Development and validation of a population pharmacokinetic model to guide perioperative tacrolimus dosing after lung transplantation.

Background: Tacrolimus therapy is standard of care for immunosuppression after lung transplantation. However, tacrolimus exposure variability during the early postoperative period may contribute to poor outcomes in this population. Few studies have examined tacrolimus pharmacokinetics (PK) during this high-risk time period. Methods: We conducted a retrospective pharmacokinetic study in lung transplant recipients at the University of Pennsylvania who were enrolled in the Lung Transplant Outcomes Group (LTOG) cohort. We derived a model in 270 patients using NONMEM (version 7.5.1) and examined validity in a separate cohort of 114 patients. Covariates were examined with univariate analysis and multivariable analysis was developed using forward and backward stepwise selection. Performance of the final model in the validation cohort was examined with calculation of mean prediction error (PE). Results: We developed a one-compartment base model with a fixed rate absorption constant. Significant covariates in multivariable analysis were postoperative day, hematocrit, transplant type, CYP3A5 genotype, total body weight, and time-varying postoperative day, hematocrit, and CYP inhibitor drugs. The strongest predictor of tacrolimus clearance was postoperative day, with median predicted clearance increasing more than threefold over the 14 day study period. In the validation cohort, the final model showed a mean PE of 36.4% (95%CI 30.8%-41.9%) and a median PE of 7.2% (IQR -29.3%-70.53%). Conclusion: Postoperative day was the strongest predictor of tacrolimus exposure in the early post-lung transplant period. Future multicenter studies employing intensive sampling to examine a broad set of variables related to critical illness physiology are needed to understand determinants of clearance, volume of distribution and absorption in this population.

A light-exploiting insectivorous bat shows no melatonin disruption under lights with different spectra.

Natural light-dark cycles synchronize an animal's internal clock with environmental conditions. The introduction of artificial light into the night-time environment masks natural light cues and has the potential to disrupt this well-established biological rhythm. Nocturnal animal species, such as bats, are adapted to low light conditions and are therefore among the most vulnerable to the impacts of artificial light at night (ALAN). The behaviour and activity of insectivorous bats is disrupted by short-wavelength artificial light at night, while long-wavelength light is less disruptive. However, the physiological consequences of this lighting have not been investigated. Here, we examine the effect of LEDs with different spectra on urinary melatonin in an insectivorous bat. We collected voluntarily voided urine samples from Gould's wattled bats (Chalinolobus gouldii) and measured melatonin-sulfate under ambient night-time conditions (baseline) and under red (λP 630 nm), amber (λP 601 nm), filtered warm white (λP 586 nm) and cool white (λP 457 nm) LEDs. We found no effect of light treatment on melatonin-sulfate irrespective of spectra. Our findings suggest that short-term exposure to LEDs at night do not disrupt circadian physiology in the light-exploiting Gould's wattled bat.

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

Nest boxes do not cause a shift in bat community composition in an urbanised landscape.

Nest boxes are often used to provide supplementary roosts for cavity-dependent wildlife, but little is known about if they influence faunal community composition. Long-term monitoring of bat boxes in south-eastern Australia indicated that their use was dominated by one generalist species (Chalinolobus gouldii), causing concern that installing bat boxes could cause a shift toward less diverse bat communities. To test this, we conducted a large-scale before-after control-impact experiment at 18 sites, over five years. Sites were either: (1) those with existing bat boxes, (2) those where boxes were added during the study, or (3) controls without boxes. We used echolocation call data from 9035 bat detector nights to compare community composition, diversity, and species' relative activity between the sites. Chalinolobus gouldii continued to dominate the use of existing boxes, but we found little difference in community composition between sites based on the presence, absence, or addition of boxes. Our study is the first to explore the influence installing artificial hollows has on localized faunal assemblages over spatio-temporal scales relevant to management. We conclude that there is cause for optimism that bat boxes might not have perverse outcomes on local community composition in the short- to medium-term, as we had feared.

