Users can specify nearly unlimited numbers of virtual diabetic patients and test how different types of treatments, doses and dosing regimens, and even lifestyle dietary changes affect the daily BG profile Figure 2 c. If a simulated regimen is found unable to keep glycaemia within desired limits, users can experiment with alternative management regimens to try and improve the daily BG pattern [ 19 ].
People with diabetes, ideally, would be aiming for an HbA 1c of 6. The AIDA software and underlying model have been previously described in detail elsewhere in the literature [ 10 , 22 , 23 ]. Based on the large number of downloads, user comments clearly demonstrate that the AIDA educational software has so far stood the test of time [ 24 — 26 ].
MATERIALS AND METHODS
Like other software products more than 14 years after their original launch, however, the time is ripe to consider potential revisions to the existing AIDA program. Developments both in the clinical and computational arena clearly point to the need to revise and extend the current software. From a clinical perspective the existing AIDA v4 software does not cater for the latest insulin analogue preparations which have become increasingly used in the therapy of people with insulin-dependent type 1 diabetes mellitus.
Furthermore, the existing program is unable to simulate either non-insulin-dependent type 2 diabetic patients with endogenous insulin secretion or management regimens involving insulin infusions in addition to subcutaneous boluses of insulin. New insights into the processes involving carbohydrate metabolism should also appear in an updated version of the educational simulator in order to fully reflect the complexity of modern day diabetes therapy. For instance, in clinical practice the regulation of the BG concentration is mainly achieved by the action of three control variables: This implies a need to extend the scope of input variables included in the underlying AIDA model.
There are also a number of technical issues to be resolved about the current software. Furthermore, it is necessary to respond to some technical requests of AIDA users and resolve certain Turbo Pascal display problems that seem to manifest themselves on the latest notebook computers. The flexibility and user friendliness of the user interface could also clearly be improved.
The main features of the current and future planned versions of the AIDA software are contrasted in Table 1. It is evident that AIDA should remain a user-friendly program that implements a novel physiological model of the glucose-insulin system. This paper aims to present both the clinical and technical results achieved to date in the current phase of the revision process. The first update to the AIDA v4. These novel developments will be overviewed in turn. The underlying AIDA model consists of glucose and insulin submodels.
BG levels are controlled by various glucose fluxes into and out of the blood stream. These fluxes are complex functions of glucose and insulin levels, some of which vary according to a diurnal rhythm [ 23 ]. As a first stage to updating the AIDA simulator, a decision was taken to focus on the appearance of insulin in the plasma following a subcutaneous injection—thereby incorporating more novel insulin analogues into the program. Following a subcutaneous injection, soluble insulin forms a subcutaneous depot, where it is present in several multimeric, primarily hexameric and dimeric, forms.
The subcutaneous depot is cleared by absorption of dimeric insulin molecules into the vasculature [ 28 ]. Although absorption of hexameric insulin has been reported, it is considered not significant as compared to dimeric insulin [ 29 , 30 ]. Various insulin absorption models have been proposed which vary in their degree of complexity. Virtually all of them handle short-acting regular insulin preparations, while a few handle intermediate-acting insulin and novel insulin analogues [ 28 , 31 , 32 ].
However, this model was developed at the end of the s and thus insulin analogues were not included. A more comprehensive model, which has focused more on physiology and pharmacokinetics, has been described by Mosekilde and colleagues [ 34 ]. This approach was later modified by Trajanoski et al. The model was also extended to support monomeric insulin analogues. Being more physiologically based, the model proposed by Mosekilde et al.
The Trajanoski model was extended to deal with the long-acting insulin analogue glargine, thereby covering the whole range of insulin preparations currently used in medical therapy. Later works have subsequently appeared in the literature that also consider insulin glargine, but where the diffusion process is neglected or approximated [ 42 — 44 ].
The generic insulin absorption model planned for inclusion in AIDA v4 [ 37 , 38 ] represents diffusion of insulin through the subcutaneous depot, transformation between the different insulin states—hexameric, dimeric, and crystallized in the case of the insulin glargine —and absorption through capillary walls.
AIDA-based real-time fault-tolerant broadcast disks
Due to diffusion, the model is no longer a set of ordinary differential equations, but partial differential equations PDEs dependent on both time and space. Since the diffusion process is considered homogeneous and isotropic, the system of PDEs is unidimensional in space distance from the injection site. The generic model is based on published data from the literature. In the study by Mosekilde et al. Typical values for insulin injection into the thigh of a fasting person with type 1 diabetes are summarized in Table 2.
