plasma requires time and delays treatment in patients who need fresh frozen plasma. We have devised a technique to provide early estimation of postbypass coagulation status.

The OxyArm - a new minimal contact oxygen delivery system for mouth or nose breathing.

PURPOSE: To describe the development and performance of a new minimal contact oxygen (O(2)) delivery system for both nasal and oral breathing, with capnographic capabilities. METHODS: The development and design challenges of the OxyArm (OA) prototype are described. The innovative design utilizes a headset with a semi-rigid boom and an O(2) diffuser. The OA was compared to the Venturi mask in eight healthy adults after informed consent. Inspired O(2) fractions were measured in the hypopharynx using continuous gas sampling at low to high O(2) flow rates. Mean data were compared using two-tailed paired t tests with significance set at 0.05. RESULTS: The measured inspired O(2) concentration was higher in the OA at 2 (26.3 +/- 2.5 vs 23.3 +/- 0.5, P <0.01) and 6 L x min(-1) (33.5 +/- 3.3 vs 28.8 +/- 1.2, P <0.01) flow rates. At 12 L x min(-1), the O(2) concentration was less in the OA (39.2 +/- 6.3 vs 46.0 +/- 2.7, P <0.02). All subjects found both systems comfortable for the short duration of the study. CONCLUSIONS: The OA delivered predictable concentrations of O(2) over low to medium flow rates. This system is comfortable, easy to use, non-obtrusive, odorless, and latex-free. The ability to monitor capnography makes this device ideal for monitored anesthesia care or in other settings (intensive care) where monitoring of respiration is warranted. This device does not contact the face and thus may be ideal for pediatric patients and those on long-term home O(2) therapy. Further clinical trials in these areas are warranted.

Timing and heritability of theNasobemia transformation inDrosophila.

Nasobemia (Ns) is a dominant homeotic mutant ofDrosophila melanogaster which converts parts or all of the antenna to mesothoracic leg.Ns has a temperature sensitive period between 48 and 60 h. The hypothesis thatNs acts during this period and is not required thereafter to maintain the homeotic transformation to leg was tested by removingNs fromNs/+ cells at different stages of development through X-ray induced somatic recombination. The expression of theNs homeotic transformation in recombinant wild type (+/+) cells increased sharply between 48 and 65 h. In clones induced after 65 h the expression of the leg transformation was equal in large and small +/+ clones. We interpret these results as supporting the hypothesis that transient action ofNs between 48 and 65 h switches antennal cells to a clonally stable leg determined state whose maintenance does not require futherNs action.