A high-resolution voxel model for predicting local tissue temperatures in humans subjected to warm and hot environments.

This work describes and presents results from a new three-dimensional whole-body model of human thermoregulation. The model has been implemented using a version of the "Brooks Man" anatomical data set, consisting of 1.3x10(8) cubic volume elements (voxels) measuring 0.2 cm/side. The model simulates thermoregulation through passive mechanisms (metabolism, blood flow, respiration, and transpiration) and active mechanisms (vasodilatation, vasoconstriction, sweating, and shivering). Compared with lumped or compartment models, a voxel model is capable of high spatial resolution and can capture a level of anatomical detail not achievable otherwise. A high spatial resolution model can predict detailed heating patterns from localized or nonuniform heating patterns, such as from some radio frequency sources. Exposures to warm and hot environments (ambient temperatures of 33-48 degrees C) were simulated with the current voxel model and with a recent compartment model. Results from the two models (core temperature, skin temperature, metabolic rate, and evaporative cooling rate) were compared with published experimental results obtained under similar conditions. Under the most severe environmental conditions considered (47.8 degrees C, 27% RH for 2 h), the voxel model predicted a rectal temperature increase of 0.56 degrees C, compared with a core temperature increase of 0.45 degrees C from the compartment model and an experimental mean rectal temperature increase of 0.6 degrees C. Similar, good agreement was noted for other thermal variables and under other environmental conditions. Results suggest that the voxel model is capable of predicting temperature response (core temperature and skin temperature) to certain warm or hot environments, with accuracy comparable to that of a compartment model. In addition, the voxel model is able to predict internal tissue temperatures and surface temperatures, over time, with a level of specificity and spatial resolution not achievable with compartment models. The development of voxel models and related computational tools may be useful for thermal dosimetry applications involving mild temperature hyperthermia and for the assessment of safe exposure to certain nonionizing radiation sources.

Determining localized garment insulation values from manikin studies: computational method and results.

The localized thermal insulation value expresses a garment's thermal resistance over the region which is covered by the garment, rather than over the entire surface of a subject or manikin. The determination of localized garment insulation values is critical to the development of high-resolution models of sensible heat exchange. A method is presented for determining and validating localized garment insulation values, based on whole-body insulation values (clo units) and using computer-aided design and thermal analysis software. Localized insulation values are presented for a catalog consisting of 106 garments and verified using computer-generated models. The values presented are suitable for use on volume element-based or surface element-based models of heat transfer involving clothed subjects.

Manipulating the folding of membrane proteins: using the bilayer to our advantage.

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.

Can we identify the forces that drive the folding of integral membrane proteins?

Protein folding has been at the forefront of molecular cell biology research for several years. However, integral membrane proteins have eluded detailed molecular level study until recently. One reason is the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is nevertheless a large body of information on lipid properties and in particular on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to design efficient in vitro, lipid-bilayer folding systems for membrane proteins. Bacteriorhodopsin has been used as a model system for our initial studies: we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency seem to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer and the lateral pressure that the lipid chains exert on their neighbouring folding protein. These are generic properties of the bilayer that can be achieved with simple mixtures of many types of biological lipid and seem to be important in vivo.

Outpatient anterior cruciate ligament reconstruction: an analysis of charges and perioperative complications.

All outpatient anterior cruciate ligament (ACL) reconstructions using patellar tendon autograft performed at an accredited outpatient surgical center between 1994 and 1998 were prospectively studied. Hospital charges pertaining to the procedures were examined, and perioperative morbidities that might be attributed to an outpatient procedure were evaluated. The study group comprised 284 patients; average patient age at surgery was 28.7 years. Patients were subgrouped into group 1 (isolated ACL reconstructions; n=163), group 2 (ACL reconstructions and meniscal repair; n=48), and group 3 (ACL reconstructions and partial meniscectomy; n=73). Surgicenter facility charges, reoperation rate, complication rate, motion, pain management, hospital emergency room visits, hospital admission, and outpatient surgical facility visits were analyzed. Historical controls from our hospital and our initial outpatient pilot study (May 1994 through November 1995) were used as financial controls. The average surgical center charge for all patients was $3,443. On average, there was a $600 increase for all subgroups from May 1994 through November 1995 compared to December 1995 through August 1998. In the latter time interval, the fixed facility charges were $3,150, $4,075, and $4,275 for groups 1, 2, and 3, respectively. Overall, 19 (7%) patients required a reoperation including 7 (2.5%) patients who required arthroscopic debridement for symptomatic motion deficits. This study expands on our initial published report regarding hospital charges pertaining to an outpatient ACL reconstruction. Extended over another 4 years, we noted slight increases reflective of regional inflationary increases. Compared to our initial inpatient study (1988-1993), significant charge reductions were maintained. This study demonstrated a low complication rate and high patient subjective satisfaction level.

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.

Simultaneous ipsilateral femoral and tibial lengthening with the Ilizarov method.

Eight pediatric patients who underwent nine simultaneous ipsilateral femoral and tibial lengthenings with the Ilizarov external fixator were reviewed. The patient's demographics, diagnoses, corticotomy levels, mechanical axes, healing indices, amounts of lengthening, and complications were recorded. The patients' average age was 8 years 10 months (5 years 4 months-15 years 10 months) with an average follow-up of 49 months (30-88 months). The percentage of femoral lengthening averaged 16.7% (8-23%) with an average healing index of 28 days/cm (20-38 days/cm). The percentage of tibial lengthening averaged 18% (9.6-23.6%) with an average healing index of 29 days/cm (1940 days/cm). Four complications in three patients occurred as a direct result of the lengthening process. Three of the complications involved soft-tissue contractures, which were each successfully treated with one additional surgical procedure, whereas the fourth complication involved poor bone regeneration and required bone grafting and additional immobilization.

Membrane protein folding.

Investigating the in vitro refolding of proteins that naturally reside in biological membranes is a notoriously difficult task. Biophysical studies on model systems are beginning to provide a sound physical basis for membrane protein folding that should help to alleviate this problem. Highlights of these studies include insights into the interaction of transmembrane alpha helices, as well as into the important role that membrane lipids play in folding.

Sensing isothermal changes in the lateral pressure in model membranes using di-pyrenyl phosphatidylcholine.

In this work we present data from a homologous series of di-pyrenyl phosphatidylcholine (dipyPC) probes which can sense lateral pressure variations in the chain region of the amphiphilic membrane (lateral pressures are tangential to the interface). The dipyPC has pyrene moieties attached to the ends of equal length acyl chains on a phosphatidylcholine molecule. Ultraviolet stimulation produces both monomer and excimer fluorescence from pyrene. At low dilutions of dipyPC in model membranes the excimer signal is entirely intra-molecular and since it depends on the frequency with which the pyrene moieties are brought into close proximity, the relative intensity of the excimer to monomer signal, eta, is a measure of the pressure. We synthesised or purchased dipyPC probes with the pyrene moieties attached to acyl chains having 4, 6, 8 and 10 carbon atoms and then measured eta in fully hydrated bilayers composed of dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine (DOPC and DOPE respectively). Although the resolution of our measurements of lateral pressure as a function of distance into the monolayer was limited, we did observe a dip in the excimer signal in the region of the DOPC/DOPE cis double bond. As we isothermally increased the DOPE composition, and hence the desire for interfacial curvature, we observed, as expected, that the net excimer signal increased. However this net increase was apparently brought about by a transfer of pressure from the region around the glycerol backbone to the region near the chain ends, with the lateral pressure dropping above the cis double bond but increasing at a greater rate beyond the double bond.

Evidence that bilayer bending rigidity affects membrane protein folding.

The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH.