Counting cell number in situ by quantification of dimethyl sulphide in culture headspace.

A novel, non-invasive technique is reported for determining the numbers of cells in a culture by quantifying dimethyl sulphide (DMS) in the culture headspace as produced by the cellular enzymatic reduction of dissolved dimethyl sulphoxide (DMSO). Measured DMS concentrations, as performed using selected ion flow tube mass spectrometry (SIFT-MS), in the headspace of 2D and 3D cultures of four cell lines, viz. HEK293 (kidney), MG63 (bone), hepG2 (liver) and CALU-1 (lung), linearly correlate with starting cell number. Clear differences in the rates of production of DMS by the four cell types in both the 2D and 3D situations are seen. This novel analytical technique for cell enumeration offers a significant contribution to quality assessment across cell-based research and industry, including analysis of large scale culture systems, and for routine cell biology research.

Reactions of the selected ion flow tube mass spectrometry reagent ions H3O(+) and NO(+) with a series of volatile aldehydes of biogenic significance.

RATIONALE: It has been shown that aldehydes are often present in biogenic media. For their analysis by selected ion flow tube mass spectrometry (SIFT-MS), the rate coefficients and the product ion distributions for the reactions of the analyte ions H3O(+) and NO(+) with volatile aldehydes in the presence of water vapour are required. METHODS: The reactions of H3O(+) and NO(+) ions have been studied with a series of n-aldehydes ranging from acetaldehyde (designated as C2), through undecanal (C11) under the conditions used for SIFT-MS analyses (1 Torr He, 0.1 Torr air sample, 300 K) and over a range of sample gas absolute humidity from 1% to 7%. For comparison, the C5 pentanal isomer 3-methyl butanal, the unsaturated trans-2-pentenal and trans-2-octenal and the aromatic benzaldehyde were also included in the study. RESULTS: The H3O(+) reactions led to the formation of protonated molecules MH(+) and their hydrates MH(+)(H2O)0,1,2,3 , and (MH(+)-H2O). The NO(+) reactions resulted in the production of NO(+)M adduct ions and of [M-H](+) fragment ions. The percentages of the different product ions for each aldehyde are seen to be dependent on the air sample humidity. Kinetic modelling was used to quantitatively explain these observations and to obtain rate coefficients for the association reactions producing NO(+) M adduct ions. CONCLUSIONS: This detailed study has provided the kinetics data, in particular the product ion distributions, for the reactions of a number of volatile aldehydes, which allows their analyses by SIFT-MS in humid air, including exhaled breath, food emanations and other biogenic media.

Does tuition for journal referees work? A quantitative evaluation of a half-day tuition course.

AIM: Most consultants participating as referees in the peer review process of papers submitted to scholarly journals have had no training or tuition. This study attempted to evaluate the effect on reviewing of a half-day course held at the Royal Society of Medicine. METHOD: Registered consultant delegates were sent two 'doctored' papers, a case report and an original paper, well before the meeting to review at home using the standard computerized score sheet issued with referee requests by Colorectal Disease. At the start of the meeting the scores were entered into a computer as 'Before'. After each paper had been presented and then discussed, it was re-marked to give the 'After' score. The Before and After scores were compared with the post-meeting feedback forms. RESULTS: The Before and After scores provided by the participants for the two papers each reviewed were not significantly different for the questions relating to the publication/rejection decision. The Before score was higher than the After score for questions relating to the Abstract, Introduction and Method sections. Feedback forms regarding the tuition were universally positive and appreciative. CONCLUSION: Consultants already have the expertise to decide whether a paper should be rejected. The study day appears to give an additional insight that may change an initial opinion. In general a paper scored before the meeting was scored lower after it was presented and discussed at the meeting (the tuition).

Quantification by SIFT-MS of acetaldehyde released by lung cells in a 3D model.

Our previous studies have shown that both lung cancer cells and non-malignant lung cells release acetaldehyde in vitro. However, data from other laboratories have produced conflicting results. Furthermore, all these studies have been carried out in 2D models which are less physiological cell growth systems when compared to 3D models. Therefore, we have carried out further work on the release of acetaldehyde by lung cells in 3D collagen hydrogels. Lung cancer cells CALU-1 and non-malignant lung cells NL20 were seeded in these hydrogels at different cell concentrations and the release of acetaldehyde was measured with the Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) technique. The data obtained showed that the amount of acetaldehyde released by both cell types grown in a 3D model is higher when compared to that of the same cells grown in 2D models. More importantly, acetaldehyde from the headspace of lung cancer cells could be measured even at a low cell concentration (10(5) cells per hydrogel). The differential of acetaldehyde release could be, depending on the cell concentration, more than 3 fold higher for cancer cells when compared to non-malignant lung cells. This pilot study is the first to study acetaldehyde emission from albeit only two cell types cultured in 3D scaffolds. Clearly, from such limited data the behaviour of other cell types and of tumour cells in vivo cannot be predicted with confidence. Nevertheless, this work represents another step in the search for volatile biomarkers of tumour cells, the ultimate goal of which is to exploit volatile compounds in exhaled breath and other biological fluids as biomarkers of tumours in vivo.

