David A. Gough

Predicting protein–protein interactions from primary structure

MOTIVATION An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. The expectation is that this will provide a fuller appreciation of cellular processes and networks at the protein level, ultimately leading to a better understanding of disease mechanisms and suggesting new means for intervention. This paper addresses the question: can protein-protein interactions be predicted directly from primary structure and associated data? Using a diverse database of known protein interactions, a Support Vector Machine (SVM) learning system was trained to recognize and predict interactions based solely on primary structure and associated physicochemical properties. RESULTS Inductive accuracy of the trained system, defined here as the percentage of correct protein interaction predictions for previously unseen test sets, averaged 80% for the ensemble of statistical experiments. Future proteomics studies may benefit from this research by proceeding directly from the automated identification of a cells gene products to prediction of protein interaction pairs.

A telemetry-instrumentation system for chronically implanted glucose and oxygen sensors

An implantable potentiostat-telemetry system for in vivo operation of glucose and oxygen sensors is described. The device conveys signals from implanted chemical-specific sensors to a remote receiver via radio telemetry. Reference signals encoded in the analog FM transmission allow the receiver to automatically compensate for variability between simultaneously operated transmitters. The implant has several programmable operating modes that provide different signal gain and power consumption. All-CMOS circuitry is used, allowing operation for up to three months on a single lithium cell. Design, fabrication, and operation of the device are described.<<ETX>>

APPLICATION OF CHRONIC INTRAVASCULAR BLOOD GLUCOSE SENSOR IN DOGS

An intravenous glucose sensor was implanted in six dogs for 1–15 wk. The glucose sensor is a flexible cylinder, ∼0.2 cm diam and 30 cm long, with a tip containing immobilized glucose oxidase and catalase coupled to a potentiostatic O2 sensor. The sensor and a similar O2 reference sensor were implanted in the superior vena cava near the entrance of the right atrium. The sensor response was conveyed externally either by a telemetry system implanted nearby, surgically accessed leads, or chronically maintained percutaneous leads. Summing over the six implants, there was a total implantation period of 333 days during which glucose sensors were functional on demand. The sensor response showed agreement with conventionally assayed blood samples after accounting for a response lag. Sensor response to glucose showed little change over the implant period. Biocompatibility, enzyme lifetime, O2 availability, O2 sensor stability, and biochemical interference were not limitations. Results demonstrated that this sensor can function effectively as an implant in dogs for a period of months and has the potential for long-term operation.

Function of an implanted tissue glucose sensor for more than 1 year in animals

An implanted tissue glucose sensor can provide stable readings of glucose concentrations for more than a year. Sweet Sensor The need for an automatic, long-term implanted glucose sensor for use in diabetes therapy has been acknowledged by the diabetes care community for several decades. However, it was previously unclear that a sensor-telemetry system could be developed that functions long enough (1 year or more) to justify implantation of such a device, and that the implant could avoid encapsulation by tissues and rejection by the body. Gough et al. describe long-term studies in animals of a continuous, totally implanted glucose sensor that wirelessly transmits glucose concentration values to an external receiver. When available for use in humans, the implant will allow people with diabetes to monitor tissue glucose continuously and report via telemetry to an external receiver that displays the blood glucose information or relays it to a caregiver. Control of blood sugar, or blood glucose, is essential for normal daily activities. For people not having diabetes, blood glucose levels remain remarkably constant during the day, except for a brief modest rise after eating followed by a rapid return to a baseline. However, for people with diabetes, blood glucose levels can remain significantly elevated for long periods after eating and are only occasionally found at the ideal baseline. Further, for people who must inject insulin to bring blood glucose back toward baseline levels after eating, there is the real possibility of blood glucose levels becoming too low. High blood glucose is linked to a number of metabolic problems and can cause serious long-term consequences such as kidney disease, blindness, heart disease, and other problems that can reduce the quality of life, whereas blood glucose levels that are too low are immediately dangerous and can lead to temporary mental impairment, loss of consciousness, and accidents. All treatments for diabetes (insulin, oral medications, and potential new treatments in the research pipeline) are intended in some way to reestablish normal control of blood glucose. People with diabetes should measure their blood glucose concentration many times during the day. The sensor system described by Gough et al. provides an alternative to the most common means for measurement of blood glucose, which involves blood collection by “fingersticking” and glucose detection by placing a drop of blood in a handheld device. This method is inconvenient and only minimally acceptable to most people with diabetes, and is rarely performed frequently enough to follow rapid blood glucose changes. The new sensor system would also be an alternative to other forms of continuous glucose monitoring used by some people with diabetes, in which sensors are inserted into subcutaneous tissues by introducer needles and remain for 3 to 7 days before being replaced. Gough et al. reported on long-term glucose monitoring with the sensor-telemetry system implanted in subcutaneous tissues of pigs. Monitoring was carried out while the pigs were initially nondiabetic (for 3 weeks in one animal and nearly 1 year in the other) and, after the pigs had been made diabetic by administration of a laboratory drug, the monitoring continued in each animal for an additional 6 months, with diabetes being managed by frequent insulin injections and diet. These studies show that by proper design of the sensor system, previous reservations can be overcome. The long-term animal results reported by Gough et al. provide a foundation for human trials, which may require several years. An implantable sensor capable of long-term monitoring of tissue glucose concentrations by wireless telemetry has been developed for eventual application in people with diabetes. The sensor telemetry system functioned continuously while implanted in subcutaneous tissues of two pigs for a total of 222 and 520 days, respectively, with each animal in both nondiabetic and diabetic states. The sensor detects glucose via an enzyme electrode that is based on differential electrochemical oxygen detection, which reduces the sensitivity of the sensor to encapsulation by the body, variations in local microvascular perfusion, limited availability of tissue oxygen, and inactivation of the enzymes. After an initial 2-week stabilization period, the implanted sensors maintained stability of calibration for extended periods. The lag between blood and tissue glucose concentrations was 11.8 ± 5.7 and 6.5 ± 13.3 minutes (mean ± standard deviation), respectively, for rising and falling blood glucose challenges. The lag resulted mainly from glucose mass transfer in the tissues, rather than the intrinsic response of the sensor, and showed no systematic change over implant test periods. These results represent a milestone in the translation of the sensor system to human applications.

Whole-proteome interaction mining

MOTIVATION A major post-genomic scientific and technological pursuit is to describe the functions performed by the proteins encoded by the genome. One strategy is to first identify the protein-protein interactions in a proteome, then determine pathways and overall structure relating these interactions, and finally to statistically infer functional roles of individual proteins. Although huge amounts of genomic data are at hand, current experimental protein interaction assays must overcome technical problems to scale-up for high-throughput analysis. In the meantime, bioinformatics approaches may help bridge the information gap required for inference of protein function. In this paper, a previously described data mining approach to prediction of protein-protein interactions (Bock and Gough, 2001, Bioinformatics, 17, 455-460) is extended to interaction mining on a proteome-wide scale. An algorithm (the phylogenetic bootstrap) is introduced, which suggests traversal of a phenogram, interleaving rounds of computation and experiment, to develop a knowledge base of protein interactions in genetically-similar organisms. RESULTS The interaction mining approach was demonstrated by building a learning system based on 1,039 experimentally validated protein-protein interactions in the human gastric bacterium Helicobacter pylori. An estimate of the generalization performance of the classifier was derived from 10-fold cross-validation, which indicated expected upper bounds on precision of 80% and sensitivity of 69% when applied to related organisms. One such organism is the enteric pathogen Campylobacter jejuni, in which comprehensive machine learning prediction of all possible pairwise protein-protein interactions was performed. The resulting network of interactions shares an average protein connectivity characteristic in common with previous investigations reported in the literature, offering strong evidence supporting the biological feasibility of the hypothesized map. For inferences about complete proteomes in which the number of pairwise non-interactions is expected to be much larger than the number of actual interactions, we anticipate that the sensitivity will remain the same but precision may decrease. We present specific biological examples of two subnetworks of protein-protein interactions in C. jejuni resulting from the application of this approach, including elements of a two-component signal transduction systems for thermoregulation, and a ferritin uptake network.

Development of the Implantable Glucose Sensor: What Are the Prospects and Why Is It Taking So Long?

The development of an implantable glucose sensor for use in diabetes was first suggested in the 1960s (1–3). The sensor would provide an alternative to the present discrete methods of glucose determination that are based on intermittent blood sampling. Continuous glucose sensing would be particularly important in the detection and management of hypoglycemia. It would also allow early detection of hyperglycemia and provide a basis for insulin administration at more appropriate dosages and timing or for automatic insulin delivery from a pump. An implantable glucose sensor could also be used in parallel with other existing or potential forms of insulin replacement, such as transplantation or hybrid islet devices.

Novel biodegradable lipid nano complex for siRNA delivery significantly improving the chemosensitivity of human colon cancer stem cells to paclitaxel.

BACKGROUND Targeting of a specific subset of cells is mandatory for the successful application of siRNA mediated silencing in anticancer therapy. A recent theory suggests that colon cancer is sustained by a small subpopulation of cells, termed cancer stem cells (CSCs). These cells are characterized by their innate drug resistance properties, which is one of the key factors of chemotherapy failure. The goal of this study was to assess whether a novel siRNA delivery carrier, with an appropriate siRNA, targeted to CD133+ cells has the potential to improve the efficacy of conventional chemotherapy. METHODS In this study, a novel synthetic siRNA carrier platform was designed and synthesized. This carrier was composed of a cationic oligomer (PEI(1200)), a hydrophilic polymer (polyethylene glycol) and a biodegradable lipid-based crosslinking moiety. Libraries of polymers were synthesized by varying their lipid composition. Their transfection efficacy was evaluated in vitro using CHOK1 cells. The polymer was characterized using molecular weight, particle encapsulation assay, particle size and surface charge analysis. RESULTS It was demonstrated that the lipid composition in the polymer plays a critical role in transfection. Optimizing the physicochemical properties of the polymers is crucial in achieving favorable knockdown. Lipid nano complex with composition PEI-Lipid(1:16) was the optimum ratio for gene silencing. Additionally, silencing of multidrug resistance gene (MDR1) and treatment with paclitaxel play a synergistic role in increasing the efficacy as compared to the drug alone. CONCLUSIONS In the present study a novel siRNA delivery carrier system with an MDR1-targeting siRNA (siMDR1) effectively reduced the expression of MDR1 in human colon CSCs (CD133+ enriched cell population), resulting in significantly increasing the chemosensitivity to paclitaxel.

Frequency Characterization of Blood Glucose Dynamics

Examples of the frequency range of blood glucose dynamics of normal subjects and subjects with diabetes are reported here, based on data from the literature. The frequency band edge was determined from suitable, frequently sampled blood glucose recordings using two methods: frequency domain estimation and signal reconstruction. The respective maximum acceptable sampling intervals, or Nyquist sampling periods (NSP), required to accurately represent blood glucose dynamics were calculated. Preliminary results based on the limited data available in the literature indicate that although blood glucose NSP values are higher in most diabetic subjects, values in some diabetic subjects are indistinguishable from those of normal subjects. High fidelity monitoring sufficient to follow the intrinsic blood glucose dynamics of all diabetic subjects requires a NSP of ~ 10 min, corresponding to a continuous frequency band edge of ~ 1 × 10−3 Hz. This analysis provides key information for the design of clinical studies that include blood glucose dynamics and for the design of new glucose monitoring systems.

Role of xanthine oxidase in hydrogen peroxide production.

Increased production of oxygen free radicals may play a role in many diseases such as hypertension. As evidence indicates that xanthine oxidase may be involved in creating these reactive oxygen species, experiments were performed to additionally characterize hydrogen peroxide (H2O2) production in xanthine oxidase catalyzed reactions. In vitro measurements of hydrogen peroxide production from the xanthine/xanthine oxidase reaction were performed in buffered saline using an electrochemical technique, and the effect of allopurinol on inhibition of xanthine oxidase was determined. Experiments were also performed in blood plasma to characterize endogenous hydrogen peroxide producing capability and xanthine oxidase activity. In the presence of sodium azide, an inhibitor of catalase, peroxide production was measured in plasma after adding xanthine or xanthine oxidase and the results were similar to those obtained in buffered saline. When only sodium azide was added to plasma, hydrogen peroxide was produced at a level of 36.1 +/- 7.6 microM (n = 5). From these measurements, endogenous xanthine oxidase activity was estimated to be 6.5 +/- 0.3 mU/ml (n = 5). These results suggests that sufficient substrate exists in plasma to produce micromolar levels of hydrogen peroxide and xanthine oxidase may catalyze these reactions.

Erogress Toward a Potentially Implantable, Enzyme-Based Glucose Sensor

Our efforts toward the development of a potentially implantable, enzyme-based glucose sensor have concentrated on understanding in sufficient detail the most important component, the enzyme-containing membrane. We describe here some features that this membrane must have to operate as a constituent of a chronically implanted sensor. A model of reaction and diffusion within the membrane is outlined and methods of membrane characterization are reviewed.