Ventricular Catheter Tract Hemorrhage as a Risk Factor for Ventriculostomy-Related Infection.

BACKGROUND: Ventriculostomy-related infection (VRI) is a feared complication of external ventricular drain (EVD) placement. Although many contributing factors to VRI have been examined, little is known whether there is an association between ventriculostomy-related catheter tract hemorrhage (VCTH) and VRI. OBJECTIVE: To evaluate risk factors for VRI and assess possible correlations with VCTH. METHODS: We performed a retrospective analysis of patients with EVD placement in a neurocritical care unit between 2011 and 2015. VRI was defined as clinical signs of infection with a positive cerebrospinal fluid gram stain and isolation of cerebrospinal fluid culture. VCTH was diagnosed by computed tomography immediately after EVD insertion. RESULTS: A total of 247 patients with EVD were identified during the 5-yr study period. An association between VCTH and gram-negative VRI was identified (P = .02). Ten percent (25 of 247 patients) developed a VRI, and 7% (18 of 247 patients) had a VCTH. Of the 25 patients with VRI, 20% (n = 5) had a VCTH, compared to 6% (n = 13) of 222 patients who had an EVD placed but did not develop VRI. There were no significant differences in demographic and clinical factors except for multiple EVD insertions (P < .00001), EVD duration (P < .001), and hospital length of stay (P < .001). CONCLUSION: VCTH is a potentially significant risk factor for VRI. Further analysis will be needed to confirm the strength of this association, and to delineate the possible mechanisms by which tract hemorrhage may serve as a nidus for bacterial penetration into the central nervous system.

The EMCOOLs surface cooling system for fever control in neurocritical care patients: A pilot study.

OBJECTIVES: Fever occurs in up to 50% of critically-ill patients with acute neurological injury. Small temperature elevations have been correlated with increased morbidity and mortality in this patient population. We sought to evaluate a novel single-use surface cooling system for the treatment of fever in patients with acute brain injury. PATIENTS AND METHODS: We conducted a retrospective analysis of a prospective product evaluation using the EMCOOLS Flex.Pad™ system for acute fever (≥38.3 °C) in our 16-bed neuro-ICU. Four refrigerated pads (-18 °C) were applied to the chest, back, and anterior thighs. Core temperature (bladder) was continuously recorded over 4 h, and the highest Bedside Shivering Assessment Scale (BSAS) score was recorded hourly. RESULTS: Twelve subjects were included in the analysis. Mean age was 55 ±â€¯9 years, 9 patients were men, and mean weight was 85 ±â€¯12 kg. The most common primary diagnoses were subarachnoid (N = 5) and intracerebral (N = 4) hemorrhage. Application of the EMCOOLS system resulted in a linear 1.3 ±â€¯0.6 °C drop (T0avg = 38.9 °C, T90avg = 37.6 °C, P = 0.0032) in mean temperature over 90 min, followed by a plateau with only one subject rebounding to >38 °C within 4 h. Normothermia (<38.0 °C) was achieved in all but one patient (92%) in an average of 65 min. Comatose patients displayed a non-significantly higher degree of cooling at 90 min than did awake subjects (ΔTcoma = 1.74 °C vs ΔTawake = 0.74 °C hr-1, P = 0.067). There was no observed skin irritation upon removal of the device for any patients. CONCLUSION: The EMCOOLs system is a well-tolerated, safe and effective short-term intervention for control of fever in neurological patients. Future studies are needed to compare efficacy of the EMCOOLs to other devices and interventions.

Evaluation of a system for enhancing mobile telephone communication for people with hearing loss.

