Association of modifiable factors with the development of physical fitness of Austrian primary school children: A 4-year longitudinal study.

Physical fitness (PF) shows favourable associations with several health indicators in children. Children's PF depends on a variety of non-modifiable (e.g., sex and age) and modifiable factors (e.g., weight status and sports participation). The aim of this study was to evaluate the impact of modifiable and non-modifiable factors on the development of PF during the 4 years of primary school. A longitudinal study was carried out with 265 children. PF was assessed using the German Motor Performance Test 6-18, whereas modifiable and non-modifiable factors with parent's and children's questionnaires. Total PF z-score increased by 1.4 standard deviations from 1st to 4th year and raw values of subtests improved by an average of about 40%. The variables "parents' physical activity", "never being overweight", "sports club participation", and "playing outside" were positively associated with PF development. The present study highlights that a variety of modifiable factors, both from children and their parents, are significantly associated with the development of children's PF during primary education. Interventions should not only focus on direct actions, such as proposing specific exercise programs, but also aim at increasing parents' awareness of their role model function in endorsing their children's healthy active lifestyle, especially at early ages.

A proposal for a new PhD level curriculum on quantitative methods for drug development.

This paper provides an overview of "Improving Design, Evaluation and Analysis of early drug development Studies" (IDEAS), a European Commission-funded network bringing together leading academic institutions and small- to large-sized pharmaceutical companies to train a cohort of graduate-level medical statisticians. The network is composed of a diverse mix of public and private sector partners spread across Europe, which will host 14 early-stage researchers for 36 months. IDEAS training activities are composed of a well-rounded mixture of specialist methodological components and generic transferable skills. Particular attention is paid to fostering collaborations between researchers and supervisors, which span academia and the private sector. Within this paper, we review existing medical statistics programmes (MSc and PhD) and highlight the training they provide on skills relevant to drug development. Motivated by this review and our experiences with the IDEAS project, we propose a concept for a joint, harmonised European PhD programme to train statisticians in quantitative methods for drug development.

Multi-arm group sequential designs with a simultaneous stopping rule.

Multi-arm group sequential clinical trials are efficient designs to compare multiple treatments to a control. They allow one to test for treatment effects already in interim analyses and can have a lower average sample number than fixed sample designs. Their operating characteristics depend on the stopping rule: We consider simultaneous stopping, where the whole trial is stopped as soon as for any of the arms the null hypothesis of no treatment effect can be rejected, and separate stopping, where only recruitment to arms for which a significant treatment effect could be demonstrated is stopped, but the other arms are continued. For both stopping rules, the family-wise error rate can be controlled by the closed testing procedure applied to group sequential tests of intersection and elementary hypotheses. The group sequential boundaries for the separate stopping rule also control the family-wise error rate if the simultaneous stopping rule is applied. However, we show that for the simultaneous stopping rule, one can apply improved, less conservative stopping boundaries for local tests of elementary hypotheses. We derive corresponding improved Pocock and O'Brien type boundaries as well as optimized boundaries to maximize the power or average sample number and investigate the operating characteristics and small sample properties of the resulting designs. To control the power to reject at least one null hypothesis, the simultaneous stopping rule requires a lower average sample number than the separate stopping rule. This comes at the cost of a lower power to reject all null hypotheses. Some of this loss in power can be regained by applying the improved stopping boundaries for the simultaneous stopping rule. The procedures are illustrated with clinical trials in systemic sclerosis and narcolepsy. © 2016 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.

Preoperative abnormalities in serum sodium concentrations are associated with higher in-hospital mortality in patients undergoing major surgery.

BACKGROUND: Abnormal serum sodium concentrations are common in patients presenting for surgery. It remains unclear whether these abnormalities are independent risk factors for postoperative mortality. METHODS: This is a secondary analysis of the European Surgical Outcome Study (EuSOS) that provided data describing 46 539 patients undergoing inpatient non-cardiac surgery. Patients were included in this study if they had a recorded value of preoperative serum sodium within the 28 days immediately before surgery. Data describing preoperative risk factors and serum sodium concentrations were analysed to investigate the relationship with in-hospital mortality using univariate and multivariate logistic regression techniques. RESULTS: Of 35 816 (77.0%) patients from the EuSOS database, 21 943 (61.3%) had normal values of serum sodium (138-142 mmol litre(-1)) before surgery, 8538 (23.8%) had hyponatraemia (serum sodium ≤137 mmol litre(-1)) and 5335 (14.9%) had hypernatraemia (serum sodium ≥143 mmol litre(-1)). After adjustment for potential confounding factors, moderate to severe hypernatraemia (serum sodium concentration ≥150 mmol litre(-1)) was independently associated with mortality [odds ratio 3.4 (95% confidence interval 2.0-6.0), P<0.0001]. Hyponatraemia was not associated with mortality. CONCLUSIONS: Preoperative abnormalities in serum sodium concentrations are common, and hypernatraemia is associated with increased mortality after surgery. Abnormalities of serum sodium concentration may be an important biomarker of perioperative risk resulting from co-morbid disease.