As reported by Trajanoski et al. Further model simplification like linearization or aggregation of distributed effects cannot be performed, since the essential characteristics of the model would be lost if linearization was done. On the other hand, model decomposition is not possible, as it is impossible to measure insulin with different association states in the subcutaneous depot. Therefore, a parameter set has been chosen from published in vivo and in vitro experiments as shown in Table 3. According to the assumptions of Trajanoski et al.
Therefore, for the absorption rate constant for soluble insulin, the final slope of the absorption curve of Kang et al. Since even large changes of do not alter the results significantly, in the study by Trajanoski et al. As the insulin flow into the blood stream is markedly slower than the elimination of insulin from the plasma, plasma insulin kinetics are typically described as a single compartment model representing the blood pool and some extravascular space from where there is a first-order elimination of insulin.
This is the modeling approach that has been used for the insulin model [ 33 ] incorporated within the existing AIDA v4 program. Model equations are integrated by separately computing the glucose and insulin submodels. For simulating type 1 diabetic patients the insulin submodel is considered independent of the glucose part of the model. As none of the parameters associated with the kinetics are assumed to be patient-specific, the insulin concentrations can be precomputed for any possible insulin delivery and stored prior to use by AIDA.
Insulin levels resulting from any particular insulin regimen are computed by summing up the precomputed individual contributions corresponding to the preparations and doses as used in the particular insulin regimen. This technique allows the glucose subsystem exhibiting slower dynamics to be simulated with a minute step size.
It is noted that integrating insulin equations in real time would require a much smaller step size less than 1 minute due to the short halftime about 5 minutes of insulin in the plasma. Although insulin levels corresponding to different types and doses of subcutaneous insulin preparations need to be determined only once, these calculations are still time-consuming because of the complexity of the generic insulin absorption model.
As the coupled PDEs have no closed solution, integration should be done numerically. It is considered that insulin after the injection forms a spherical volume in the adipose tissue and starts to diffuse out symmetrically. Hence, a numerical implementation has been carried out by means of a spatial discretisation consisting of spherical shells with equal volume, based on the work of Trajanoski et al.
Initial conditions are provided by the amount dose and type of the injected insulin. For rapidly acting, short-acting, and intermediate-acting insulin, it is considered that all the insulin is in the inner shell in chemical equilibrium between hexameric and dimeric forms. The volume of the inner shell corresponds to the injected volume. For the outermost spherical shell it is considered that the insulin concentration is null outside the considered spherical depot. Thus, a varying number of shells is considered here depending on the dose and type of insulin.
AIDA-based real-time fault-tolerant broadcast disks
To calculate the required number of shells, the absorbed insulin flow is computed in two different ways that become equivalent for a large enough spherical depot Figure 4: In the first case a , if the insulin concentration outside the considered spherical depot is significant, the computed value will underestimate the actual insulin absorption flow, since this insulin will be neglected.
However, in the second case b , the insulin concentration will be considered as absorbed, yielding an overestimation. Profiles computed by each of the methods will converge as the number of shells is increased, and thus the radius of the spherical depot increases. As computational time will increase with the number of shells considered, the solution is accepted as a compromise between efficiency and precision. The number of shells is considered adequate when the area under the curve of the calculated absorption profile i. One percent can be considered adequate given that these losses only occur for small doses that are hardly ever administered.
This represents a good compromise between speed and precision. For smaller doses the injected volume is small, too. This automatically leads to rather small shell radii. Since the volume is kept constant, the further from the injection site, the thinner the shell will be. Furthermore, the smaller the radii the higher the diffusion speed and the higher the ratio between shell surface and volume.
Therefore, for small injection doses, the insulin diffuses very quickly from the inner to the outermost shell and beyond, which leads to the loss if the number of shells is not sufficient. Although for future paediatric use such issues about fractional insulin injection dosages may potentially become of greater significance.
As can be seen, the radii are within a reasonable range with respect to the thickness of the subcutaneous tissue. Regarding time discretisation, the Euler method is applied with a time step of 0. The computational burden to actually calculate insulin absorption flows is not trivial but does not exceed the capabilities of modern personal computers either.
This remains true for the vast majority of insulin injection doses. It is only when calculating extremely low doses that much longer computational times are observed. As can be seen, the number of required shells may increase dramatically for low insulin doses Figure 5. For this reason, AIDA v4 relies on precomputed insulin profiles. Packed with a handy installer, it comes as a 1. It is freely available from http: Installing and starting DOSBox is a straightforward process.