Objective: To evaluate a system - "the service" - designed to improve mobile telephone communication for people with hearing loss. Design: Each participant registered online and took an online hearing test using their own mobile telephone held in the position of their choice. The test assessed the combined effects of the telephone, the hearing loss of the participant and the hearing aid of the participant (if worn). The results were stored as a "hearing profile" and used to set up frequency-dependent amplification and multichannel amplitude compression. The signal processing was performed in the telephone network and applied to all calls of the participant. Participants completed four questionnaires about their listening experiences using the telephone, one before using the service, two while using the service and one after using the service. Also, the ability to understand speech was measured using challenging sentences. Study sample: Fifty-three participants with varying degrees of hearing loss. Results: The great majority of participants indicated that call clarity was improved and that listening difficulty was reduced when using the service. Speech intelligibility scores were significantly improved when using the service. Conclusions: The service appears to be effective in improving phone communication for people with hearing loss.

Addressing spirituality during critical illness: A review of current literature.

OBJECTIVES: The purpose of this review is to provide an overview of research on spirituality and religiosity in the intensive care setting that has been published since the 2004-2005 American College of Critical Care Medicine (ACCM) Clinical Practice Guidelines for the Support of Family in the Patient-Centered Intensive Care Unit with an emphasis on its application beyond palliative and end-of-life care. MATERIALS AND METHODS: ACCM 2004-2005 guidelines emphasized the importance of spiritual and religious support in the form of four specific recommendations: [1] assessment and incorporation of spiritual needs in ICU care plan; [2] spiritual care training for doctors and nurses; [3] physician review of interdisciplinary spiritual need assessments; and [4] honoring the requests of patients to pray with them. We reviewed 26 studies published from 2006 to 2016 and identified whether studies strengthened the grade of these recommendations. We further categorized findings of these studies to understand the roles of spirituality and religiosity in surrogate perceptions and decision-making and patient and family experience. CONCLUSIONS: Spiritual care has an essential role in the treatment of critically ill patients and families. Current literature offers few insights to support clinicians in navigating this often-challenging aspect of patient care and more research is needed.

Surface reflectance drives nest box temperature profiles and thermal suitability for target wildlife.

Thermal properties of tree hollows play a major role in survival and reproduction of hollow-dependent fauna. Artificial hollows (nest boxes) are increasingly being used to supplement the loss of natural hollows; however, the factors that drive nest box thermal profiles have received surprisingly little attention. We investigated how differences in surface reflectance influenced temperature profiles of nest boxes painted three different colors (dark-green, light-green, and white: total solar reflectance 5.9%, 64.4%, and 90.3% respectively) using boxes designed for three groups of mammals: insectivorous bats, marsupial gliders and brushtail possums. Across the three different box designs, dark-green (low reflectance) boxes experienced the highest average and maximum daytime temperatures, had the greatest magnitude of variation in daytime temperatures within the box, and were consistently substantially warmer than light-green boxes (medium reflectance), white boxes (high reflectance), and ambient air temperatures. Results from biophysical model simulations demonstrated that variation in diurnal temperature profiles generated by painting boxes either high or low reflectance colors could have significant ecophysiological consequences for animals occupying boxes, with animals in dark-green boxes at high risk of acute heat-stress and dehydration during extreme heat events. Conversely in cold weather, our modelling indicated that there are higher cumulative energy costs for mammals, particularly smaller animals, occupying light-green boxes. Given their widespread use as a conservation tool, we suggest that before boxes are installed, consideration should be given to the effect of color on nest box temperature profiles, and the resultant thermal suitability of boxes for wildlife, particularly during extremes in weather. Managers of nest box programs should consider using several different colors and installing boxes across a range of both orientations and shade profiles (i.e., levels of canopy cover), to ensure target animals have access to artificial hollows with a broad range of thermal profiles, and can therefore choose boxes with optimal thermal conditions across different seasons.

A global synthesis of survival estimates for microbats.

