Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology.

Antarctic permafrost soils have not received as much geocryological and biological study as has been devoted to the ice sheet, though the permafrost is more stable and older and inhabited by more microbes. This makes these soils potentially more informative and a more significant microbial repository than ice sheets. Due to the stability of the subsurface physicochemical regime, Antarctic permafrost is not an extreme environment but a balanced natural one. Up to 10(4) viable cells/g, whose age presumably corresponds to the longevity of the permanently frozen state of the sediments, have been isolated from Antarctic permafrost. Along with the microbes, metabolic by-products are preserved. This presumed natural cryopreservation makes it possible to observe what may be the oldest microbial communities on Earth. Here, we describe the Antarctic permafrost habitat and biodiversity and provide a model for martian ecosystems.

Calibration of a subcutaneous amperometric glucose sensor. Part 1. Effect of measurement uncertainties on the determination of sensor sensitivity and background current.

The calibration of a continuous glucose monitoring system, i.e. the transformation of the signal I(t) generated by the glucose sensor at time (t) into an estimation of glucose concentration G(t), represents a key issue. The two-point calibration procedure consists of the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The estimation of G(t) is subsequently given by G(t) = (I(t)-I(o))/S. A glucose sensor was implanted in the subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). For each individual trial, S and I(o) were determined by taking into account the values of two sets of sensor output and blood glucose concentration distant by at least 1 h, the procedure being repeated for each consecutive set of values. S and I(o) were found to be negatively correlated, the value of I(o) being sometimes negative. Theoretical analysis demonstrates that this phenomenon can be explained by the effect of measurement uncertainties on the determination of capillary glucose concentration and of sensor output.

Calibration of a subcutaneous amperometric glucose sensor implanted for 7 days in diabetic patients. Part 2. Superiority of the one-point calibration method.

UNLABELLED: Calibration, i.e. the transformation in real time of the signal I(t) generated by the glucose sensor at time t into an estimation of glucose concentration G(t), represents a key issue for the development of a continuous glucose monitoring system. OBJECTIVE: To compare two calibration procedures. In the one-point calibration, which assumes that I(o) is negligible, S is simply determined as the ratio I/G, and G(t) = I(t)/S. The two-point calibration consists in the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The subsequent estimation of G(t) is given by G(t) = (I(t)-I(o))/S. RESEARCH DESIGN AND METHODS: A glucose sensor was implanted in the abdominal subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). The one-point calibration was performed a posteriori either once per day before breakfast, or twice per day before breakfast and dinner, or three times per day before each meal. The two-point calibration was performed each morning during breakfast. RESULTS: The percentages of points present in zones A and B of the Clarke Error Grid were significantly higher when the system was calibrated using the one-point calibration. Use of two one-point calibrations per day before meals was virtually as accurate as three one-point calibrations. CONCLUSION: This study demonstrates the feasibility of a simple method for calibrating a continuous glucose monitoring system.

Orbitally induced oscillations in the East Antarctic ice sheet at the Oligocene/Miocene boundary.

Between 34 and 15 million years (Myr) ago, when planetary temperatures were 3-4 degrees C warmer than at present and atmospheric CO2 concentrations were twice as high as today, the Antarctic ice sheets may have been unstable. Oxygen isotope records from deep-sea sediment cores suggest that during this time fluctuations in global temperatures and high-latitude continental ice volumes were influenced by orbital cycles. But it has hitherto not been possible to calibrate the inferred changes in ice volume with direct evidence for oscillations of the Antarctic ice sheets. Here we present sediment data from shallow marine cores in the western Ross Sea that exhibit well dated cyclic variations, and which link the extent of the East Antarctic ice sheet directly to orbital cycles during the Oligocene/Miocene transition (24.1-23.7 Myr ago). Three rapidly deposited glacimarine sequences are constrained to a period of less than 450 kyr by our age model, suggesting that orbital influences at the frequencies of obliquity (40 kyr) and eccentricity (125 kyr) controlled the oscillations of the ice margin at that time. An erosional hiatus covering 250 kyr provides direct evidence for a major episode of global cooling and ice-sheet expansion about 23.7 Myr ago, which had previously been inferred from oxygen isotope data (Mi1 event).