Preoperative anaemia is associated with poor clinical outcome in non-cardiac surgery patients.

BACKGROUND: Retrospective studies suggest that preoperative anaemia is associated with poor outcomes after surgery. The objective of this study was to describe mortality rates and patterns of intensive care resource use for patients with anaemia undergoing non-cardiac and non-neurological in-patient surgery. METHODS: We performed a secondary analysis of a large prospective study describing perioperative care and survival in 28 European nations. Patients at least 16 yr old undergoing in-patient surgery during a 7 day period were included in the study. Data were collected for in-hospital mortality, duration of hospital stay, admission to intensive care, and intensive care resource use. Multivariable logistic regression analysis was performed to understand the effects of preoperative haemoglobin (Hb) levels on in-hospital mortality. RESULTS: We included 39 309 patients in the analysis. Preoperative anaemia had a high prevalence in both men and women (31.1% and 26.5%, respectively). Multivariate analysis showed that patients with severe [odds ratio 2.82 (95% confidence interval 2.06-3.85)] or moderate [1.99 (1.67-2.37)] anaemia had higher in-hospital mortality than those with normal preoperative Hb concentrations. Furthermore, hospital length of stay (P<0.001) and postoperative admission to intensive care (P<0.001) were greater in patients with anaemia than in those with normal Hb concentrations. CONCLUSIONS: Anaemia is common among non-cardiac and non-neurological surgical patients, and is associated with poor clinical outcome and increased healthcare resource use. CLINICAL TRIAL REGISTRATION: NCT01203605 (ClinicalTrials.gov).

A novel method to estimate the response of habitat types to nitrogen deposition.

Increasing nitrogen depositions adversely affect European landscapes, including habitats within the Natura2000 network. Critical loads for nitrogen deposition have been established to quantify the loss of habitat quality. When the nitrogen deposition rises above a habitat-specific critical load, the quality of the focal habitat is expected to be negatively influenced. Here, we investigate how the quality of habitat types is affected beyond the critical load. We calculated response curves for 60 terrestrial habitat types in the Netherlands to the estimated nitrogen deposition (EMEP-data). The curves for habitat types are based on the occurrence of their characteristic plant species in North-Western Europe (plot data from the European Vegetation Archive). The estimated response curves were corrected for soil type, mean annual temperature and annual precipitation. Evaluation was carried out by expert judgement, and by comparison with gradient deposition field studies. For 39 habitats the response to nitrogen deposition was judged to be reliable by five experts, while out of the 41 habitat types for which field studies were available, 25 showed a good agreement. Some of the curves showed a steep decline in quality and some a more gradual decline with increasing nitrogen deposition. We compared the response curves with both the empirical and modelled critical loads. For 41 curves, we found a decline already starting below the critical load.

Elevated levels of soluble total and hyperphosphorylated tau result in early behavioral deficits and distinct changes in brain pathology in a new tau transgenic mouse model.

