Towards monitoring real-time cellular response using an integrated microfluidics-matrix assisted laser desorption ionisation/nanoelectrospray ionisation-ion mobility-mass spectrometry platform.

The combination of microfluidic cell trapping devices with ion mobility-mass spectrometry offers the potential for elucidating in real time the dynamic responses of small populations of cells to paracrine signals, changes in metabolite levels and delivery of drugs and toxins. Preliminary experiments examining peptides in methanol and recording the interactions of yeast and Jurkat cells with their superfusate have identified instrumental set-up and control parameters and online desalting procedures. Numerous initial experiments demonstrate and validate this new instrumental platform. Future outlooks and potential applications are addressed, specifically how this instrumentation may be used for fully automated systems biology studies of the significantly interdependent, dynamic internal workings of cellular metabolic and signalling pathways.

Audit of operating theatre time utilization in neurosurgery.

Utilization of operating theatre time is an important issue in neurosurgery, in a National Health Service Hospital. NHS Trusts are under ever increasing pressure to meet specified 'targets' in relation to admissions and operations. We performed a retrospective audit on the utilization of neurosurgical operating theatres at Royal Preston Hospital, analysed the times required for various common neurosurgical operations, and broke them down into clinical (operating and anaesthetic) and non-clinical times. We have also looked at the adequacy of available theatre sessions, and the under or over-running of available theatre sessions. A detailed time-based evaluation of 810 procedures over a 16-month period is presented. The mean and 80th centile of the time taken for anaesthesia, surgery and other non-clinical activities are described along with the total time spent in theatre for common neurosurgical procedures. The mean times for transit, preparation for anaesthesia, preparation for surgery, recovery in theatre and time between cases were 16, 13, 14, 15 and 8 minutes, respectively. The mean time duration between the end of one surgical procedure and the beginning of the next was 101 minutes. It was found that actual operating time was surprisingly only 56% of the time available. These data could be used to schedule operating theatre sessions for neurosurgery in the UK, as we believe our practice to be representative of a majority of units in the country.

Dietary restraint, exercise, and bone density in young women: are they related?

PURPOSE: Women with high scores for dietary restraint have been found to have higher 24-h urinary cortisol excretion and a higher prevalence of subclinical ovulatory disturbances, both of which may be risk factors for bone loss. The purpose of this study was to explore relationships between dietary restraint and bone health in regularly menstruating young women. METHODS: 62 women (age: 21.7 +/- 2.5 yr) had body composition and total body and lumbar spine bone mineral density (BMD) and content (BMC) assessed using dual-energy x-ray absorptiometry. Dietary restraint was assessed using the restraint subscale from the Three-Factor Eating Questionnaire: 29 women had low restraint (LR; restraint score 0--5), 33 had high restraint (HR; restraint score 13--21). Exercise (h x wk(-1)) was assessed by questionnaire on two occasions. RESULTS: LR and HR women were similar in age and body composition (fat mass = 15.0 +/- 4.7 kg, lean mass = 40.9 +/- 4.9 kg), but HR women exercised more (3.4 +/- 1.7 vs 2.2 +/- 1.8 h x wk(-1), P < 0.05). Exercise was correlated with BMD and BMC, and when it was included as a covariate, total body BMC was significantly lower in HR than LR women. In multiple regression analysis, weekly hours of exercise and restraint score were significant predictors of total body BMD and BMC. CONCLUSION: The observations of this cross-sectional study suggest that high levels of cognitive dietary restraint, or associated factors such as higher cortisol, may attenuate the positive effects of exercise on bone in young women.

Cognitive dietary restraint is associated with higher urinary cortisol excretion in healthy premenopausal women.

BACKGROUND: Cognitive dietary restraint, assessed by the Three-Factor Eating Questionnaire restraint subscale, is associated with subclinical menstrual cycle disturbances. This association may be mediated by stress-activated cortisol release. OBJECTIVE: We assessed whether 24-h urinary cortisol excretion differs between women with high and low restraint scores. DESIGN: Participants (aged 21.6+/-2.5 y; n = 62) with normal-length menstrual cycles and high (n = 33) or low (n = 29) restraint scores completed a questionnaire describing weight history, dietary practices, and exercise. Cortisol, calcium, and creatinine were measured in urine collected over 24 h on a day when all food and beverages were provided and measured. Previously, 3-d food records and anthropometric measurements were obtained. RESULTS: Age, height, weight, body mass index, and length of menstrual cycle were similar between groups. The reported amount of exercise was higher (3.4+/-1.7 compared with 2.2+/-1.8 h/wk; P<0.05) and energy intakes (assessed from 3-d and 24-h food records) were lower in the high- than in the low-restraint group. Ratios of urinary cortisol (nmol) to creatinine (mmol) were higher in the high-restraint than in the low-restraint group (42.9+/-12.9 compared with 36.3+/-8.9; P<0.05), whereas ratios of urinary calcium (mmol) to creatinine were lower (0.3+/-0.1 compared with 0.4+/-0.2; P<0.05) in the high-restraint group. Urinary cortisol was not associated with exercise, nutrient intakes, or anthropometric measurements. CONCLUSIONS: High dietary restraint scores are associated with urinary cortisol, a biological marker of stress, and high cortisol excretion may affect bone health. Our results suggest that further research is warranted to clarify these associations and to determine whether they persist over time.

Effects of body mass and reproduction on the basal metabolic rate of brown long-eared bats (Plecotus auritus).

We measured basal metabolic rate (BMR) of nonreproductive and of breeding (pregnant and lactating) female brown long-eared bats (Plecotus auritus) to investigate the effects of intra- and interindividual variation in body mass and of reproduction on metabolism. The BMR of six nonreproductive females was measured between five and seven times at approximately 2-wk intervals over a period of 2.5 mo. There was a highly significant effect (P<0.001) of body mass on BMR of these nonreproductive females. The pooled within-individual scaling exponent (1.88) significantly exceeded the established mammalian interspecific exponent (0.75). In addition, we made single observations on 14 nonreproductive females to establish the effects of differences in mass between individuals. The mean BMR across all 14 individuals was 82 mW (+/-24 SD). There was a significant positive relationship between BMR and body mass across these individuals (r2=0.39), with a between-individual scaling exponent of 0.75. Inter- and intraindividual effects of mass on BMR were combined in a regression analysis that included mean body mass and deviation from mean mass on any given day as predictors. This regression model explained 55% of the variation in BMR. We made longitudinal measurements of BMR throughout reproduction and compared these with the predicted BMR of nonreproductive bats of the same body mass. Reproductive females exhibited temporal flexibility in BMR. BMR during pregnancy increased on a whole-animal basis but was significantly lower (by, on average, 15%) than BMR predicted for nonreproductive females of the same mass. Over a period of 1-75 d following birth, whole-animal BMR was greater than that during pregnancy, even though body mass declined after parturition. Hence, postbirth BMR was greater than the level predicted for nonreproductive females of the same mass. This study indicates that the scaling of BMR with body mass differs significantly within and between individuals and that there is a reduction of BMR in pregnancy and an elevation of BMR during lactation.

Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial cells: formation of intracellular lead-inclusion bodies and induction of apoptosis.

Occupational exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate, has been associated with lung cancer and respiratory tract toxicity. We have previously shown that apoptosis is a major mode of death in cultured rodent cells treated with soluble sodium chromate and particulate lead chromate. Here we report the cellular and molecular effects of lead chromate and sodium chromate in normal human lung small airway epithelial (HSAE) cells, which may be one of the targets for Cr(VI)-induced lung cancer and respiratory tract toxicity. Phagocytosed lead chromate particles and intracellular lead-inclusion bodies (LIB) were observed by transmission electron microscopy and confirmed by X-ray analysis. HSAE cells exposed to lead chromate and sodium chromate underwent dose-dependent apoptosis. The cellular uptake and genomic interactions of both Cr and lead (Pb) were examined by inductively coupled plasma mass spectrometry (ICPMS) coupled with a novel, direct-injection high-efficiency nebulizer (DIHEN). Using this approach, we have quantitated a dose-dependent formation of Cr-DNA adducts and DNA-associated Pb in lead chromate-treated HSAE cells. The formation of LIB in normal human lung cells exposed to lead chromate indicates that ionic Pb is released from the particles and thus might contribute to the cell toxicity caused by lead chromate. Internalization and dissolution of lead chromate particles and the interaction of ionic Cr and Pb with DNA, may be components of the mechanism of lead chromate carcinogenesis. Lead chromate-induced apoptosis may be a mechanism to eliminate cells with chromium- and/or lead-damaged DNA.

Ultratrace and isotope analysis of long-lived radionuclides by inductively coupled plasma quadrupole mass spectrometry using a direct injection high efficiency nebulizer.

The direct injection high efficiency nebulizer (DIHEN) was explored for the ultrasensitive determination of long-lived radionuclides ((226)Ra, (230)Th, (237)Np, (238)U, (239)Pu, and (241)Am) and for precise isotope analysis by inductively coupled plasma mass spectrometry (ICPMS). The DIHEN was used at low solution uptake rates (1-100 µL/min) without a spray chamber. Optimal sensitivity (e.g., (238)U, 230 MHz/ppm; (230)Th, 190 MHz/ppm; and (239)Pu, 184 MHz/ppm) was achieved at low nebulizer gas flow rates (0.16 L/min), high rf power (1450 W), and low solution uptake rates (100 µL/min). The optimum parameters varied slightly for the two DIHENs tested. The detection limits of long-lived radionuclides in aqueous solutions varied from 0.012 to 0.11 ng/L. The sensitivity of the DIHEN was improved by a factor of 3 to 5 compared with that of a microconcentric nebulizer (MicroMist used with a minicyclonic spray chamber at a solution uptake rate of 85 µL/min) and a factor of 1.5 to 4 compared with that of a conventional nebulizer (cross-flow used with a Scott type spray chamber at a solution uptake rate of 1 mL/min). The precision of the DIHEN ranged from 0.5 to 1.7% RSD (N = 3) for all measurements at the 10 ng/L concentration level (∼3 pg sample size). The sensitivity decreased to 10 MHz/ppm at a solution uptake rate of 1 µL/min. The precision was about 5% RSD at a sample size of 30 fg for each long-lived radionuclide by the DIHEN-ICPMS method. The oxide to atom ratios were less than 0.05 (except ThO(+)/Th(+) ) and decreased under the optimum conditions in the following sequence: ThO(+)/Th(+) > UO(+)/U(+) > NpO(+)/Np(+) > PuO(+)/Pu(+) > AmO(+)/Am(+) > RaO(+)/Ra(+). Atomic and oxide ions were used as analyte ions for ultratrace and isotope analyses of long-lived radionuclides in environmental and radioactive waste samples. The analytical methods developed were applied to the determination of long-lived radionuclides and isotope ratio measurements in different radioactive waste and environmental samples using the DIHEN in combination with quadrupole ICPMS. For instance, the (240)Pu/(239)Pu isotope ratio was measured in a radioactive waste sample at a plutonium concentration of 12 ng/L. This demonstrates a main advantage of DIHEN-ICPMS compared with α-spectrometry, which cannot be used to selectively determine (239)Pu and (240)Pu because of similar α energies (5.244 and 5.255 MeV, respectively).

A direct injection high-efficiency nebulizer for inductively coupled plasma mass spectrometry.

A simple, relatively low-cost direct injection high-efficiency nebulizer (DIHEN) is introduced for argon inductively coupled plasma (Ar ICP) spectrometry. The DIHEN may be operated at solution uptake rates of 1-100 microL/min. Analytical performance indexes for the DIHEN and fundamental characteristics of the aerosol produced are obtained using an ICP mass spectrometer (ICPMS) and a two-dimensional phase Doppler particle analyzer (2D PDPA), respectively. Results are compared to those obtained with a conventional crossflow pneumatic nebulizer (PN), equipped with a Scott-type spray chamber. Droplet sizes and velocities produced with the DIHEN are smaller than those reported for the direct injection nebulizer (DIN). The DIHEN offers optimal sensitivity at low injector gas flow rates (approximately 0.25 L/min) and high rf power (approximately 1.5 kW). For the 17 elements tested, detection limits (ppt) and sensitivities achieved with the DIHEN (at 85 microL/min) are similar to, or better than, those obtained on the same instrument using the PN (at 1 mL/min). However, because the primary aerosol is injected directly into the plasma, oxide-to-metal ion ratios (MO+/M+) are high, as in the case of the DIN. The utility of the DIHEN for the analysis of small-volume samples is demonstrated by microscale flow injection analysis (muFIA) of Cr bound to human lung DNA. Detection of Cr at the femtogram level is feasible.

Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer.

A novel method is described for the sensitive detection of chromium-DNA adducts. Chromium-DNA adducts were determined in 1 microgram of DNA from normal human lung fibroblasts exposed to sodium chromate using microscale flow injection analysis with a direct injection high-efficiency nebulizer and inductively coupled plasma mass spectrometry detection. The frequency of Cr-DNA adducts increased in a dose-dependent sigmoidal manner, indicating saturation and toxicity. The low detection limits (on the order of parts per trillion) allows the detection of as few as two Cr adducts per 10,000 bases, which, coupled with the small DNA sample requirement, makes this technique suitable for measuring metal-DNA adducts as biomarkers of exposure to toxic and carcinogenic metals such as Cr, in cultured cells, animals, and humans.

Improvement of hemispatial neglect with cold-water calorics: an electrophysiological test of the arousal hypothesis of neglect.

Introducing ice-water into the left ear of right-brain-damaged patients attenuates unilateral neglect symptoms. By examining EEG changes over each hemisphere during this procedure, we were able to test a hypothesis concerning the mechanism of cold-water calorics and the attention-arousal hypothesis of hemispatial neglect. We present a case study of an 83-year-old woman with a massive right-hemisphere CVA exhibiting severe hemispatial neglect. Caloric stimulation produced a leftward eye deviation to central position, and a temporary partial remission of neglect symptoms. Significant changes in EEG activation indicated a central mechanism associated with the regularization of eye gaze. Caloric stimulation also produced a significant interaction between EEG frequency band and hemisphere, indicating that while both hemispheres increased in cortical activation, the right hemisphere increase was significantly greater. This supports the activation-arousal hypothesis of neglect over the mutual inhibition model.

Elimination rate of 65Zn as a measure of food intake: a validation study in the mouse (Mus sp.).

We measured elimination of 65Zn in white mice (Mus musculus) using daily whole body counting. Thirteen male mice were randomly divided into three groups, each maintained at a different temperature. Each animal was labeled with 65Zn on day 0 and monitored over days 0-48 postinjection. Daily food intake and body masses of all the animals were measured. We evaluated the ability of derived components of the 65Zn elimination curves to predict food intake over different phases of the measurement period. Food intake was significantly different between temperature groups; temporal variation in food intake was not intercorrelated between groups. Whole body elimination of 65Zn involved a rapid decline over days 0-1, followed by a biexponential decline in counts over days 1-48. Components of the first phase of the biexponential elimination curve were not significantly related to food intake. The rate (k2) of isotope elimination in the second phase was significantly related to mean food intake over days 25-48, 13-24, and 37-48. Rate of turnover in the second phase of elimination, incorporating the variation in zinc body pool size (k2 x 1/N2), where N2 is the constant of the second phase of elimination, was the best predictor of food intake and accounted for 60% of the variability over days 37-48.

Immediate hypersensitivity reaction to cyclophosphamide.

We report a case of an acute anaphylactic reaction to intravenous cyclophosphamide in a patient with systemic lupus erythematosus. Skin testing for reactivity to cyclophosphamide and one of its metabolites, 4-hydroperoxycyclophosphamide, was performed on the patient and on 6 controls. The patient exhibited positive skin test responses; all controls had negative responses. The results suggest an acute hypersensitivity reaction related to sensitivity to cyclophosphamide and to 4-hydroperoxycyclophosphamide, through a shared antigenic determinant or dual sensitization.

Direct muscle neurotization recovers gastrocnemius muscle function.

This case report describes a devastating injury in which a distal motor nerve had been avulsed from the muscle belly and was not available for reconstruction. This otherwise irreparable posterior tibial nerve injury was successfully treated by direct neurotization of the muscle belly. The patient did have one small, intact, muscular branch to the medial gastrocnemius muscle; however, electrodiagnostic and clinical examination and recovery pattern suggested the neurotization procedure was responsible for the functional recovery. In the rare situation where no distal nerve is available, and tendon transfers or arthrodesis are inappropriate, direct muscle neurotization can be considered as a salvage technique.

An evaluation of the Deltatrac indirect calorimeter by gravimetric injection and alcohol burning.

We evaluated a 'state of the art' open hood type of indirect calorimeter (Deltatrac, Datex) to determine its stability and precision, and related these to in vivo measurements made with the same type of calorimeter. Three Deltatrac Metabolic Monitors were investigated at two centres over a period of twelve months by two methods of recovery: (i) gravimetric injection of a nitrogen/CO2 gas mix and (ii) ethanol burning using the manufacturers approved apparatus. We compared machine variation with in vivo variation in measurements of oxygen uptake and carbon dioxide production at rest in healthy subjects. The Deltatrac recovery of both oxygen and carbon dioxide was on average 4.8% and 4.7% higher respectively for the alcohol burning method. We suspected that this apparent over-recovery was because the alcohol burner produced a resistance to the airflow generated across the canopy. By gravimetrically injecting the gas mix into the canopy hose whilst the alcohol burner was attached we were able to confirm this error. There was no significant change in recovery for any of the Deltatracs by either method over the year of evaluation. The between-study variation for the machine and in vivo measurements were less than 4%, and the within-study variations were less than 6% and less than 7%, respectively. These studies re-emphasise that careful evaluation of commercially produced indirect calorimeters is essential before clinical measurements are made.

Efficacy of feedback training in long-standing facial nerve paresis.

An efficacious treatment has not been available to patients with aberrant regeneration of the facial nerve as a result of Bell's palsy or after acoustic neuroma excision. This prospective controlled trial examines the efficacy of electromyographic feedback versus mirror feedback as treatment strategies for patients suffering from long-standing (18 months minimum) facial nerve paresis. Twenty-five patients were randomly assigned to electromyography with mirror feedback or mirror feedback alone. Seven rural patients who did not undergo treatment served as controls. At 0, 6, and 12 months, facial motor function was objectively quantified by linear measurement of facial movement, visual assessment of voluntary movement, and electrical measurement of facial nerve response to maximal stimulation. Statistically significant improvements were noted in both electromyography and mirror-feedback groups with respect to symmetry of voluntary movement (P less than .03) and linear measurement of facial expression (P less than .01). The positive results of this controlled trial demonstrate that feedback training in combination with a structured home rehabilitation program is a clinically efficacious treatment for patients with facial nerve paresis.

Flight behavior of scolytid beetle in response to semiochemicals at different wind speeds.

The response of the striped ambrosia beetle,Trypodendron lineatum (Olivier) (Coleoptera: Scolytidae), to a semiochemical-baited funnel trap was studied in a wind tunnel in the first of two experiments. Wind speeds were tested over a range of 0.0-0.9 m/sec. Percent beetle capture decreased linearly with increasing wind speed within the range tested. A second experiment showed that in the presence of wind, beetles flew upwind to a semiochemical-baited substrate. In still air, they tended to fly randomly and erratically; yet in close proximity to the baited substrate, a greater proportion of the beetles were arrested in response to the chemical stimuli and landed on the substrate than when an airflow was present. These results suggest thatT. lineatum are capable of responding to semiochemicals under varied wind conditions typically present in a forest where they use wind to orient to olfactory stimuli. However, greater numbers are arrested in response to the stimuli under relatively still conditions.

Driving experience and the risk of traffic accident among motorcyclists.

In a case-control study on the risk of traffic accidents among motorcyclists in Singapore, we demonstrated an inverse monotonic relationship between accident risk and driving experience. As compared to drivers with less that 1 yr of driving experience, the odds ratios (adjusted for race, age and frequency of riding) for those with driving experience of 1-4, 5-9 and 10 yr or more were 0.60, 0.50 and 0.36, respectively. We propose that intensive in-circuit training of learner motorcyclists should replace the conventional on-the-road training on the basis that the former serves to increase their driving experience without subjecting the learner motorcyclists to the risk of sustaining road accident.

Roles of minor components in pheromone-mediated behavior of western spruce budworm male moths.

The behavior of male western spruce budworm moths,Choristoneura occidentalis Freeman, was observed in a flight tunnel in response to virgin females and synthetic sex pheromone components, alone and in blends. Pheromone blends were also compared in the field using sticky trap bioassays. Pheromones were incorporated into small rods of polyvinyl chloride. The blend of 92∶8 (E/Z)-11-tetradecenal-89∶11 (E/Z)-11-tetradecenyl acetate-85∶15 (E/Z)-11-tetradecenol (Ald∶Ac∶OH) that approximated that released from a virgin female moth elicited levels of response similar to those elicited by the female. This blend induced a significantly greater percentage of moths to fly upwind and land at the lure than did the Ald lure. In contrast to the flight-tunnel bioassays, the numbers of moths caught in Ald-baited sticky traps in the field were not significantly increased by the addition of Ac and OH lures. The net upwind groundspeed of flight in response to the 0.05% Ald lure was lower than that in response to the virgin females and was significantly increased by the addition of Ac + OH lures in two of three bioassays. The flight-tunnel bioassays support the hypothesis that the natural blend of major (Ald) and minor (Ac + OH) components stimulates the precopulatory behavior of western spruce budworm male moths at long range (> 1 m downwind) as well as at close range.

Effect of sex-pheromone concentration on behavior of three strains of western spruce budworm male moths.

The responses of male western spruce budworm moths,Choristoneura occidentalis Freeman, to a range of concentrations of the major sex pheromone, 92∶8 (E/Z)-11-tetradecenal (Ald), in polyvinyl chloride lures, were observed using the electroantennogram technique, a flight tunnel, and field-trapping bioassays. The responses to virgin female moths were also observed in the flight tunnel and field bioassays. The moths were from three strains: a nondiapausing laboratory colony; field-collected wild budworm; and laboratory-wild crosses. The mean peak amplitude of antennal response and the time required for the electroantennogram signal to return to the baseline after stimulation (lag) increased with Ald concentration in both laboratory and wild moths. However, at Ald concentrations of 0.005% and greater, the lag period of the wild male's antennae was significantly shorter than that of the laboratory male's. The mean number of moths caught in the field in delta sticky traps increased with Ald concentration, but the number of moths caught per trap was not significantly different between concentrations of 0.005 % and 0.5 %. The threshold concentration required to elicit upwind flight in the flight tunnel was between 0.0005 and 0.005% Ald; peak response occurred to 0.05 % Ald but was not significantly different from that to 0.005 % or 0.5% Ald. Moths from all three populations significantly reduced their net upwind groundspeed as they approached the pheromone lure. When pheromone concentration was increased, the net upwind groundspeed of laboratory and lab-wild moths, but not wild moths, was significantly reduced between 2 m and 1 m downwind from the pheromone lure. The three populations of moths differed significantly in the percentage of wing-fanning and copulatory attempts, and in the net upwind groundspeed of flight from 2 m to 1 m downwind from the lure.