Electrochemical biosensors: recommended definitions and classification.

Two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission 1.7 on Biophysical Chemistry formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) have prepared recommendations on the definition, classification and nomenclature related to electrochemical biosensors: these recommendations could, in the future, be extended to other types of biosensors. An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device that is both disposable after one measurement, i.e. single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration, should be designated a single use biosensor. Biosensors may be classified according to the biological specificity-conferring mechanism or, alternatively, to the mode of physico-chemical signal transduction. The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been isolated, engineered or present in their original biological environment. In the latter cases. equilibrium is generally reached and there is no further, if any, net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor. Biosensors may be further classified according to the analytes or reactions that they monitor: direct monitoring of analyte concentration or of reactions producing or consuming such analytes; alternatively, an indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be achieved. A rapid proliferation of biosensors and their diversity has led to a lack of rigour in defining their performance criteria. Although each biosensor can only truly be evaluated for a particular application, it is still useful to examine how standard protocols for performance criteria may be defined in accordance with standard IUPAC protocols or definitions. These criteria are recommended for authors. referees and educators and include calibration characteristics (sensitivity, operational and linear concentration range, detection and quantitative determination limits), selectivity, steady-state and transient response times, sample throughput, reproducibility, stability and lifetime.

MicroITIES detection of nitrate by facilitated ion transfer.

A microITIES array, created by laser photoablation of a 12-microm polyester film, was used to investigate electroassisted anion transfer between two immiscible electrolyte solutions. Besides measuring directly the transfer of nitrate to the organic phase, the enhancement of transfer of the cation (K+) by facilitated anion (counterion) transfer was measured as well. In the presence of a triamide derived from tris(2-aminoethyl)amine (tren), which is known to function as a host for nitrate, the current responses for both nitrate and potassium transfer were monitored. The linear relationships between the current responses and nitrate concentration formed the basis of an anion sensor with a dynamic range from 0.1 to 5 mM. A dual facilitated transfer mechanism is proposed to explain the enhancement phenomenon.

Synthesis of bifunctional antibodies for immunoassays.

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.

The thickness of the human precorneal tear film: evidence from reflection spectra.

PURPOSE: Interferometric methods have considerable potential for studying the thickness of layers of the human tear film and cornea because of their ability to make noninvasive, accurate, and rapid measurements. However, previous interferometric studies by Prydal and Danjo yielded tear thickness values near 40 and 11 microm, respectively, considerably greater than estimates made by invasive methods of 4 to 8 microm. Using a modified version of Danjo's method, interference effects from the tear film and cornea were studied, with the aim of correlation with known structure and optical properties of the cornea and hence determining the most probable value of tear film thickness. METHODS: Reflectance spectra from the human cornea were measured at normal incidence. These spectra show oscillations whose maxima correspond to constructive interference between light reflected from the air surface and from some deeper surface. The frequency of these spectral oscillations is proportional to the thickness of the layer between the air surface and the second surface. Therefore, Fourier analysis of reflectance spectra can be used to determine the thickness of layers of the tear film and cornea. In the main experiment, 36 low-resolution spectra were obtained from six normal eyes for measuring thickness up to 100 microm. Control experiments included measurements of the time course of thickness changes and high-resolution spectra for measuring thickness up to 1000 microm. RESULTS: For the main experiment, in the thickness range 1 to 100 microm, the strongest peak in the Fourier transform was near 3 microm (range, 1.5-4.7 microm) beneath the air surface. In the range 20 to 100 microm, the strongest peak was near 55 microm (range, 50-59 microm) for all 36 spectra; none were in Prydal's range near 40 microm. This 55-microm peak is consistent with a reflection from the basement membrane of the epithelium. Time course measurements after a blink show that the 3-microm peak is not an artifact. High-resolution spectra gave a peak near 510 microm, corresponding to the complete thickness of the cornea (plus tear film). This peak had a contrast similar to that of the 3-microm peak. CONCLUSIONS: These studies did not confirm Prydal's estimate of approximately 40 microm. Nor were there prominent peaks near Danjo's value of approximately 11 microm, except in cases of probable reflex tears. Because the reflection at the aqueous-mucus boundary would be expected to be weaker than that from the epithelial surface, the 3-microm peak is unlikely to correspond to the aqueous layer (rather than the complete tear film). The proposal that the 3-microm peak corresponds to a reflection from the front of the cornea is supported by the demonstration of a peak of similar contrast from the back of the cornea. Thus, the current evidence consistently supports a value of approximately 3 microm for the thickness of the human precorneal tear film.

