Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

Pseudoxanthoma elasticum: biopsy of clinically normal skin in the investigation of patients with angioid streaks.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by fragmentation and calcification of elastic fibres with resultant pathological changes in the dermis, Bruch's membrane and blood vessels. Defects in Bruch's membrane produce angioid streaks on the retina but this appearance is not pathognomonic of PXE. Biopsy of clinically normal skin or scar tissue in patients with angioid streaks may show the histological features of PXE. OBJECTIVES: To test the hypothesis that biopsy of clinically normal skin is a useful investigation in patients with angioid streaks. METHODS: This prospective study investigated 18 consecutive patients with angioid streaks. Each patient underwent a full dermatological examination and was investigated for diseases known to be associated with angioid streaks. Axillary skin biopsies were taken from 14 consenting patients. RESULTS: Typical PXE was found in 11 patients. No other diseases associated with angioid streaks were identified. Five patients had angioid streaks in the absence of systemic disease. Two patients had nondiagnostic dermatological features which were not clarified by histology. Two of the 11 patients with PXE showed histological evidence of PXE from clinically normal axillary skin. However, in both cases flexural skin elsewhere showed the typical clinical and histological features of PXE. CONCLUSIONS: This study demonstrates the association between angioid streaks and PXE. However, it does not support the hypothesis that biopsy of normal-looking skin is helpful in the investigation of adult patients with angioid streaks.

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

Nickel oxide nanotubes: synthesis and electrochemical performance for use in lithium ion batteries.

Uniform and aligned Nickel Oxide (NiO) nanotube bundles have been synthesized by a template process. Individual nanotubes are 60 microm long with a 200 nm outer diameter and wall thickness of 20-30 nm. The synthesis involved forming Ni(OH)2 nanotubes that were subsequently heated to 350 degrees C in order to fully convert the product to NiO nanotubes. NiO nanotube powder was used in lithium-ion cells for assessment of lithium storage ability and electrochemical performance. Discharge capacity of the NiO nanotube electrode was in excess of 30% higher than that of the standard NiO nanocrystalline powder electrode after 20 cycles. Impedance data suggests the NiO nanotube electrode provides more controlled and sustainable Li diffusion when compared to the NiO reference powder electrode system.

Laparoscopic peritoneal lavage in staging gastric and oesophageal cancer.

AIMS: Accurate staging of gastric, oesophageal and oesophagogastric cancer is essential to avoid unnecessary laparotomies in patients where only palliation is appropriate. This requires a multimodal approach utilizing endoscopy, computed tomography and laparoscopy. Previous authors have found that the presence of free peritoneal tumour cells (FPTCs) detected at laparoscopy or laparotomy confers a poorer prognosis. However, various methods of peritoneal lavage are described. The aim of this study was to evaluate the prognostic value of our technique of peritoneal lavage. MATERIALS AND METHODS: 88 staging laparoscopies with peritoneal lavage were carried out between March 1997 and February 1999 on patients eligible for attempted curative resection of a gastric, oesophageal or oesophagogastric cancer. During laparoscopy the pelvis was irrigated with 200 ml of normal saline, with 100 ml aspirated and examined cytologically. Patients were followed-up until September, 1999. RESULTS: 11 patients had FPTC-positive cytology with a median survival following laparoscopy of 122 days (95% CI 82-161) with only a single patient surviving more than one year. In the FPTC-negative group, median survival was 378 days (95% CI 256,-). Log-rank Chi(2)=16.7, P<0.001. CONCLUSIONS: The presence of FPTCs detected by our technique is a contraindication to attempted curative resection - palliation only (medical or surgical) is appropriate.

Performance of a pentafluorophenylpropyl stationary phase for the electrospray ionization high-performance liquid chromatography-mass spectrometry-mass spectrometry assay of cocaine and its metabolite ecgonine methyl ester in human urine.

A pentafluorophenylpropyl (PFPP) bonded silica column has been used for the high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry assay (HPLC-ESI-MS-MS) of cocaine (COC) and its metabolite, ecgonine methyl ester (EME) in human urine. COC and EME were used as model basic solutes to demonstrate that a PFPP phase yields excellent results for the assay and validation of drugs in biological fluids. The assay was linear over three orders of magnitude (1.0-1000 ng/ml) and precision and accuracy of the assay was 4 and 15%, respectively. The limit of detection (LOD) for COC and EME was 1.6 and 2.8 pg on column, respectively. In addition, only a simple 1:10 dilution of the urine was necessary for the sample preparation procedure thus saving time on a laborious extraction step. The major advantage of the PFPP phase was the enhancement of the ESI-MS signal by providing good retention and good peak shape of COC and EME with a mobile phase of 90% acetonitrile. The MS signal for COC was a factor of 12 times greater on the PFPP phase than on the C18 phase.

The high performance liquid chromatography electrospray ionization mass spectrometry analysis of diverse basic pharmaceuticals on cyanopropyl and pentafluorophenylpropyl stationary phases.

Cyanopropyl (CN) and pentafluorophenylpropyl (PFPP) modified silica columns give good retention and good peak shape for the high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) analysis of several classes of basic drugs. These phases enhance the ESI-MS signal by providing good retention of basic drugs with a mobile phase containing 90% acetonitrile. With C18 columns, in order to achieve good retention of basic drugs, only a mobile phase containing less than 40% acetonitrile can be used. Higher concentrations of acetonitrile produce a larger MS signal in the ESI process; the MS signal was a factor of 9 and 12 times greater on the CN and PFPP phases when compared with the C18 phase for the analysis of codeine. The C18 phase required only 4.0-6.0% acetonitrile to obtain the same retention time for codeine. The CN and PFPP stationary phases can be used for the analysis of a range of basic drugs, including many compounds which are poorly retained on the popular C18 and C8 stationary phases. Applications of CN and PFPP columns in the HPLC/ESI/MS of basic drugs include the analysis of antimalarials, such as quinine, bronchodilators, such as salbutamol and tulobuterol, cardioactive drugs, such as procainamide and beta-blockers, tricyclic antidepressants (TCAs), such as protriptyline and trimipramine and alkaloids, such as morphine and codeine. The CN and PFPP phases are also useful for the analysis of bufuralol and its metabolite, hydroxy-bufuralol. All the above analyses were performed using the same mobile phase, 90% acetonitrile; thus the HPLC method development process was expedited. The CN and PFPP phases also gave reproducible retention times and peak shape after more than 8 h of analyses.

Optimized stationary phases for the high-performance liquid chromatography-electrospray ionization mass spectrometric analysis of basic pharmaceuticals.

Stationary phases were investigated for HPLC coupled with electrospray ionization mass spectrometry (ESI-MS) for the analysis of basic drugs. Tricyclic antidepressants (TCAs) and beta-blockers were used as model solutes. The functional groups, pentafluorophenyl (PFP), OH, CN or CH3 were attached to the silica via a propyl chain. The effects of these stationary phases as well as C8 and C18 phases on retention and peak shape of the basic drugs were studied. The CN and PFP phases adequately retained (tR of 2 to 6 min) the basic drugs when the mobile phase was composed of 90% acetonitrile, whereas with the C4, C8 and C18 phases, less than 40% acetonitrile had to be used to provide adequate retention of the basic drugs. Because acetonitrile provides better desolvation in ESI than an aqueous solvent, it produces an increased MS signal. As an example of the HPLC-ESI-MS analysis of the beta-blocker, pindolol, on a CN phase, the use of 90% acetonitrile in the mobile phase increased the ESI-MS signal by 790% when compared to a C18 phase which could use only 5% acetonitrile in the mobile phase for retention of the solute. In addition, the CN and PFP phases provided better peak shape than the OH phase and the hydrophobic phases (C4, C8 and C18) and ion-pairing or ion-suppressing agents were not required. The retention behavior of the TCAs and beta-blockers on each of the phases is described.

Rapid confirmation/quantitation of cocaine and benzoylecgonine in urine utilizing high performance liquid chromatography and tandem mass spectrometry.

A rapid, but sensitive and selective method for confirmation and quantitation of benzoylecgonine (BZE) and cocaine (COC) in urine by fast-gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) is described. The chromatographic separation was performed on a reversed phase column employing fast-gradient techniques. Matrix prepared standards, blanks, and QC's were filtered then aliquots were transferred into a 96 well plate. Injection volumes of 25 microL were made onto the analytical column, with the flow diverted from the atmospheric pressure ionization source for the first 0.5 min of the analysis. Simultaneous multiple reaction monitoring (MRM) of three discrete transitions for each compound were used to identify BZE and COC. Quantitation was achieved utilizing the most prominent parent-daughter transition and internal standard calibration techniques. The coefficients of variation (CV) for the analysis of these drugs ranged from 0.6% to 6.8% at a concentration of 150 ng/mL (n = 155). This method suggests that fast-gradient LC/MS/MS may be suitable for routine confirmation of immunoassay cocaine-positive samples.

Direct plasma injection for high-performance liquid chromatographic-mass spectrometric quantitation of the anxiolytic agent CP-93 393.

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC-ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC-ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10-1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 microl) were performed per column with unattended, automated analysis.

Clinical features and prognosis of children assessed for isolated small bowel or combined small bowel and liver transplantation.

The hepatic histology and clinical status of 37 children on long-term parenteral nutrition (PN) referred for consideration of small bowel transplantation were determined. Seventy five percent of the children had splenomegaly and plasma bilirubin level of greater than 100 mumol/L. All of 21 children who underwent liver biopsy, had increased fibrosis, but only half had established cirrhosis. Thirty-one children were considered to be in need of transplantation (combined liver and bowel transplant, 29; isolated bowel transplant, 2), but only 13 were stable enough to be placed on the transplant list. Seven out of the thirteen children waiting have died because of lack of size-matched organs, and the overall mortality rate of the 37 children was 70%. The main risk factors for death within 6 months were bilirubin level of greater than 100 mumol/L, splenomegaly, and cirrhosis (P = .01). The natural history of PN-associated liver disease is that of progressive liver failure and death 6 to 12 months after onset of cholestasis, defined as bilirubin level of greater than 100 mumol/L. The development of cirrhosis occurs after the onset of jaundice, so early referral may also permit some children to be offered isolated bowel transplantation, which has better outcome than combined liver and bowel transplantation.

From hydralazine to CGRP to man?

Attempts to selectively reduce tumour blood flow have, in the past, concentrated on the use of hydralazine. However, although this vasodilator can be highly effective in experimental animals, it is only at such high concentration as to result in a severe and clinically unacceptable reduction in systemic blood pressure. At clinically acceptable levels, the drug appears to produce a small increase in tumour blood flow. We have used the techniques of magnetic resonance spectroscopy as indicators of metabolism and blood flow in a search for vasoactive drugs that would produce an effective reduction in tumour blood flow without causing severe hypotension or other serious side effects. Single injections of either prazosin or CGRP are shown to be substantially more effective than hydralazine in causing a reduction in tumour blood flow without massive reduction in blood pressure. Even more effective was CGRP given by continuous infusion. In this case a three-fold reduction in tumour blood flow could be obtained with a reduction of only 15-20% in systemic blood pressure. All these studies, however, have been made with transplanted animal tumours. Using high-dose hydralazine and primary tumours that were either radiation or chemically induced, we obtained a success rate of only about a 35% in causing selective reduction in blood flow. In contrast, in a transplanted tumour line derived from one of the non-responding radiation-induced primary lesions, the success rate was about 95%, consistent with the majority of animal studies using transplanted tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in vascular response between primary and transplanted tumours.

The vast majority of studies on tumour vasculature are performed on transplanted tumours in rodents. However, it is known that there may be differences between primary and transplanted lesions. The purpose of this study is to test whether a specific vascular response is similar in primary tumours and in transplanted tumours derived from them. The technique used was to give an intraperitoneal injection of 5 mg kg-1 hydralazine, which is known to result in hypoxia in transplanted tumours. Changes in perfusion were indicated by changes in metabolism, monitored using 31P Magnetic Resonance Spectroscopy. The primary tumours were induced by local irradiation many months previously and only 4/11 (36%) of these responded to hydralazine. One of the non responders was subsequently transplanted into isogeneic mice to produce a tumour line which was histologically very similar to the primary. Of these 16/17 (94%) responded. The difference is statistically significant (P = 0.001). The reasons for this difference are not known. A number of possibilities are discussed and in the authors' opinion, the most likely cause is that it results from an artefact of transplantation.

Indium-111-labelled granulocyte accumulation in respiratory tract of patients with bronchiectasis.

18 patients with severe bronchiectasis and persistent purulent sputum production were investigated, by the use of gamma scans, between acute exacerbations of their symptoms to determine localisation and traffic of 111In-labelled granulocytes in the lungs. Labelled granulocytes appeared in the respiratory tract of 16 patients within 24 h of their injection. Uptake occurred almost exclusively in areas demonstrated by computed tomography (CT) to be bronchiectatic. However, 111In uptake was not demonstrated in 21 of the 55 segments showing bronchiectasis on CT, and these were therefore thought to be not actively inflamed. The whole-body loss of radioactivity up to 4-7 days following injection of the labelled cells ranged from 0 to 53% and correlated with 24 h purulent sputum volume (r = 0.45, p less than 0.01) and with CT extent of moderate and severe bronchiectasis (r = 0.40, p less than 0.05), but not with overall CT extent of bronchiectasis, age, forced expiratory volume in one second, or duration of purulent sputum production in years.