Tauopathies, characterized by hyperphosphorylation and aggregation of tau protein, include frontotemporal dementias and Alzheimer's disease. To explore disease mechanisms and investigate potential treatments, we generated a transgenic (tg) mouse line overexpressing human tau441 with V337M and R406W mutations. Biochemical characterization of these TMHT (Thy-1 mutated human tau) mice showed a significant increase in human transgene expression relative to endogenous murine tau by Western blot and multi-array immunosorbent assay. Only soluble total tau and phosphorylated tau (ptau at residue Thr(181), Ser(199), Thr(231) and Thr(235)), but not insoluble total tau and ptau were increased. Application of the Phospho-Tau SRM assay revealed that phosphorylation at Ser(396) and Ser(404) in soluble tau in the presence of the R406W mutation was at baseline levels in the cortex of TMHT mice compared to non-tg littermates. Histological analyses showed a progressive increase in human tau protein in the amygdala over age, while hippocampal tau levels remained constant from 2 months onwards. Behavioral testing of TMHT mice in the Morris water maze revealed a distinct progressive spatial learning impairment starting already at 5 months of age. Furthermore, we showed that the TMHT mice have early olfactory deficits. These impairments are unbiased by any motor disturbance or lack of motivation. Our results prove that combination of the V337M and R406W mutations of tau accelerates human tau phosphorylation and induces tau pathology as well as cognitive deficits, making this model a suitable tool for basic research on tau as well as in vivo drug testing.

Are early post-discharge physician contacts associated with 30-day psychiatric re-hospitalisation? A nationwide claims data based retrospective cohort study in Austria free of immortal time bias.

OBJECTIVES: Cost containment and quality of care considerations have increased research interest in the potential preventability of early re-hospitalisations. Various registry-based retrospective cohort studies on psychiatric re-hospitalisation have focused on the role of early post-discharge service contacts, but either did not consider their time-dependent nature ('immortal time bias') or evaded the issue by analysing late re-hospitalisations. The present study takes care of the immortal time bias in studying early psychiatric re-hospitalisations. METHODS: In a retrospective cohort study using nationwide electronic claims data in Austria, 10,689 adults discharged from acute psychiatric inpatient wards were followed up for 30 days. Cox regression analyses were performed with post-discharge psychiatric and general practitioner contacts as time-dependent covariates and time to first psychiatric re-hospitalisation as outcome. RESULTS: Post-discharge ambulatory physician contacts were significantly associated with a decreased psychiatric re-hospitalisation rate (hazard ratio 0.77 [95% CI 0.69; 0.87], p < 0.0001), with similarly strong contributions to this association by general practitioners and psychiatrists. CONCLUSIONS: Despite avoiding the immortal time bias and controlling for several confounders, we suggest to be cautious with a causal interpretation of the identified association, since potentially relevant confounders, such as disease severity, were unavailable in our claims data base.

Long-term efficacy and respective potencies of botulinum toxin A and B: a randomized, double-blind study.

BACKGROUND: Mouse units (mU) are used for quantification of the biological activity of botulinum A and B toxin preparations. However, in human tissue, mU values between preparations are not equivalent and lack of clarity concerning efficacy and safety remains with regard to their respective potencies, duration of drug effect and diffusion qualities. OBJECTIVES: To compare short-term and long-term effects of Botox(®) (BOT; Allergan Inc., Irvine, CA, U.S.A.) and Neurobloc(®)/Myobloc(®) (NBC; Solstice Neurosciences Inc., Malvern, PA, U.S.A.) in different doses and dilutions in a human skin model. METHODS: In this prospective randomized double-blind study, 18 healthy volunteers (eight women and 10 men; mean ± SD age 28·4 ± 5·7 years) were injected intradermally with pure saline, BOT and NBC at 10 points in the abdomen in random order, using the BOT/NBC conversion ratio 1 : 75 and different dilution schemes. For an objective outcome, the ninhydrin sweat test was used to compare the anhidrotic areas (action halos). Ten measurements were taken during a time period of 54 weeks. RESULTS: Both preparations showed a peak effect at week 3, with significantly larger anhidrotic areas for NBC. Thereafter, however, the rate of decline was lower in BOT and after week 24, mean BOT areas were larger. The effect of dilution was higher in NBC and the mean dose equivalence conversion ratio (BOT/NBC) was 1 : 29 (area under the curve). Gender effects were seen in both products, with smaller action halos in women. CONCLUSIONS: These results have important implications in clinical routine, especially for autonomic indications.

Use of dynamic soil-vegetation models to assess impacts of nitrogen deposition on plant species composition: an overview.

Field observations and experimental data of effects of nitrogen (N) deposition on plant species diversity have been used to derive empirical critical N loads for various ecosystems. The great advantage of such an approach is the inclusion of field evidence, but there are also restrictions, such as the absence of explicit criteria regarding significant effects on the vegetation, and the impossibility to predict future impacts when N deposition changes. Model approaches can account for this. In this paper, we review the possibilities of static and dynamic multispecies models in combination with dynamic soil-vegetation models to (1) predict plant species composition as a function of atmospheric N deposition and (2) calculate critical N loads in relation to a prescribed protection level of the species composition. The similarities between the models are presented, but also several important differences, including the use of different indicators for N and acidity and the prediction of individual plant species vs. plant communities. A summary of the strengths and weaknesses of the various models, including their validation status, is given. Furthermore, examples are given of critical load calculations with the model chains and their comparison with empirical critical N loads. We show that linked biogeochemistry-biodiversity models for N have potential for applications to support European policy to reduce N input, but the definition of damage thresholds for terrestrial biodiversity represents a major challenge. There is also a clear need for further testing and validation of the models against long-term monitoring or long-term experimental data sets and against large-scale survey data. This requires a focused data collection in Europe, combing vegetation descriptions with variables affecting the species diversity, such as soil acidity, nutrient status and water availability. Finally, there is a need for adaptation and upscaling of the models beyond the regions for which dose-response relationships have been parameterized, to make them generally applicable.

On the ropivacaine-reducing effect of low-dose sufentanil in intrathecal labor analgesia.

BACKGROUND: Combining ropivacaine with sufentanil for intrathecal (i.t.) analgesia in labor is well recognized, but information on dosing is limited. This study aimed to determine the ED 50 of i.t. ropivacaine and to assess the effect of adding defined low doses of sufentanil. METHODS: This was a two-phase, double-blind, randomized and prospective study. One hundred and fifteen parturients receiving combined spinal epidural analgesia were allocated to one of four groups to receive ropivacaine or sufentanil alone or in combination. In phase one, sufentanil dose-response was calculated using logistic regression. In phase two, ED 50 of ropivacaine and of the combination with a fixed dosage of sufentanil at ED 20 and ED 40 was evaluated using the technique of up-down sequential allocation. Analgesic effectiveness was assessed 15 min after injection using a 100 mm visual analog scale, with <10 mm lasting for 45 min defined as effective. Furthermore, side effects and duration were recorded. RESULTS: The ED 50 of i.t. ropivacaine was 4.6 mg [95% confidence intervals (95% CI) 4.28, 5.31]. Adding sufentanil at ED 20 significantly decreased the ED 50 of i.t. ropivacaine to 2.1 mg (95%CI 1.75, 2.5) (P<0.005); at ED 40, the reduction was similar (P<0.005). Combining sufentanil with ropivacaine resulted in a dose-independent prolongation of analgesia. Besides pruritus, which was well tolerated, there were no differences in side effects. CONCLUSION: Adding sufentanil at ED 20 results in a more than 50% dose-sparing effect of ropivacaine and considerably prolongs analgesia. Increasing dosage implicates no clinical benefit.

Threshold effects of air pollution and climate change on understory plant communities at forested sites in the eastern United States.

Forest understory plant communities in the eastern United States are often diverse and are potentially sensitive to changes in climate and atmospheric inputs of nitrogen caused by air pollution. In recent years, empirical and processed-based mathematical models have been developed to investigate such changes in plant communities. In the study reported here, a robust set of understory vegetation response functions (expressed as version 2 of the Probability of Occurrence of Plant Species model for the United States [US-PROPS v2]) was developed based on observations of forest understory and grassland plant species presence/absence and associated abiotic characteristics derived from spatial datasets. Improvements to the US-PROPS model, relative to version 1, were mostly focused on inclusion of additional input data, development of custom species-level input datasets, and implementation of methods to address uncertainty. We investigated the application of US-PROPS v2 to evaluate the potential impacts of atmospheric nitrogen (N) and sulfur (S) deposition, and climate change on forest ecosystems at three forested sites located in New Hampshire, Virginia, and Tennessee in the eastern United States. Species-level N and S critical loads (CLs) were determined under ambient deposition at all three modeled sites. The lowest species-level CLs of N deposition at each site were between 2 and 11 kg N/ha/yr. Similarly, the lowest CLs of S deposition, based on the predicted soil pH response, were less than 2 kg S/ha/yr among the three sites. Critical load exceedance was found at all three model sites. The New Hampshire site included the largest percentage of species in exceedance. Simulated warming air temperature typically resulted in lower maximum occurrence probability, which contributed to lower CLs of N and S deposition. The US-PROPS v2 model, together with the PROPS-CLF model to derive CL functions, can be used to develop site-specific CLs for understory plants within broad regions of the United States. This study demonstrates that species-level CLs of N and S deposition are spatially variable according to the climate, light availability, and soil characteristics at a given location. Although the species niche models generally performed well in predicting occurrence probability, there remains uncertainty with respect to the accuracy of reported CLs. As such, the specific CLs reported here should be considered as preliminary estimates.