DOSBox also offers a convenient way to configure itself. Among these are the keyboard layout, the emulation speed, and all the steps to be executed automatically right after starting DOSBox. In this way, DOSBox can be tailored to suit the hardware and software environments found on the host computer. All possible settings are documented at the DOSBox website http: The possibility to configure the keyboard layout is especially important as AIDA is used internationally. Interestingly, the display problems, AIDA v4.
As neither special sound output nor game-relevant hardware support e. This is an advanced open source installation system that can be used for free. NSIS is especially suited for the AIDA installation as it allows more complex tasks to be performed than mere file unpack and copy routines Figure 6 a. Instead, via its inherent script system, it can, for instance, call any operating system application programming interface API functions. In this way, it is possible to query, for example, the keyboard layout of the computer on which AIDA is to be installed.
Moreover, NSIS supports several handy script functions to edit text files. Thus, it can perform all the configurations of the DOSBox environment. Furthermore, being open source software and freeware it is very compatible with the AIDA freeware ethos. The stability of the heterologous passenger domain on the bacterial cell surface and the efficiency of translocation across the outer membrane are crucial issues in the process of autodisplay.
The outer membrane protease OmpT is known to degrade heterologous passenger proteins displayed by autotransporters on the cell surface 21 , 24 , and the presence of the periplasmic oxidoreductase DsbA, required for the efficient formation of disulfide bonds in the periplasm 1 , has been shown to hamper the translocation of passenger domains containing stable tertiary structures that contain disulfide bonds Interestingly, full-length FP77 was expressed in all E. The differences observed might reflect distinct phenotypes of the outer membrane.
The protease-resistant core of the AIDA-I autotransporter domain, migrating at 37 kDa, remains embedded in the outer membrane after trypsin treatment, confirming previous findings obtained with other heterologous passenger domains Surface targeting and protease accessibility of FP77 and FP Membrane preparations of E. Integrity of the outer membrane of FPexpressing cells. The integrity of the outer membrane was assessed by a control experiment using the outer membrane protein OmpA as a marker 14 , In cells displaying a high degree of membrane disorder, the periplasmic C-terminal domain of OmpA becomes sensitive to trypsin when whole cells are treated.
We subjected JK cells expressing FP77 to trypsin digestion in order to monitor membrane integrity. This experiment was also performed with JK pJM cells, leading to the same results data not shown. These results clearly indicate that the periplasmic domain of OmpA was not affected by the action of trypsin, demonstrating that the outer membranes of JK cells expressing FP50 or FP77 are physiologically intact.
Protease accessibility of OmpA. Functional surface expression of Bla. To distinguish between periplasmic and surface-located activity of this enzyme, we set up an in vivo assay for the cleavage of penicillin G using physiologically intact JK pLAT cells, since penicillin G penetrates the outer membrane poorly The following strains were employed as controls: Control ii allows periplasmic and surface-exposed Bla activity to be distinguished, while control iii permits the detection of potential membrane disorders caused by the artificial AIDA-I fusion proteins.
Such disorders might lead to enhanced diffusion of penicillin G into the periplasm and subsequent degradation by the action of prematurely folded but not exported Bla. Table 2 shows that JK pLAT , displaying Bla on the cell surface, has high whole-cell Bla activity, leading to the rapid cleavage of penicillin G. However, the Bla activity of JK pJM does not differ significantly from that of JK pLAT83 despite the higher copy number of the former plasmid and the expression of large amounts of FP50 in the outer membrane.
This could also be demonstrated using cephaloridine as the substrate. Whole-cell penicillinase activity of E. Assessing the enzymatic activity of surface-displayed Bla.
According to the model of autodisplay established by Pohlner et al. Consequently, it is important to determine what percentage of the molecules in which the passenger domain is displayed on the bacterial surface are functional. To compare the enzymatic activity of the surface-displayed Bla with the purified periplasmic enzyme, the Bla activity of JK pLAT cells was determined with cephaloridine as the substrate Table 2. Subsequently, a stock solution of the purified periplasmic enzyme was diluted until the activity of the diluted Bla solution was equal to the activity obtained with the amount of JK pLAT cells present in 1 ml of a suspension with an OD of 0.
The amount of soluble Bla present in the diluted Bla solution was compared semiquantitatively by Western blotting to the amount of FP77 present in 2. Figure 5 shows that the amount of Bla in JK pLAT cells lane 2 is significantly higher than the amount in the Bla solution with the same enzymatic activity lane 1.