A study of enzymatic activity in cell cultures via the analysis of volatile biomarkers.

Aldehyde dehydrogenase (ALDH) enzymes are responsible for the metabolism of aldehydes, including acetaldehyde (AA), and are linked to disease. We describe a method to study ALDH activity in cell cultures involving the measurement of AA concentrations in the gas/vapour phase. This has been achieved using selected ion flow tube mass spectrometry (SIFT-MS), developed for the rapid quantification of trace gases in humid media. Human cells of the hepG2 hepatocellular carcinoma cell line and primary bone marrow-derived mesenchymal stem cells (hMSCs) depleted AA from the culture media, but the application of ALDH inhibitors diethylaminobenzaldehyde (DEAB) and disulfiram (DSF), suppressed this depletion or in some cases resulted in elevated AA concentrations. Further, the cells were shown to reduce the dimethyl sulphoxide (DMSO) to dimethyl sulphide, which is mediated by methionine sulfoxide reductase A (MsrA) enzymes. Interestingly, this process was also inhibited by DEAB and DSF. The results of this study indicate that SIFT-MS gas phase analysis could be applied to the study of volatile metabolites of intracellular enzyme reactions, this having potential utility in disease research and drug discovery.

Breath acetone concentration; biological variability and the influence of diet.

Previous measurements of acetone concentrations in the exhaled breath of healthy individuals and the small amount of comparable data for individuals suffering from diabetes are briefly reviewed as a prelude to the presentation of new data on the sporadic and wide variations of breath acetone that occur in ostensibly healthy individuals. Data are also presented which show that following a ketogenic diet taken by eight healthy individuals their breath acetone concentrations increased up to five times over the subsequent 6 h. Similarly, the breath acetone increased six and nine times when a low carbohydrate diet was taken by two volunteers and remained high for the several days for which the diet was continued. These new data, together with the previous data, clearly indicate that diet and natural intra-individual biological and diurnal variability result in wide variations in breath acetone concentration. This places an uncertainty in the use of breath acetone alone to monitor blood glucose and glycaemic control, except and unless the individual acts as their own control and is cognizant of the need for dietary control.

Time-resolved selected ion flow tube mass spectrometric quantification of the volatile compounds generated by E. coli JM109 cultured in two different media.

Preliminary measurements have been made of the volatile compounds emitted by the bacterium E. coli JM109 cultured in the commonly used media Dulbecco's modified Eagle's medium (DMEM) and lysogeny broth (LB) using selected ion flow tube mass spectrometry, SIFT-MS, as a step towards the real time, non-invasive monitoring of accidental infections of mammalian cell cultures. In one procedure, the culture medium alone and the E. coli cells/medium combination were held at 37 °C in bottles sealed with septa for a given time period, usually overnight, to allow the bacterium to proliferate, after which the captured headspace was analysed directly by SIFT-MS. Several compounds were seen to be produced by the E. coli cells that depended on the liquid medium used: when cultured in DMEM, copious amounts of ethanol, acetaldehyde and hydrogen sulphide were produced; in LB ammonia is the major volatile product. In a second procedure, to ensure aerobic conditions prevailed in the cell culture, selected volatile compounds were monitored by SIFT-MS in real time for several hours above the open-to-air E. coli/DMEM culture held at close to 37 °C. The temporal variations in the concentrations of some compounds, which reflect their production rates in the culture, indicate maxima. Thus, the maxima in the ethanol and acetaldehyde production are a reflection of the reduction of glucose from the DMEM by the vigorous E. coli cells and the maximum in the hydrogen sulphide level is an indication of the loss of the sulphur-bearing amino acids from the DMEM. Serendipitously, emissions from DMEM inadvertently infected with the bacterium C. testosteroni were observed when large quantities of ammonia were seen to be produced. The results of this preliminary study suggest that monitoring volatile compounds might assist in the early detection of bacterial infection in large-scale bioreactors.

Determination of the deuterium abundances in water from 156 to 10,000 ppm by SIFT-MS.

In response to a need for the measurement of the deuterium (D) abundance in water and aqueous liquids exceeding those previously recommended when using flowing afterglow mass spectrometry (FA-MS) and selected ion flow tube mass spectrometry (SIFT-MS) (i.e. 1000 parts per million, ppm), we have developed the theory of equilibrium isotopic composition of the product ions on which these analytical methods are based to encompass much higher abundances of D in water up to 10,000 ppm (equivalent to 1%). This has involved an understanding of the number density distributions of the H, D, (16)O, (17)O and (18)O isotopes in the isotopologues of H(3)O(+)(H(2)O)(3) hydrated ions (i.e. H(9)O (4) (+) cluster ions) at mass-to-charge ratios (m/z) of 73, 74 and 75, the relative ion number densities of which represent the basis of FA-MS and SIFT-MS analyses of D abundance. Specifically, an extended theory has been developed that accounts for the inclusion of D atoms in the m/z 75 ions, which increasingly occurs as D abundance in the water is increased, and which is used as a reference signal for the m/z 74 ions in the measurement of D abundance. In order to investigate the efficacy of this theory, experimental measurements of deuterium abundance in standard mixtures were made by the SIFT-MS technique using two similar instruments and the results compared with the theory. It is demonstrated that the parameterization of experimental data can be used to formulate a simple calculation algorithm for real-time SIFT-MS measurements of D abundance to an accuracy of 1% below 1000 ppm and degrades to about 2% at 10,000 ppm.

Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes.

INTRODUCTION: Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. MATERIALS AND METHODS: Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real-time polymerase chain reaction (QRT-PCR) analysis. RESULTS AND DISCUSSION: The mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety-three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p < 0.05) in the expression of the same 11 of 93 and two of 93 apoptotic genes in individual SLE and RA patients tested as compared with controls. CONCLUSION: We propose that similarly altered expression of specific apoptotic regulatory genes (e.g., the death effector domain-containing DNA-binding protein and apoptosis-associated speck-like protein containing a CARD) occurs in the lymphocytes of individual patients with SLE or RA that may influence the extent and rate of spontaneous apoptosis in these autoimmune conditions.

[Course system of the Working Group for Gastro-intestinal Surgery Davos]. / Kurssystem der Arbeitsgemeinschaft für Gastrointestinale Chirurgie Davos.

Training in technical skills is essential for advanced surgical education. Training is moving more and more from the operating room to surgical training laboratories. A crucial impulse for this development came from Davos, where the first skills course was organized in 1984 after the formation of the Working Group for Gastro-intestinal (GI) Surgery (AGC Davos). Since this first course more than 5,000 residents have successfully completed the GI skills training course in Davos and many of the alumni are themselves teaching surgery today. The level and quality of this course has remained stable for 27 years on a high quality level although teaching has continuously been adjusted to modern techniques. The language of this international workshop is English. The number of applications exceeds the course capacity every year, which is an indication for the need of such training courses and should be principally included into the skills curriculum for surgeons.

Ensuring a graduated integrated progression of learning in a changing environment.

Surgical training is under threat in the changing environment of today. In the past, training consisted of an apprenticeship, which is no longer feasible or practical within the time limitation imposed on trainees currently, and so a new and innovative approach is required to train the surgeons of tomorrow. There is therefore a need for an explicit curriculum that ensures a graduated and integrated progression of learning in which both trainees and trainers are aware of what is required for each stage in training and for each surgical specialty. Such a curriculum has now been developed in the UK.

Teaching and assessing surgical competence.

Surgical competence and its assessment is one of the most hotly debated topics engaging the profession. In the current climate of diminishing working hours and shorter training, the surgical profession is having to address the complex issue as to how surgery as a craft specialty should be taught, and how to assess when an individual is competent within their chosen sphere as well as how that competence should be maintained. Internationally, there is political pressure upon the professional to achieve contracted activity to comply with political imperatives and, at the same time, to achieve a greater degree of specialisation. Within Europe, the working time directive has led to a shift system of rotas and this, along with a shorter overall period of training, has led to reduced time available to surgical trainees in which to learn their craft.

Assessing the technical skills of surgical trainees.

BACKGROUND: The aims were to determine whether tests of technical skill on simple simulations can predict competence in the operating theatre and whether objective assessment in the operating theatre by direct observation and video recording is feasible and reliable. METHODS: Thirty-three general surgical trainees undertook five simple skill simulations (knotting, skin incision and suturing, tissue dissection, vessel ligation and small bowel anastomosis). The operative competence of each trainee was then assessed during two or three saphenofemoral disconnections (SFDs) by a single surgeon. Video recordings of the operations were also assessed by two surgeons. RESULTS: The inter-rater reliability between direct observation and blinded videotape assessment was high (alpha = 0.96 (95 per cent confidence interval 0.92 to 0.98)). Backward stepwise regression analysis revealed that the best predictors of operative competence were the number of SFDs performed previously plus the simulation scores for dissection and ligation, the key components of SFD (64 per cent of variance explained; P = 0.001). CONCLUSION: Deconstruction of operations into their component parts enables trainees to practise on simple simulations representing each component, and be assessed as competent, before undertaking the actual operation. Assessment of surgical competence by direct observation and video recording is feasible and reliable; such assessments could be used for both formative and summative assessment.

Differential effects of primary hyperparathyroidism on ultrasound properties of bone.

Primary hyperparathyroidism (PHPT) may result in greater cortical than trabecular bone loss. Ultrasound is able to predict osteoporotic fracture risk independent of densitometric measurements, but little is known about the changes in ultrasound variables with PHPT. The aim of our study was to examine the effect of PHPT on ultrasound variables and bone density measurements at cortical (hand) and trabecular (lumbar spine and heel) sites, and to evaluate their reversibility following surgical treatment. We recruited 25 postmenopausal women diagnosed with PHPT ages 51-76 years (mean 62 years) and 95 postmenopausal controls ages 57-80 years (mean 67 years). Measurements were made at baseline and 1 year. Speed of sound (SOS) and broadband ultrasound attenuation (BUA) of the heel were measured using the Lunar Achilles (LA+) and McCue CUBA Clinical (CC). Amplitude-dependent speed of sound (AD-SoS) and ultrasound bone profile index (UBPI) of the fingers were measured using the IGEA DBM Sonic. Bone mineral density (BMD) of the hand and lumbar spine (LS) were measured by dual-energy X-ray absorptiometry (DXA). At baseline, hand BMD, LS BMD and heel BUA were significantly lower and finger UBPI significantly higher in the PHPT patients compared with controls ( p<0.001). There were no differences in Stiffness Index, heel SOS or finger AD-SoS between control and PHPT subjects. At 1 year postoperatively, there was a mean (+/-SD) increase in LS and hand BMD of 3+/-1% ( p<0.01). BUA at the heel increased (11+/-5%, p<0.001), and UBPI of the fingers decreased (17+/-7%, p<0.001) probably reflecting different modes of attenuation in trabecular (scattering) and cortical (absorption) bone. Stiffness Index, SOS of the heel and AD-SoS of the fingers did not change. BUA, UBPI and BMD returned towards normal postmenopausal values following surgery. There were no changes in BMD or QUS variables at 1 year in the control group. Quantitative ultrasound (QUS) measurements provide different information about bone structure than densitometric measurements and cannot be regarded as simply reflecting bone density. With further research the combined use of BMD and QUS could improve the assessment of skeletal status in patients with PHPT before and after surgery.

Structural insights into the mechanical regulation of molecular recognition sites.

Intriguing experimental and computational data are emerging to suggest that mechanical forces regulate the functional states of some proteins by stretching them into nonequilibrium states. Using the extracellular matrix protein fibronectin as an example, we discuss molecular design principles that might control the exposure of a protein's recognition sites, and/or their relative distances, in a force-dependent manner. Fibronectin regulates many cellular functions by binding directly to integrins. Although integrins have a key role in the transduction of force across the cell membrane by coupling the extracellular matrix to the cytoskeleton, the studies reviewed here suggest that fibronectin might be one of the molecules responsible for the initial transformation of mechanical force into a biochemical signal.

In vitro responses of gallbladder muscle from patients with acalculous biliary pain.

BACKGROUND: Diagnosis and identification of patients with acalculous biliary pain, who would benefit from surgery, remains a significant clinical problem. The cholecystokinin (CCK) provocation test helps diagnosis, but lack of consistency limits its usefulness. AIM: To characterize the response of gallbladder muscle strips, from patients with acalculous biliary pain, to hormonal and muscarinic stimulation and to compare these with strips from gallstone patients and normal controls. PATIENTS: Eleven patients with acalculous biliary pain were studied, 5 had a positive CCK test. Eight gallbladders from gallstone patients and 6 from partial hepatectomies were used for comparison. METHODS: Muscle strips from the body and neck of the gallbladder were suspended in organ baths and dose-response curves were constructed for CCK-8 and carbachol. RESULTS: In the acalculous group the strips from the body were less sensitive to carbachol than those of the neck. CONCLUSION: Since we found no differences in the CCK responses for the groups, it casts doubt over the effectiveness of the CCK test to diagnose acalculous biliary pain. Since carbachol sensitivity was different, it might be that a similar test using muscarinic stimulation would help in the diagnosis of this difficult group of patients.