Prediction of plant species occurrence as affected by nitrogen deposition and climate change on a European scale.

Plant species occurrence in Europe is affected by changes in nitrogen deposition and climate. Insight into potential future effects of those changes can be derived by a model approach based on field-based empirical evidence on a continental scale. In this paper, we present a newly developed empirical model PROPS, predicting the occurrence probabilities of plant species in response to a combination of climatic factors, nitrogen deposition and soil properties. Parameters included were temperature, precipitation, nitrogen deposition, soil pH and soil C/N ratio. The PROPS model was fitted to plant species occurrence data of about 800,000 European relevés with estimated values for pH and soil C/N ratio and interpolated climate and modelled N deposition data obtained from the Ensemble meteo data set and EMEP model results, respectively. The model was validated on an independent data set. The test of ten species against field data gave an average Pearson's r-value of 0.79. PROPS was applied to a grassland and a heathland site to evaluate the effect of scenarios for nitrogen deposition and climate change on the Habitat Suitability Index (HSI), being the average of the relative probabilities, compared to the maximum probability, of all target species in a habitat. Results for the period 1930-2050 showed that an initial increase and later decrease in nitrogen deposition led to a pronounced decrease in HSI, and with dropping nitrogen deposition to an increase of the HSI. The effect of climate change appeared to be limited, resulting in a slight increase in HSI.

T cell senescence and contraction of T cell repertoire diversity in patients with chronic obstructive pulmonary disease.

Pathogenetic mechanisms leading to chronic obstructive pulmonary disease (COPD) remain poorly understood. Because clonogenic T cells (CD4(+)CD28(null)) were shown to be increased in autoimmune diseases we hypothesized that CD4(+)CD28(null) T cells play a role in COPD. Here we describe that enhanced presence of CD4(+)CD28(null) cells is associated with impaired lung function. Sixty-four patients and controls were included. T cell phenotype was analysed using flow cytometry. Enzyme-linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non-parametric group comparisons and correlations. A logistic regression model was used to determine predictive values of CD4(+)CD28(null) in the diagnosis of COPD. Populations of CD4(+) T cells lacking surface co-stimulatory CD28 were enlarged significantly in evaluated patients when compared with controls. Natural killer (NK)-like T cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4(+)CD28(null) cells. Cytokine production after triggering of peripheral blood mononuclear cells (PBMCs) was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4(+)CD28(null) T cells has a diagnostic value. These CD4(+)CD28(null) T cells show increased expression of NK-like T cell receptors (CD94, 158) and intracellular perforin and granzyme B. Furthermore, triggering of PBMCs obtained from patients with mild COPD led to increased interferon-gamma and tumour necrosis factor-alpha production in vitro compared with controls. Our finding of increased CD4(+)CD28(null) T cells in COPD indicates that chronic antigen exposure, e.g. through contents of smoke, leads to loss of CD28 and up-regulation of NK cell receptors expression on T cells in susceptible patients.

Statistical analysis of Goal Attainment Scaling endpoints in randomised trials.

Goal Attainment Scaling is an assessment instrument to evaluate interventions on the basis of individual, patient-specific goals. The attainment of these goals is mapped in a pre-specified way to attainment levels on an ordinal scale, which is common to all goals. This approach is patient-centred and allows one to integrate the outcomes of patients with very heterogeneous symptoms. The latter is of particular importance in clinical trials in rare diseases because it enables larger sample sizes by including a broader patient population. In this paper, we focus on the statistical analysis of Goal Attainment Scaling outcomes for the comparison of two treatments in randomised clinical trials. Building on a general statistical model, we investigate the properties of different hypothesis testing approaches. Additionally, we propose a latent variable approach to generate Goal Attainment Scaling data in a simulation study, to assess the impact of model parameters such as the number of goals per patient and their correlation, the choice of discretisation thresholds and the type of design (parallel group or cross-over). Based on our findings, we give recommendations for the design of clinical trials with a Goal Attainment Scaling endpoint. Furthermore, we discuss an application of Goal Attainment Scaling in a clinical trial in mastocytosis.

