EON: a component-based approach to automation of protocol-directed therapy

Provision of automated support for planning protocol-directed therapy requires a computer program to take as input clinical data stored in an electronic patient-record system and to generate as output recommendations for therapeutic interventions and laboratory testing that are defined by applicable protocols. This paper presents a synthesis of research carried out at Stanford University to model the therapy-planning task and to demonstrate a component-based architecture for building protocol-based decision-support systems. We have constructed general-purpose software components that (1) interpret abstract protocol specifications to construct appropriate patient-specific treatment plans; (2) infer from time-stamped patient data higher-level, interval-based, abstract concepts; (3) perform time-oriented queries on a time-oriented patient database; and (4) allow acquisition and maintenance of protocol knowledge in a manner that facilitates efficient processing both by humans and by computers. We have implemented these components in a computer system known as EON. Each of the components has been developed, evaluated, and reported independently. We have evaluated the integration of the components as a composite architecture by implementing T-HELPER, a computer-based patient-record system that uses EON to offer advice regarding the management of patients who are following clinical trial protocols for AIDS or HIV infection. A test of the reuse of the software components in a different clinical domain demonstrated rapid development of a prototype application to support protocol-based care of patients who have breast cancer.     

Children conceived by in vitro fertilisation after fresh embryo transfer

AIMS: To compare the outcome in in vitro fertilisation (IVF) children (after fresh embryo transfer) from multiple and singleton births with one another, and with normally conceived control children. METHODS: A cohort of 278 children (150 singletons, 100 twins, 24 triplets and four quadruplets), conceived by IVF after three fresh embryos had been transferred, born between October 1984 and December 1991, and 278 normally conceived control children (all singletons), were followed up for four years after birth. They were assessed for neonatal conditions, minor congenital anomalies, major congenital malformations, cerebral palsy and other disabilities. Control children, all born at term, were matched for age, sex and social class. RESULTS: The ratio of male:female births was 1.03. Forty six per cent of IVF children were from multiple births; 34.9% were from preterm deliveries; and 43.2% weighed less than 2500 g at birth. The IVF singletons were on average born one week earlier than the controls, weighed 400 g less, and had a threefold greater chance of being born by caesarean section. The higher percentage of preterm deliveries was largely due to multiple births and they contributed to neonatal conditions in 45.0% of all IVF children. The types of congenital abnormalities varied: 3.6% of IVF children and 2.5% of controls had minor congenital anomalies, and 2.5% of IVF children and none of the controls had major congenital malformations. The numbers of each specific type of congenital abnormality were small and were not significantly related to multiple births. IVF children (2.1%) and 0.4% of the controls had mild/moderate disabilities. They were all from multiple births, including two children with cerebral palsy who were triplets. CONCLUSIONS: The outcome of IVF treatment leading to multiple births is less satisfactory than that in singletons because of neonatal conditions associated with preterm delivery and disabilities in later childhood. A reduction of multiple pregnancies by limiting the transfer of embryos to two instead of three remains a high priority.     

Progesterone protects against lipid peroxidation following traumatic brain injury in rats

Large scale use of lysozyme for periplasmic release has been impeded by the cost of the pure enzyme and its subsequent presence as a contaminant in later downstream processing steps. In this paper, we discuss the use of lysozyme for pilot scale recovery of a periplasmic enzyme from E. coli. The effects of concentration of sucrose, lysozyme and cells on periplasmic enzyme release were examined. Lysozyme concentration can be reduced 5-fold from previous reports and a reduction in sucrose concentration from 20 to 15% (w/v) allows an improvement in centrifugal harvesting by reducing viscosity. High levels of release were still achieved using this technique and further improvements in yield were obtained by optimising other components of the releasing mixture. Results show that some release is still achieved in circumstances where no lysozyme use is possible. Results also indicate that a substantial proportion (up to 70%) of lysozyme remains bound to the cellular debris after its action and is removed with this material.     

The Target Preparation Laboratory at Daresbury

The Target Preparation Laboratory at Daresbury Laboratory is described. This laboratory provides targets for the UK Nuclear Physics community whose experimental programme is performed in several laboratories worldwide. Details are given of its present capabilities and range of targets produced.     

Merocyanine 540 as a fluorescence indicator for molecular packing stress at the onset of lamellar-hexagonal transition of phosphatidylethanolamine bilayers

The fluorescence of Merocyanine 540 (MC 540) is sensitive to the molecular packing of membrane lipids. Therefore, the fluorescence of MC 540 is expected to be sensitive to the curvature-related packing stress at the onset of the lamellar-hexagonal phase transition. We measured the fluorescence intensity of MC 540 when the temperatures of lipid bilayers approached their lamellar-hexagonal phase transitions. The fluorescence of MC 540 in the presence of egg and dioleoylphosphatidylethanolamine bilayers increased at the respective lamellar-hexagonal phase transitions of these lipids. Furthermore, increases in fluorescence intensity were also observed at temperatures just below their phase transitions. The enhanced fluorescence was not due to the specific interaction of the dye with the ethanolamine headgroup, because no such increase was observed when the probe was exposed to phosphatidylethanolamines which do not form hexagonal phase within the range of applied temperature. In addition, when the temperature of the lamellar-hexagonal phase transition was shifted, by the addition of a small amount of phosphatidylcholine, the dependence of the fluorescence intensity on temperature was modified accordingly. We postulate that the change of MC 540 fluorescence intensity at temperatures approaching the lamellar-hexagonal phase transition reflects changes in the partition of MC 540 into the fluid lipid phase. The change in partition is influenced by the curvature stress in bilayers at temperatures just below the lamellar-hexagonal phase transition.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.     

Differential regulation of angiotensinogen and AT1A receptor mRNA within the rat subfornical organ during dehydration

The present study describes the differential rostro-caudal patterning of angiotensinogen (AoGen) and AT1A receptor mRNAs in the rat SFO using specific and validated oligodeoxynucleotide probes for in situ hybridization. Highest levels of AoGen-specific gene expression were observed in the rostral region of the SFO with gradually decreasing intensity towards the caudal region of this sensory circumventricular organ lacking blood-brain barrier function. AoGen-related hybridization signals proved to be specifically prominent above cells in lateral aspects of the SFO, surrounding septal venules. Maximal expression of the AT1A receptor-specific gene, on the other hand, could be detected in the neuron-enriched, ventro-medial core region and dorsal annulus of the SFO, with low-intensity hybridization signals in its rostral and caudal parts. Water deprivation for 48 h, leading to extracellular hypertonic hypovolemia with elevated circulating AngII concentrations within the physiological range, caused a significant increase in AoGen-specific hybridization signals in the rostral and medial SFO regions. AT1A receptor gene expression and AngII receptor binding were markedly stimulated in the medial and caudal regions of the SFO (core and annulus) as compared to euhydrated animals. These data indicate, that mild dehydration differentially up-regulates AoGen- and AT1A receptor-specific mRNA formation as well as AT1 receptor binding in distinct regions of the SFO, and supports the involvement of different cellular subgroups in the expression of two major components of the central nervous renin-angiotensin system in this sensory circumventricular organ.