The general practice perspective on cancer services in Lothian.

OBJECTIVES: The aim of the present study was to obtain views from general practices about current and potential improvements to services for patients with suspected lung, large bowel, non-melanoma skin and breast cancer. METHOD: A questionnaire study was carried out of 134 general practices within the Lothian Health Board boundary. Information was sought about referral choices, communication, quality of care, liaison between community and hospital, health promotion, treatment outcomes and palliative care. Main outcome measures were determinants of primary care referral behaviour and clinical investigation strategies, and perception of quality in secondary care and health promotion services. RESULTS: Seventy-nine general practices (59%) returned completed questionnaires. One-fifth of practices maintained a cancer register, and 85% provide patient information about cancer prevention. Initial management was disease dependent. Most cases of suspected lung cancer, about half of suspected colorectal cancer cases and very few cases of suspected breast cancer were investigated in primary care before referral to hospital. Hospital referral depended on knowledge of local services. A minority of practices wanted referral guidelines. It was estimated that 92% of lung and breast cancer cases, 68% of colorectal cancers and 35% of skin cancers are seen within 4 weeks. Breast cancer care was rated more highly than that for other cancers. One-third ranked community nursing support as 'excellent' and 10-15% described it as 'fair' or 'poor'; 77% describe palliative care as 'excellent' or 'good'. Fifty-one percent believe that communication with hospital is 'excellent' or 'good'. Practices were sometimes unaware of the hospital's post-diagnosis management plan; communication was often too slow and practices often received 'poor' advice about symptom control. Eighty percent thought that hospital follow-up for breast, colorectal and lung cancer should be routine; 20% thought that it was indicated for non-melanomatous skin cancer. CONCLUSIONS: Communication problems between primary and secondary sectors need to be tackled innovatively and the perceived quality variation in services addressed-perhaps by developing local guidelines. Practices would welcome further education about health promotion resources and cancer epidemiology.

Catalytic antibodies for complex reactions: hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction.

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.

Interstitial glucose concentration and glycemia: implications for continuous subcutaneous glucose monitoring.

The changes in plasma glucose concentration and in interstitial glucose concentration, determined with a miniaturized subcutaneous glucose sensor, were investigated in anesthetized nondiabetic rats. Interstitial glucose was estimated through two different calibration procedures. First, after a glucose load, the magnitude of the increase in interstitial glucose, estimated through a one-point calibration procedure, was 70% of that in plasma glucose. We propose that this is due to the effect of endogenous insulin on peripheral glucose uptake. Second, during the spontaneous secondary decrease in plasma glucose after the glucose load, interstitial glucose decreased faster than plasma glucose, which may also be due to the effect of insulin on peripheral glucose uptake. Third, during insulin-induced hypoglycemia, the decrease in interstitial glucose was less marked than that of plasma glucose, suggesting that hypoglycemia suppressed transfer of glucose into the interstitial tissue; subsequently, interstitial glucose remained lower than plasma glucose during its return to basal value, suggesting that the stimulatory effect of insulin on peripheral glucose uptake was protracted. If these observations obtained in rats are relevant to human physiology, such discrepancies between plasma and interstitial glucose concentration may have major implications for the use of a subcutaneous glucose sensor in continuous blood glucose monitoring in diabetic patients.

An independently addressable microbiosensor array: what are the limits of sensing element density?

A microdisc sensor array, prepared by thin film technology, has been used as a model for miniaturized multi-functional biosensors. It consists of a series of wells, 20 microns in diameter, possessing a 1000 A Pt layer at the bottom that serves as the indicating electrode. The depth of the wells ranged from 2.3-24 microns, depending on the photoresist employed and the spinning speed used to coat the electrode interconnect grid. Ten such wells were arranged in a circular array within an area of radius 130 microns. The center to center distance between any two of the discs ranged from 30 to 155 microns. Each disc is connected by a conductive film line to corresponding pads on the side of the sensor chip. A cylinder placed on top of the chip array formed the electrochemical cell into which a common reference and counter electrode were placed. The reference electrode was operated at ground potential. Prior to the evaluation of enzyme sensors, an assessment of "chemical cross-talk", the perturbation of sensor response resulting from the overlap of proximal diffusion layers, was made using Fe(CN)6(4-). The preliminary conclusion is that the sensing elements probably must be separated by about 100 microns in order to avoid interference from adjacent sensors. A technique was developed for the precision delivery of enzyme and cross-linking agent to the 2.3 microns cavity, having a capacity of 4 pL. This procedure makes possible the preparation of sensor arrays capable of detecting different analytes by employing different enzymes. The sensors gave reasonably rapid (2-4 s) response with linearity (up to about 10 mM. However, the sensors in the center of the array clearly showed the effects of depletion of substrates by the surrounding sensors.

Purified protein derivative (PPD) as an immunogen carrier elicits high antigen specificity to haptens.

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1'-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.

High sensitivity ELISA determination of taxol in various human biological fluids.

Taxol (paclitaxel)--the natural product isolated from Pacific yew (Taxus brevifolia)--is a novel agent with high activity in the treatment of patients with several malignant tumors including those resistant to other cytotoxic drugs. The therapeutic index of this promising anticancer drug could be further increased by the exploration of its pharmacokinetic pharmacodynamic relationship in cancer patients. Since taxol is highly protein bound, a very specific and highly sensitive analytical method is required in order to determine free, protein unbound and biologically active taxol species in human physiological fluids: plasma; plasma ultrafiltrate; and salivary fluids. In order to accomplish this, a new indirect competitive enzyme-linked immunosorbent assay (ELISA), for quantitating such a low bioactive taxol concentration level, has been developed in our laboratories. This method uses taxol competitive inhibition of mouse anti-taxol antibodies binding to the solid phase coated antigen 7-succinyltaxol-bovine serum albumin. This indicates recognition of the active taxol in the solution phase, where a diluted horseradish peroxidase labeled goat anti-mouse enzyme conjugate is used. While employing this technique, after systematic optimization of the experimental conditions, we are able to detect the anticipated taxol in plasma ultrafiltrate and salivary fluids at the concentration level of subpicogram per milliliter. The working range of the assay is approximately five orders in magnitude, i.e. from pg ml(-1) to 100 ng ml(-1). The clinical part of this study verified the working range of the ELISA method using samples of physiological fluids from a cancer patient treated with 3 h intravenous (i.v.) infusion of this drug. Our results of taxol determination in plasma, plasma ultrafiltrate and saliva demonstrate the applicability of the newly developed ELISA method for further pharmacokinetic studies of free, biologically active taxol species in cancer patients.

Using routine accident and emergency department data to describe local injury epidemiology.

All 17330 Accident and Emergency Department (A and E) attendances following injury (67% of all A and E attendances by residents of the EH54 postcode (the town of Livingston) at St John's Hospital during 1995 and 1996 were examined to study local accident epidemiology. The overall annual injury attendance rate for males (245.7/1000) and females (148.0/1000) and sex and age group analyses show recognised patterns reflecting occupation and domestic circumstances. Higher attendance rates were associated with greater deprivation and living close to the hospital. The unique injury coding system used by the hospital offers the potential to highlight particular injury types occurring within population sub-groups. When linked with primary care and out-of-hours centre data, this could be useful in targeting preventive activities; this will be facilitated in this hospital, which will become part of a 'combined' acute and primary care trust from April 1999.