Modelling and mapping long-term risks due to reactive nitrogen effects: an overview of LRTAP convention activities.

Long-range transboundary air pollution has caused severe environmental effects in Europe. European air pollution abatement policy, in the framework of the UNECE Convention on Long-range Transboundary Air Pollution (LRTAP Convention) and the European Union Clean Air for Europe (CAFE) programme, has used critical loads and their exceedances by atmospheric deposition to design emission abatement targets and strategies. The LRTAP Convention International Cooperative Programme on Modelling and Mapping Critical Loads and Levels and Air Pollution Effects, Risks and Trends (ICP M&M) generates European critical loads datasets to enable this work. Developing dynamic nitrogen flux models and using them for a prognosis and assessment of nitrogen effects remains a challenge. Further research is needed on links between nitrogen deposition effects, climate change, and biodiversity.

Advanced Methods for Dose and Regimen Finding During Drug Development: Summary of the EMA/EFPIA Workshop on Dose Finding (London 4-5 December 2014).

Inadequate dose selection for confirmatory trials is currently still one of the most challenging issues in drug development, as illustrated by high rates of late-stage attritions in clinical development and postmarketing commitments required by regulatory institutions. In an effort to shift the current paradigm in dose and regimen selection and highlight the availability and usefulness of well-established and regulatory-acceptable methods, the European Medicines Agency (EMA) in collaboration with the European Federation of Pharmaceutical Industries Association (EFPIA) hosted a multistakeholder workshop on dose finding (London 4-5 December 2014). Some methodologies that could constitute a toolkit for drug developers and regulators were presented. These methods are described in the present report: they include five advanced methods for data analysis (empirical regression models, pharmacometrics models, quantitative systems pharmacology models, MCP-Mod, and model averaging) and three methods for study design optimization (Fisher information matrix (FIM)-based methods, clinical trial simulations, and adaptive studies). Pairwise comparisons were also discussed during the workshop; however, mostly for historical reasons. This paper discusses the added value and limitations of these methods as well as challenges for their implementation. Some applications in different therapeutic areas are also summarized, in line with the discussions at the workshop. There was agreement at the workshop on the fact that selection of dose for phase III is an estimation problem and should not be addressed via hypothesis testing. Dose selection for phase III trials should be informed by well-designed dose-finding studies; however, the specific choice of method(s) will depend on several aspects and it is not possible to recommend a generalized decision tree. There are many valuable methods available, the methods are not mutually exclusive, and they should be used in conjunction to ensure a scientifically rigorous understanding of the dosing rationale.

Modelling the effect of climate change on recovery of acidified freshwaters: relative sensitivity of individual processes in the MAGIC model.

The MAGIC model was used to evaluate the relative sensitivity of several possible climate-induced effects on the recovery of soil and surface water from acidification. A common protocol was used at 14 intensively studied sites in Europe and eastern North America. The results show that several of the factors are of only minor importance (increase in pCO(2) in soil air and runoff, for example), several are important at only a few sites (seasalts at near-coastal sites, for example) and several are important at nearly all sites (increased concentrations of organic acids in soil solution and runoff, for example). In addition changes in forest growth and decomposition of soil organic matter are important at forested sites and sites at risk of nitrogen saturation. The trials suggest that in future modelling of recovery from acidification should take into account possible concurrent climate changes and focus specially on the climate-induced changes in organic acids and nitrogen retention.

"Threshold-crossing": A Useful Way to Establish the Counterfactual in Clinical Trials?

A central question in the assessment of benefit/harm of new treatments is: how does the average outcome on the new treatment (the factual) compare to the average outcome had patients received no treatment or a different treatment known to be effective (the counterfactual)? Randomized controlled trials (RCTs) are the standard for comparing the factual with the counterfactual. Recent developments necessitate and enable a new way of determining the counterfactual for some new medicines. For select situations, we propose a new framework for evidence generation, which we call "threshold-crossing." This framework leverages the wealth of information that is becoming available from completed RCTs and from real world data sources. Relying on formalized procedures, information gleaned from these data is used to estimate the counterfactual, enabling efficacy assessment of new drugs. We propose future (research) activities to enable "threshold-crossing" for carefully selected products and indications in which RCTs are not feasible.

Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts.

Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability.