Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA.

A methodology is reported to conjugate human O6-alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O4-thymidine-alkylene-O4-thymidine and the (5'→3') O6-2'-deoxyguanosine-alkylene-O4-thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O4-thymidine-alkylene-O6-2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

A randomised controlled trial using the Epidrum for labour epidurals.

The aim of our study was to determine if using the Epidrum to site epidurals improves success and reduces morbidity. Three hundred parturients requesting epidural analgesia for labour were enrolled. 150 subjects had their epidural sited using Epidrum and 150 using standard technique. We recorded subject demographics, operator experience, number of attempts, Accidental Dural Puncture rate, rate of failure to site epidural catheter, rate of failure of analgesia, Post Dural Puncture Headache and Epidural Blood Patch rates. Failure rate in Epidrum group was 9/150 (6%) vs 0 (0%) in the Control group (P = 0.003). There were four (2.66%) accidental dural punctures in the Epidrum group and none in the Control group (P = 0.060), and 2 epidurals out of 150 (1.33%) in Epidrum group were re-sited, versus 3/150 (2%) in the control group (P = 1.000). The results of our study do not suggest that using Epidrum improves success or reduces morbidity.

Use of the Obstetric Quality-of-Recovery score (ObsQoR-11) to measure the impact of an enhanced recovery programme for elective caesarean section.

BACKGROUND: Enhanced recovery after caesarean (ERAC) has been shown to postoperatively reduce opioid consumption, reduce pain scores, and shorten hospital stay. Arguably, none of these measures provide for a patient-centred approach. We believe that patient-reported outcome measures (PROMs) represent a more holistic approach to the reporting of outcomes. One such PROM is the Obstetric Quality-of-Recovery Score (ObsQoR-11). This has been shown to be a valid and reliable assessment of recovery after elective caesarean section. METHODS: This before-and-after quality improvement programme studied consecutive patients undergoing elective caesarean section. We implemented an ERAC pathway with the aim of improving quality of recovery and patient satisfaction. Our primary outcome was the change in the ObsQoR-11 score. RESULTS: A total of 318 medical records were reviewed (n = 93 before ERAC, n = 225 after ERAC). There was a significant improvement in ObsQoR-11 score in ERAC patients compared with pre-ERAC patients (85.0 vs 82.3, P < 0.001). Morphine consumption (MMEQ) was reduced by 10% overall in the ERAC group, with no increase in pain scores at day 1 postoperatively and a decrease in pain scores on day 2 in the ERAC group (P = 0.02). The length of hospital stay was significantly shorter in ERAC patients (63.1 h vs 79.9 h, P < 0.001). CONCLUSIONS: Our study demonstrated an improved ObsQoR-11 score after ERAC implementation. This is the first example in the literature of using ObsQoR-11 in ERAC. We believe this is a more comprehensive way to assess patient recovery and the impact of an ERAC programme.

Infrared thermographic assessment of spinal anaesthesia-related cutaneous temperature changes during caesarean section.

INTRODUCTION: Assessment of adequacy of spinal anaesthesia, prior to obstetric surgery is extremely important but can be problematic because currently available clinical assessment methods are indirect and subjective. As the sympathectomy associated with spinal anaesthesia is known to cause vasodilation and heat redistribution, we sought to assess whether spinal anaesthesia led to significant and consistent cutaneous temperature changes as measured by infrared thermography. METHODS: Following ethics committee approval, this observational study was conducted in a tertiary level obstetric centre. Participants included women undergoing elective caesarean section under spinal anaesthesia. Following consent, a Flir T540 infrared camera captured thermographic images over the feet, patella, buttock, iliac crests, xiphisternum and axilla. Temperature was measured prior to spinal needle insertion (T0) and following clinical assessment when the block was deemed adequate. RESULTS: Thirty patients were included. Baseline temperature varied considerable by site. Spinal anaesthesia altered skin temperature in all areas of interest: right and left hallux (mean of differences (MD) +4.0°C and 5.2°C respectively, P <0.0001), right and left plantar (MD +6.1°C and 6.8°C respectively, P <0.0001), patella (MD -0.33°C, P=0.0445), buttock (MD -0.5°C, P=0.009), iliac crest (MD -0.7°C, P=0.0004), xiphisternum (MD -0.95°C, P <0.0001) and axilla (MD -0.71°C, P=0.0002). CONCLUSIONS: Following spinal anaesthesia thermographic imaging identified different patterns of skin temperature changes, with pronounced temperature increases measured in the feet and cooling of a lesser amplitude in the thoracic and lumbar dermatomes. Infrared thermography has the potential to provide objective measurement of sympathectomy.

Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η.

DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O4-Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4-O4 bond on processing by human DNA polymerase η (hPol η) was studied for oligonucleotides containing O4-methylthymidine, O4-ethylthymidine, and analogs restricting the O4-methylene group in an anti-orientation. Primer extension assays revealed that the O4-alkyl orientation influences hPol η bypass. Crystal structures of hPol η•DNA•dNTP ternary complexes with O4-methyl- or O4-ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn-O4-methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O4-methylthymidine with hPol η, inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O4MedT and O4EtdT when acted upon by hPol η.

Endotracheal intubation skills of medical students.

The ability and confidence of clinical medical students to insert endotracheal tubes correctly and quickly and to recognize oesophageal misplacement was evaluated. Ten (33%) of the medical students intubated the trachea correctly at their first attempt but 14 (47%) incorrectly identified the position of the endotracheal tube. However, recognition improved by their second and third attempts (70% and 80% respectively). Ninety-three percent of students intubated correctly on their third attempt. Although medical students can obtain better results at correct tube placement with repeated attempts under optimum conditions--a practice effect--and do better at recognizing correct tube placement there is still a persistent failure to recognize endotracheal tube misplacement, ie oesophageal intubation. It is the ability to recognize oesophageal intubation promptly that is a life-saving skill. This essential skill should be taught during the introductory anaesthesia programme through the use of clinical patients.

Total intravenous anesthesia with propofol for transsternal thymectomy in myasthenia gravis.

Patients with myasthenia gravis (MG) represent a significant management problem for the anesthesiologist. Anesthetic concerns center on the MG patient's unpredictable response to muscle relaxants and increased susceptibility to postoperative respiratory failure, resulting in prolonged dependence on mechanical ventilation. We describe the first reported use of total intravenous anesthesia with propofol to provide satisfactory surgical conditions in two patients with MG undergoing trans-sternal thymectomy. Propofol is a suitable drug for intubation and continuous infusion anesthesia, allowing fine control of anesthetic depth, good operating conditions, and a recovery profile suitable for MG patients undergoing surgery.

The relationship between intact PTH and biointact PTH (1-84) with bone and mineral metabolism in pre-dialysis chronic kidney disease (CKD).

OBJECTIVES: Abnormalities in PTH are implicated in the pathogenesis of bone abnormalities in chronic kidney disease (CKD)-mineral bone disorder (CKD-MBD). PTH concentrations are important in clinical decision and management. This emphasises the importance of providing an assay which measures biologically active PTH. We compared concentrations of intact PTH with biointact PTH (1-84) in CKD and end stage renal disease (ESRD) and investigated the relationship between the 2 PTH assays with bone and mineral laboratory parameters and bone mineral density (BMD) in CKD. DESIGN AND METHODS: We assessed 140 patients (61 in ESRD and 79 with CKD stages 1-4) in this cross-sectional study. We measured biointact PTH (1-84) as well as routine biochemical parameters on all subjects. In the CKD cohort, bone turnover markers; bone alkaline phosphatase (BAP) and tartrate resistant acid phosphatase (TRACP)-5b and bone mineral density (BMD) were also determined. RESULTS: In ESRD, intact PTH concentration was significantly higher compared to biointact PTH (1-84) (422 [443] v/s 266 [251] pg/mL, (p<0.001) with an average bias of 60%. In CKD, intact PTH concentration was also higher compared to biointact PTH (1-84) (79[55] v/s 68[49] pg/mL p<0.001) with an average bias of 18%. Only the biointact PTH (1-84) assay showed any significant correlation with serum calcium concentrations (r=-0.26, p<0.05) and phosphate (r=0.25, p<0.05) in CKD. Following multilinear regression analysis and adjustment for all significant co-variables, only eGFR, BAP and 25 (OH)vitamin remained significantly associated with intact PTH and biointact PTH (1-84). The strength of association was stronger between BAP and biointact PTH (1-84) (biointact PTH (1-84): p=0.007, intact PTH: p=0.01). In adjusted analyses, only biointact PTH (1-84) was significantly associated with BMD at the fore-arm (FARM) (p=0.049). CONCLUSIONS: The study confirms the differences between intact PTH and biointact PTH (1-84) in ESRD. Whilst there may be similarities in the diagnostic ability of both intact and biointact PTH (1-84), our data suggest that biointact PTH (1-84) assay may better reflect bone metabolism and BMD in CKD. Further longitudinal studies are needed.