Accurate survival estimates are needed to construct robust population models, which are a powerful tool for understanding and predicting the fates of species under scenarios of environmental change. Microbats make up 17% of the global mammalian fauna, yet the processes that drive differences in demographics between species are poorly understood. We collected survival estimates for 44 microbat species from the literature and constructed a model to determine the effects of reproductive, feeding and demographic traits on survival. Our trait-based model indicated that bat species which produce more young per year exhibit lower apparent annual survival, as do males and juveniles compared with females and adults, respectively. Using 8 years of monitoring data for two Australian species, we demonstrate how knowledge about the effect of traits on survival can be incorporated into Bayesian survival analyses. This approach can be applied to any group and is not restricted to bats or even mammals. The incorporation of informative priors based on traits can allow for more timely construction of population models to support management decisions and actions.

Multifunctional cytomegalovirus (CMV)-specific CD8(+) T cells are not restricted by telomere-related senescence in young or old adults.

Antigen-specific multifunctional T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α simultaneously after activation are important for the control of many infections. It is unclear if these CD8(+) T cells are at an early or late stage of differentiation and whether telomere erosion restricts their replicative capacity. We developed a multi-parameter flow cytometric method for investigating the relationship between differentiation (CD45RA and CD27 surface phenotype), function (cytokine production) and replicative capacity (telomere length) in individual cytomegalovirus (CMV) antigen-specific CD8(+) T cells. This involves surface and intracellular cell staining coupled to fluorescence in situ hybridization to detect telomeres (flow-FISH). The end-stage/senescent CD8(+)  CD45RA(+)  CD27(-) T-cell subset increases significantly during ageing and this is exaggerated in CMV immune-responsive subjects. However, these end-stage cells do not have the shortest telomeres, implicating additional non-telomere-related mechanisms in inducing their senescence. The telomere lengths in total and CMV (NLV)-specific CD8(+) T cells in all four subsets defined by CD45RA and CD27 expression were significantly shorter in old compared with young individuals in both a Caucasian and an Asian cohort. Following stimulation by anti-CD3 or NLV peptide, similar proportions of triple-cytokine-producing cells are found in CD8(+) T cells at all stages of differentiation in both age groups. Furthermore, these multi-functional cells had intermediate telomere lengths compared with cells producing only one or two cytokines after activation. Therefore, global and CMV (NLV)-specific CD8(+) T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α are at an intermediate stage of differentiation and are not restricted by excessive telomere erosion.

p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8⁺ T cells.

T cell senescence is thought to contribute to immune function decline, but the pathways that mediate senescence in these cells are not clear. Here, we evaluated T cell populations from healthy volunteers and determined that human CD8+ effector memory T cells that reexpress the naive T cell marker CD45RA have many characteristics of cellular senescence, including decreased proliferation, defective mitochondrial function, and elevated levels of both ROS and p38 MAPK. Despite their apparent senescent state, we determined that these cells secreted high levels of both TNF-α and IFN-γ and showed potent cytotoxic activity. We found that the senescent CD45RA-expressing population engaged anaerobic glycolysis to generate energy for effector functions. Furthermore, inhibition of p38 MAPK signaling in senescent CD8+ T cells increased their proliferation, telomerase activity, mitochondrial biogenesis, and fitness; however, the extra energy required for these processes did not arise from increased glucose uptake or oxidative phosphorylation. Instead, p38 MAPK blockade in these senescent cells induced an increase in autophagy through enhanced interactions between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9) in an mTOR-independent manner. Together, our findings describe fundamental metabolic requirements of senescent primary human CD8+ T cells and demonstrate that p38 MAPK blockade reverses senescence via an mTOR-independent pathway.

Tree-hugging koalas demonstrate a novel thermoregulatory mechanism for arboreal mammals.

How climate impacts organisms depends not only on their physiology, but also whether they can buffer themselves against climate variability via their behaviour. One of the way species can withstand hot temperatures is by seeking out cool microclimates, but only if their habitat provides such refugia. Here, we describe a novel thermoregulatory strategy in an arboreal mammal, the koala Phascolarctos cinereus. During hot weather, koalas enhanced conductive heat loss by seeking out and resting against tree trunks that were substantially cooler than ambient air temperature. Using a biophysical model of heat exchange, we show that this behaviour greatly reduces the amount of heat that must be lost via evaporative cooling, potentially increasing koala survival during extreme heat events. While it has long been known that internal temperatures of trees differ from ambient air temperatures, the relevance of this for arboreal and semi-arboreal mammals has not previously been explored. Our results highlight the important role of tree trunks as aboveground 'heat sinks', providing cool local microenvironments not only for koalas, but also for all tree-dwelling species.

High levels of activity of bats at gold mining water bodies: implications for compliance with the International Cyanide Management Code.

Wildlife and livestock are known to visit and interact with tailings dam and other wastewater impoundments at gold mines. When cyanide concentrations within these water bodies exceed a critical toxicity threshold, significant cyanide-related mortality events can occur in wildlife. Highly mobile taxa such as birds are particularly susceptible to cyanide toxicosis. Nocturnally active bats have similar access to uncovered wastewater impoundments as birds; however, cyanide toxicosis risks to bats remain ambiguous. This study investigated activity of bats in the airspace above two water bodies at an Australian gold mine, to assess the extent to which bats use these water bodies and hence are at potential risk of exposure to cyanide. Bat activity was present on most nights sampled during the 16-month survey period, although it was highly variable across nights and months. Therefore, despite the artificial nature of wastewater impoundments at gold mines, these structures present attractive habitats to bats. As tailings slurry and supernatant pooling within the tailings dam were consistently well below the industry protective concentration limit of 50 mg/L weak acid dissociable (WAD) cyanide, wastewater solutions stored within the tailings dam posed a minimal risk of cyanide toxicosis for wildlife, including bats. This study showed that passively recorded bat echolocation call data provides evidence of the presence and relative activity of bats above water bodies at mine sites. Furthermore, echolocation buzz calls recorded in the airspace directly above water provide indirect evidence of foraging and/or drinking. Both echolocation monitoring and systematic sampling of cyanide concentration in open wastewater impoundments can be incorporated into a gold mine risk-assessment model in order to evaluate the risk of bat exposure to cyanide. In relation to risk minimisation management practices, the most effective mechanism for preventing cyanide toxicosis to wildlife, including bats, is capping the concentration of cyanide in tailings discharged to open impoundments at 50 mg/L WAD.

Hypersalinity reduces the risk of cyanide toxicosis to insectivorous bats interacting with wastewater impoundments at gold mines.

Wildlife and livestock that ingest bioavailable cyanide compounds in gold mining tailings dams are known to experience cyanide toxicosis. Elevated levels of salinity in open impoundments have been shown to prevent wildlife cyanide toxicosis by reducing drinking and foraging. This finding appears to be consistent for diurnal wildlife interacting with open impoundments, however the risks to nocturnal wildlife of cyanide exposure are unknown. We investigated the activity of insectivorous bats in the airspace above both fresh (potable to wildlife) and saline water bodies at two gold mines in the goldfields of Western Australian. During this study, cyanide-bearing solutions stored in open impoundments at both mine sites were hypersaline (range=57,000-295,000 mg/L total dissolved solids (TDS)), well above known physiological tolerance of any terrestrial vertebrate. Bats used the airspace above each water body monitored, but were more active at fresh than saline water bodies. In addition, considerably more terminal echolocation buzz calls were recorded in the airspace above fresh than saline water bodies at both mine sites. However, it was not possible to determine whether these buzz calls corresponded to foraging or drinking bouts. No drinking bouts were observed in 33 h of thermal video footage recorded at one hypersaline tailings dam, suggesting that this water is not used for drinking. There is no information on salinity tolerances of bats, but it could be assumed that bats would not tolerate salinity in drinking water at concentrations greater than those documented as toxic for saline-adapted terrestrial wildlife. Therefore, when managing wastewater impoundments at gold mines to avoid wildlife mortalities, adopting a precautionary principle, bats are unlikely to drink solutions at salinity levels ≥50,000 mg/L TDS.