The Bla sample with a fivefold-higher concentration of the enzyme lane 7 shows a signal of the same strength as that in 2. Semiquantitative analysis of the enzymatic activity of surface-displayed Bla. Increasing amounts of a solution of commercially available purified TEM-Bla were compared by Western blot analysis with the amount present in 2. Influence of cultivation conditions on whole-cell penicillinase activity. Whole-cell penicillinase activity was assessed as described above.
Interestingly, when cells displaying FP77 on the surface were grown in the absence of ampicillin, the whole-cell penicillinase activity was found to decrease significantly Table 2. Again, the amounts of FP77 expressed by JK pLAT grown in the presence and absence of ampicillin were compared by Western blotting and shown to be similar. Thus, the differences observed in whole-cell penicillinase activity are not due to down-regulation of FP77 data not shown. In this report, we describe the export of an active enzyme to the surfaces of E.
This was demonstrated by the accessibility of the Bla moiety to the exogenously added protease trypsin and by monitoring the Bla activity of physiologically intact cells using penicillin G and cephaloridine as the substrates. Surface display has become a rising focus of interest due to possible applications in biotechnology, biomedicine, and vaccine development.
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Filamentous bacteriophage have been successfully employed for the display of random peptide libraries 3 and single-chain scFv antibodies and combinatorial libraries thereof Peptide and scFv antibody libraries of high diversity have been established, and screening for appropriate ligands has become very efficient by virtue of high-throughput panning procedures 26 or the selectively infective phage technology Despite the high degree of sophistication achieved in phage display technology, bacterial systems for surface display offer distinct advantages, including the strict linkage of genotype and phenotype, constant growth under selective conditions, the high copy number of the passenger proteins on the bacterial surface 14 , 24 , and the ease of reamplification of selected bacteria expressing peptide libraries on the surface For the targeting of an scFv molecule to the surfaces of E.
The PAL-scFv fusion was located in the periplasm and bound to the murein layer, and after permeabilization of the outer membrane, the scFv became accessible to externally added antigen. Another system for bacterial surface display is based on the C-terminal fusion of a heterologous passenger protein to a genetically engineered hybrid molecule of the major E. By use of this system, export to the surface of E. Furthermore, the functionality of all three fusions was demonstrated by the degradation of externally added penicillin G by surface-displayed Bla and substrate binding for the Cex exoglucanase and the scFV antibody As discussed by Georgiou et al.
Thus, in these strains differentiation between periplasmic and surface-located enzymatic activities was not possible. A third system comprises the fusion of heterologous passenger moieties to autotransporter domains of various proteins that are members of the autotransporter family present in gram-negative species, resulting in the export of the passenger domain to the surfaces of E.
Display of functional protein domains has not been demonstrated for this system until recently A limitation, however, is the incompatibility for the translocation of passenger domains containing extensive tertiary structures such as disulfide bonds This restriction could be overcome by inactivating the dsbA gene product of E. The Bla moiety was clearly shown to be surface exposed and retained functionality on the bacterial surface.
In contrast, the differences observed between surface-located and periplasmic Bla obtained with cephaloridine as the substrate were less prominent due to the enhanced capacity of cephaloridine to penetrate the periplasm. In addition, our data indicate that the membranes of E. Thus, large amounts of AIDA-I fusion proteins inserted into the outer membrane do not appear to cause membrane disorders that might promote the influx of penicillin G into the periplasm. Additionally, the periplasmic domain of OmpA was not accessible to trypsin in JK cells expressing FP77 or FP50, providing further evidence that the outer membrane is intact in these strains.
However, it is difficult to assess the molecular basis of the reduction of enzymatic activity on the cell surface. A decrease in the enzymatic activity of surface-displayed Bla might be caused by conformational changes of the enzyme due to the C-terminal attachment of Bla to the autotransporter domain.
Nevertheless, at this point it is not clear what factors are responsible for the decrease in Bla activity on the cell surface. It is likely that in this strain enzymatically active Bla moieties with preformed disulfide bonds are translocated across the outer membrane, leading to enhanced whole-cell Bla activity. Recently Veiga et al.
Interestingly, binding of cells expressing the scFv molecule was observed only in the presence of the dsbA gene product, although the relative amount of active scFv molecules on the bacterial surface was not quantified. Based on these data, Veiga et al. In contrast, the Bla moiety of FP77 is displayed functionally on the cell surface in a dsbA background, although FP77 is also expressed functionally in the presence of the dsbA gene product.
Additionally, the highest rate of expression and stabilization of the full-length gene product was achieved in JK dsbA ompT. This is in accordance with the findings of Klauser et al.