Effects of surgical stimulation on autonomic reflex function: assessment by changes in heart rate variability.

Analysis of small oscillations in heart rate (known as heart rate variability or HRV) associated with the activity of homeostatic reflexes can provide a noninvasive measure of autonomic reflex function. We have investigated the effects of surgical stimulation on autonomic reflex function by assessment of changes in HRV. Healthy female patients undergoing laparoscopic tubal ligation were anaesthetized with either thiopentone-nitrous oxide-isoflurane (group I; n = 13) or continuous propofol infusion (group P; n = 13). Power spectral measurements of HRV (HRVtot = total HRV power; %HRVlo = percent of HRV power in the low frequency range) were obtained at the following times; control; before incision; after incision (3 min, 10 min and before skin closure). Compared with control values, measurements of HRVtot before incision were reduced significantly in both groups (group P: 16 (SEM 3)% of control; group I: 2.5 (0.7)% of control). With surgical stimulation, mean HRVtot in group P was restored to 55 (13)% of control (P < 0.01 compared with measurement before incision), whereas mean HRVtot in group I remained at less than 4% of control (ns). %HRVlo increased also in group P, from 49 (7)% to 75(3)% (P < 0.05), consistent with a shift in sympathetic-parasympathetic balance towards sympathetic dominance. These results suggest that surgical stimulation may have significant effects on the autonomic reflexes mediating HRV, and that such effects vary with anesthetic technique.

The end-tidal carbon dioxide detector. Assessment of a new method to distinguish oesophageal from tracheal intubation.

A new method to distinguish oesophageal from tracheal intubation using an end-tidal carbon dioxide detector was evaluated. In a prospective study on 50 healthy adult patients, the end-tidal carbon dioxide detector was reliably used to detect initial oesophageal intubation in 22 cases, and then to confirm tracheal intubation in all 50 patients. We conclude from this study that the end-tidal carbon dioxide detector is a reliable, rapid and easy method for the detection of oesophageal intubation.

Ventilation, thermal noise, and errors in cardiac output measurements after cardiopulmonary bypass.