Age-associated increase of low-avidity cytomegalovirus-specific CD8+ T cells that re-express CD45RA.

The mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.

Echolocating bats emit terminal phase buzz calls while drinking on the wing.

Echolocating bats are known to produce terminal buzz calls during pursuit and capture of airborne prey, however the use of buzz calls while drinking on the wing has not been previously investigated. In this study I recorded the first empirical evidence that bats produce terminal phase buzz calls while drinking on the wing. Every drinking pass recorded during this study was characterised by a terminal buzz which bats emitted immediately prior to touching the water surface with their mouth. The characteristic frequency (the frequency at the end or flattest portion of the pulse) of echolocation call sequences containing drinking buzzes varied from 25kHz to 50kHz, suggesting multiple bat species present at the study site emit buzzes while drinking on the wing. As feeding buzz calls appear to be ubiquitous among echolocating bat taxa, the prevalence of drinking buzzes clearly warrants further investigation. Drinking buzzes could potentially be used to document rates of drinking by bats in the same way that feeding buzzes are used to infer foraging activity.

Differing HLA types influence inhibitory receptor signalling in CMV-specific CD8+ T cells.

The dysregulated immune response to CMV constitutes a major force driving T cell immunosenescence and growing evidence suggests that it is not a benign virus in old age. We show here that the PD-1/L pathway defines a reversible defect in CMV specific CD8(+) T cell proliferative responses in both young and old individuals. More specifically, highly differentiated CD45RA(+)CD27(-) CMV-specific CD8(+) T cells exhibit a proliferative deficit compared their central and effector memory counterparts, which is reversed following PD-L blockade. However, we also report that HLA-B(∗)07/TPR specific CD8(+) T cells express higher levels of PD-1 than HLA-A(∗)02/NLV specific cells and HLA-A(∗)02 individuals show a higher proliferative response to PD-L blockade, than HLA-B(∗)07 individuals, which we postulate may be due to the differing functional avidities for these two CMV-specific CD8(+) T cells populations. Nevertheless data presented here demonstrate that CMV-specific CD8(+) T cells can be functionally enhanced by perturbation of the PD-1/L signalling pathway, whose manipulation may provide a therapeutic modality to combat age-associated immune decline.

Predictions of U.K. regulated power station contributions to regional air pollution and deposition: a model comparison exercise.

Contributions of the emissions from a U.K. regulated fossil-fuel power station to regional air pollution and deposition are estimated using four air quality modeling systems for the year 2003. The modeling systems vary in complexity and emphasis in the way they treat atmospheric and chemical processes, and include the Community Multiscale Air Quality (CMAQ) modeling system in its versions 4.6 and 4.7, a nested modeling system that combines long- and short-range impacts (referred to as TRACK-ADMS [Trajectory Model with Atmospheric Chemical Kinetics-Atmospheric Dispersion Modelling System]), and the Fine Resolution Atmospheric Multi-pollutant Exchange (FRAME) model. An evaluation of the baseline calculations against U.K. monitoring network data is performed. The CMAQ modeling system version 4.6 data set is selected as the reference data set for the model footprint comparison. The annual mean air concentration and total deposition footprints are summarized for each modeling system. The footprints of the power station emissions can account for a significant fraction of the local impacts for some species (e.g., more than 50% for SO2 air concentration and non-sea-salt sulfur deposition close to the source) for 2003. The spatial correlation and the coefficient of variation of the root mean square error (CVRMSE) are calculated between each model footprint and that calculated by the CMAQ modeling system version 4.6. The correlation coefficient quantifies model agreement in terms of spatial patterns, and the CVRMSE measures the magnitude of the difference between model footprints. Possible reasons for the differences between model results are discussed. Finally, implications and recommendations for the regulatory assessment of the impact of major industrial sources using regional air quality modeling systems are discussed in the light of results from this case study.