BACKGROUND: The authors observed transient increases in the amplitude of respiratory variations in pulmonary artery blood temperature in many patients after cardiopulmonary bypass (CPB). This increased "thermal noise" may significantly influence measurements of thermodilution cardiac outputs (TDCO) performed during this time. METHODS: The authors recorded the peak-to-peak amplitude of respiratory variations in pulmonary artery blood temperature in 15 patients during the first 35 min after CPB. Possible relationships between the amplitude of these variations and the magnitude of temperature differences between commonly monitored body temperature sites (nasopharyngeal, rectal, bladder, and pulmonary artery) were also examined. In ten additional patients, the authors investigated the influence of these increased respiratory variations on TDCO measurements by correlating the maximum variation in three successive TDCO measurements with the peak-to-peak amplitude of the respiratory variations in pulmonary artery blood temperature. Potential error in TDCO measurements caused by these increased respiratory variations in pulmonary artery blood temperature were also examined using model calculations of the effects of respiratory variations in pulmonary artery blood temperature on measured TDCO thermal areas. RESULTS: In the first 15 patients, the mean amplitude of respiratory variations in pulmonary artery blood temperature after CPB (mean +/- SEM) were: (1) within 5 min after CPB, 0.037 +/- 0.004 degrees C; (2) 10 min after #1, 0.025 +/- 0.003 degrees C; (3) 20 min after #1, 0.019 +/- 0.003 degrees C; and (4) 30 min after #1, 0.012 +/- 0.002 degrees C. There were no significant correlations between the magnitude of the respiratory variation in pulmonary artery blood temperature and the observed temperature differences between body sites. Four patients had pulmonary artery blood temperature variations in excess of the maximum amplitude previously reported in man (0.05 degrees C). In the next ten patients, the maximum variation between three successive TDCO measurements taken at specified times in the respiratory cycle (end inspiration, end exhalation, and 3 s after end exhalation) was significantly correlated with the amplitude of respiratory variations in pulmonary artery blood temperature (r = 0.83, P < 0.001). Four patients with increased respiratory variations in pulmonary artery blood temperature had variations in TDCO measurements exceeding 2 l/min. Subsequent model calculations demonstrated that the magnitude of potential error in TDCO measurements is dependent on both the amplitude of the respiratory variations in pulmonary artery blood temperature and the baseline cardiac output. On the basis of these thermal area calculations, potential errors of 15-50% could be caused by respiratory variations in pulmonary artery blood temperature > 0.05 degrees C. CONCLUSIONS: The authors concluded that respiratory variations in pulmonary artery blood temperature are transiently increased in many patients after CPB, and that this increased "thermal noise" may cause significant errors in TDCO measurements.

Inadvertent suppression of a fixed rate ventricular pacemaker using a peripheral nerve stimulator.

Electromagnetic interference usually produces only minor effects in patients with pacemakers. Nevertheless, the possibilities of serious and even fatal consequences of this complication must be recognised. This case reports an unusual anaesthetic source of interference, caused by activation of a popular nerve stimulator, resulting in cardiac arrest in a patient with a fixed-rate ventricular pacemaker.

Pre-oxygenation: an easy method for all elective patients.

The transparent plastic facemask has been investigated as a pleasant method of pre-oxygenation for elective non-high risk cases in 60 healthy ASA I or II patients randomly allocated to two groups. The patients in the pre-oxgenated group (n = 30) received 8 litre min-1 oxygen through a plastic facemask for 3 min whereas those in a control group (n = 30) were not pre-oxygenated. In all patients anaesthesia was induced with propofol 2.5 mg kg-1, fentanyl 1 ug kg-1 and atracurium 0.6 mg kg-1. Manual ventilation of the lungs using a Mapleson A breathing system was performed for 2 min with 50% oxygen in nitrous oxide prior to oral intubation. Arterial saturation in the pre-oxygenated group rose significantly from a mean baseline value of 96.4 (+/- 0.9)% to 99 (+/- 0.8)% (P < 0.01) and then remained stable both after induction and intubation: 99.1 (+/- 0.8)% and 98.9 (+/- 1.1)% respectively. In the control group arterial saturation dropped sharply within 20 s following induction to a mean of 89.8 (+/- 3.1)%, and it was 30 s before arterial saturation reached the equivalent value in the pre-oxygenated group as a result of manual inflation of the lungs. An 8 litre min-1 oxygen flow via a standard transparent plastic facemask is a simple, feasible and acceptable method for routine pre-oxygenation for all elective cases.

Tracheal intubation in a mannikin: comparison of the Belscope with the Macintosh laryngoscope.

Tracheal intubation carries a risk of accidental oesophageal intubation; this is increased with inexperienced trainees, and in patients with a difficult airway. The recent introduction of an angulated laryngoscope, the Belscope, may permit a better view of the vocal cords and increase the accuracy of orotracheal intubation. To determine how easy it is to learn to use the Belscope compared with the traditional Macintosh laryngoscope, a group of medical students attempted to intubate a mannikin which had been modified to simulate a difficult intubation. Time to intubation was fast with both laryngoscopes, although faster with the Macintosh, but the Belscope produced an unexpected greater incidence of failed intubation.