Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.

Kinetic modelling of glycolytic oscillations.

Glycolytic oscillations have been studied for well over 60 years, but aspects of their function, and mechanisms of regulation and synchronisation remain unclear. Glycolysis is amenable to mechanistic mathematical modelling, as its components have been well characterised, and the system can be studied at many organisational levels: in vitro reconstituted enzymes, cell free extracts, individual cells, and cell populations. In recent years, the emergence of individual cell analysis has opened new ways of studying this intriguing system.

Is distance from equilibrium a good indicator for a reaction's flux control?

By analysing a large set of models obtained from the JWS Online and Biomodels databases, we tested to what extent the disequilibrium ratio can be used as an estimator for the flux control of a reaction, a discussion point that was already raised by Kacser and Burns, and Heinrich and Rapoport in their seminal MCA manuscripts. Whereas no functional relation was observed, the disequilibrium ratio can be used as an estimator for the maximal flux control of a reaction step. We extended the original analysis of the relationship by incorporating the overall pathway disequilibrium ratio in the expression, which made it possible to make explicit expressions for flux control coefficients.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

Inhibitor titrations reveal low control of glyceraldehyde 3-phosphate dehydrogenase and high control of hexokinase on glycolytic flux in an aggressive triple-negative breast cancer cell line.

The glycolytic flux, and in particular lactate production, is strongly increased in cancer cells compared to normal cells, a characteristic often referred to as aerobic glycolysis or the Warburg effect. This makes the glycolytic pathway a potential drug target, in particular if the flux control distribution in the pathway has shifted due to the metabolic reprogramming in cancer cells. The flux response of a drug is dependent on both the sensitivity of the target to the drug and the flux control of the target, and both these characteristics can be exploited to obtain selectivity for cancer cells. Traditionally drug development programs have focused on selective sensitivity of the drug, not necessarily focussing on the flux control of the target. We determined the flux control of two steps that have been suggested to have high control in cancer cells, using two inhibitors, iodoacetic acid and 3-bromopyruvate, and measured a flux control of the glyceraldehyde 3-phosphate dehydrogenase close to zero, while the hexokinase holds 50% of all flux control in glycolysis in an invasive cancer cell line MDA-mb-231.

Antimicrobial nano-assemblies of tryptocidine C, a tryptophan-rich cyclic decapeptide, from ethanolic solutions.

Tryptocidine C (TpcC), a Trp-rich cyclodecapeptide is a minor constituent in the antibiotic tyrothricin complex from Brevibacillus parabrevis. TpcC possesses a high tendency to oligomerise in aqueous solutions and dried TpcC forms distinct self-assembled nanoparticles. High-resolution scanning electron microscopy revealed the influence of different ethanol:water solvent systems on TpcC self-assembly, with the TpcC, dried from a high concentration in 15% ethanol, primarily assembling into small nanospheres with 24.3 nm diameter and 0.05 polydispersity. TpcC at 16 µM, near its CMC, formed a variety of structures such as small nanospheres, large dense nanospheroids and facetted 3-D-crystals, as well as sheets and coarse carpet-like structures which depended on ethanol concentration. Drying 16 µM TpcC from 75% ethanol resulted in highly facetted 3-D crystals, as well as small nanospheres, while those in 10% ethanol preparation had less defined facets. Drying from 20 to 50% ethanol led to polymorphic architectures with a few defined nanospheroids and various small nanoparticles, imbedded in carpet- and sheet-like structures. These polymorphic surface morphologies correlated with maintenance of fluorescence properties and the surface-derived antibacterial activity against Staphylococcus aureus over time, while there was a significant change in fluorescence and loss in activity in the 10% and 75% preparations where 3-D crystals were observed. This indicated that TpcC oligomerisation in solutions with 20-50% ethanol leads to metastable structures with a high propensity for release of antimicrobial moieties, while those leading to crystallisation limit active moieties release. TpcC nano-assemblies can find application in antimicrobial coatings, surface disinfectants, food packaging and wound healing materials.

Uncovering the effects of heterogeneity and parameter sensitivity on within-host dynamics of disease: malaria as a case study.

BACKGROUND: The fidelity and reliability of disease model predictions depend on accurate and precise descriptions of processes and determination of parameters. Various models exist to describe within-host dynamics during malaria infection but there is a shortage of clinical data that can be used to quantitatively validate them and establish confidence in their predictions. In addition, model parameters often contain a degree of uncertainty and show variations between individuals, potentially undermining the reliability of model predictions. In this study models were reproduced and analysed by means of robustness, uncertainty, local sensitivity and local sensitivity robustness analysis to establish confidence in their predictions. RESULTS: Components of the immune system are responsible for the most uncertainty in model outputs, while disease associated variables showed the greatest sensitivity for these components. All models showed a comparable degree of robustness but displayed different ranges in their predictions. In these different ranges, sensitivities were well-preserved in three of the four models. CONCLUSION: Analyses of the effects of parameter variations in models can provide a comparative tool for the evaluation of model predictions. In addition, it can assist in uncovering model weak points and, in the case of disease models, be used to identify possible points for therapeutic intervention.

Computational modelling of the Δ4 and Δ5 adrenal steroidogenic pathways provides insight into hypocortisolism.

This study demonstrates the application of a mathematical steroidogenic model, constructed with individual in vitro enzyme characterisations, to simulate in vivo steroidogenesis in a diseased state. This modelling approach was applied to the South African Angora goat, that suffers from hypocortisolism caused by altered adrenal function. These animals are extremely vulnerable to cold stress, leading to substantial monetary loss in the mohair industry. The Angora goat has increased CYP17A1 17,20-lyase enzyme activity in comparison with hardy livestock species. Determining the effect of this altered adrenal function on adrenal steroidogenesis during a cold stress response is difficult. We developed a model describing adrenal steroidogenesis under control conditions, and under altered steroidogenic conditions where the animal suffers from hypocortisolism. The model is parameterised with experimental data from in vitro enzyme characterisations of a hardy control species. The increased 17,20-lyase activity of the Angora goat CYP17A1 enzyme was subsequently incorporated into the model and the response to physiological stress is simulated under both control and altered adrenal steroidogenic conditions.

Intercellular communication induces glycolytic synchronization waves between individually oscillating cells.

Many organs have internal structures with spatially differentiated and sometimes temporally synchronized groups of cells. The mechanisms leading to such differentiation and coordination are not well understood. Here we design a diffusion-limited microfluidic system to mimic a multicellular organ structure with peripheral blood flow and test whether a group of individually oscillating yeast cells could form subpopulations of spatially differentiated and temporally synchronized cells. Upon substrate addition, the dynamic response at single-cell level shows glycolytic oscillations, leading to wave fronts traveling through the monolayered population and to synchronized communities at well-defined positions in the cell chamber. A detailed mechanistic model with the architectural structure of the flow chamber incorporated successfully predicts the spatial-temporal experimental data, and allows for a molecular understanding of the observed phenomena. The intricate interplay of intracellular biochemical reaction networks leading to the oscillations, combined with intercellular communication via metabolic intermediates and fluid dynamics of the reaction chamber, is responsible for the generation of the subpopulations of synchronized cells. This mechanism, as analyzed from the model simulations, is experimentally tested using different concentrations of cyanide stress solutions. The results are reproducible and stable, despite cellular heterogeneity, and the spontaneous community development is reminiscent of a zoned cell differentiation often observed in multicellular organs.

Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers.

In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls. A user who follows the protocols should be able to detect clear metabolite time traces over the course of up to an hour that are indicative of dynamics on the second scale in individual cells during fast and reversible environmental adjustments. © 2018 by John Wiley & Sons, Inc.

Harmonizing semantic annotations for computational models in biology.

Life science researchers use computational models to articulate and test hypotheses about the behavior of biological systems. Semantic annotation is a critical component for enhancing the interoperability and reusability of such models as well as for the integration of the data needed for model parameterization and validation. Encoded as machine-readable links to knowledge resource terms, semantic annotations describe the computational or biological meaning of what models and data represent. These annotations help researchers find and repurpose models, accelerate model composition and enable knowledge integration across model repositories and experimental data stores. However, realizing the potential benefits of semantic annotation requires the development of model annotation standards that adhere to a community-based annotation protocol. Without such standards, tool developers must account for a variety of annotation formats and approaches, a situation that can become prohibitively cumbersome and which can defeat the purpose of linking model elements to controlled knowledge resource terms. Currently, no consensus protocol for semantic annotation exists among the larger biological modeling community. Here, we report on the landscape of current annotation practices among the COmputational Modeling in BIology NEtwork community and provide a set of recommendations for building a consensus approach to semantic annotation.

Phosphofructokinase controls the acetaldehyde-induced phase shift in isolated yeast glycolytic oscillators.

The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronisation and phase locking and can be quantified in a phase response curve (PRC). In individual, oscillating yeast cells, we characterised experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses and followed the transduction of the perturbation through the system. Subsequently, we analysed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction network. We find that our model quantitatively predicts the phase-dependent phase shift observed in the experimental data. The phase shift is in agreement with an adaptation leading to synchronisation with an external signal. Our model analysis establishes that phosphofructokinase plays a key role in the phase shift dynamics as shown in the PRC and adaptation time to external perturbations. Specific mechanism-based interventions, made possible through such analyses of detailed models, can improve upon standard trial and error methods, e.g. melatonin supplementation to overcome jet-lag, which are error-prone, specifically, since the effects are phase dependent and dose dependent. The models by Gustavsson and Goldbeter discussed in the text can be obtained from the JWS Online simulation database: (https://jjj.bio.vu.nl/models/gustavsson5 and https://jjj.bio.vu.nl/models/goldbeter1).

11-Oxygenated androgen precursors are the preferred substrates for aldo-keto reductase 1C3 (AKR1C3): Implications for castration resistant prostate cancer.

The progression of castration resistant prostate cancer (CRPC) is driven by the intratumoral conversion of adrenal androgen precursors to potent androgens. The expression of aldo-keto reductase 1C3 (AKR1C3), which catalyses the reduction of weak androgens to more potent androgens, is significantly increased in CRPC tumours. The oxidation of androgens to their inactive form is catalysed by 17ß-hydroxysteroid dehydrogenase type 2 (17ßHSD2), but little attention is given to the expression levels of this enzyme. In this study, we show that the 11-oxygenated androgen precursors of adrenal origin are the preferred substrate for AKR1C3. In particular we show that the enzymatic efficiency of AKR1C3 is 8- and 24-fold greater for 11-ketoandrostenedione than for the classic substrates androstenedione and 5α-androstanedione, respectively. Using three independent experimental systems and a computational model we subsequently show that increased ratios of AKR1C3:17ßHSD2 significantly favours the flux through the 11-oxygenated androgen pathway as compared to the classical or 5α-androstanedione pathways. Our findings reveal that the flux through the classical and 5α-androstanedione pathways are limited by the low catalytic efficiently of AKR1C3 towards classical androgens combined with the high catalytic efficiency of 17ßHSD2, and that the expression of the oxidative enzyme therefore plays a vital role in determining the steady state concentration of active androgens. Using microarray data from prostate tissue we confirm that the AKR1C3:17ßHSD2 ratio is significantly increased in patients undergoing androgen deprivation therapy as compared to benign tissue, and further increased in patients with CRPC. Taken together this study therefore demonstrates that the ratio of AKR1C3:17ßHSD2 is more important than AKR1C3 expression alone in determining intratumoral androgen levels and that 11-oxygenated androgens may play a bigger role in CRPC than previously anticipated.

Phosphoglycerate kinase acts as a futile cycle at high temperature.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Identifiers for the 21st century: How to design, provision, and reuse persistent identifiers to maximize utility and impact of life science data.

In many disciplines, data are highly decentralized across thousands of online databases (repositories, registries, and knowledgebases). Wringing value from such databases depends on the discipline of data science and on the humble bricks and mortar that make integration possible; identifiers are a core component of this integration infrastructure. Drawing on our experience and on work by other groups, we outline 10 lessons we have learned about the identifier qualities and best practices that facilitate large-scale data integration. Specifically, we propose actions that identifier practitioners (database providers) should take in the design, provision and reuse of identifiers. We also outline the important considerations for those referencing identifiers in various circumstances, including by authors and data generators. While the importance and relevance of each lesson will vary by context, there is a need for increased awareness about how to avoid and manage common identifier problems, especially those related to persistence and web-accessibility/resolvability. We focus strongly on web-based identifiers in the life sciences; however, the principles are broadly relevant to other disciplines.

The Peculiar Glycolytic Pathway in Hyperthermophylic Archaea: Understanding Its Whims by Experimentation In Silico.

Mathematical models are key to systems biology where they typically describe the topology and dynamics of biological networks, listing biochemical entities and their relationships with one another. Some (hyper)thermophilic Archaea contain an enzyme, called non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), which catalyzes the direct oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate omitting adenosine 5'-triphosphate (ATP) formation by substrate-level-phosphorylation via phosphoglycerate kinase. In this study we formulate three hypotheses that could explain functionally why GAPN exists in these Archaea, and then construct and use mathematical models to test these three hypotheses. We used kinetic parameters of enzymes of Sulfolobus solfataricus (S. solfataricus) which is a thermo-acidophilic archaeon that grows optimally between 60 and 90 °C and between pH 2 and 4. For comparison, we used a model of Saccharomyces cerevisiae (S. cerevisiae), an organism that can live at moderate temperatures. We find that both the first hypothesis, i.e., that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plus phosphoglycerate kinase (PGK) route (the alternative to GAPN) is thermodynamically too much uphill and the third hypothesis, i.e., that GAPDH plus PGK are required to carry the flux in the gluconeogenic direction, are correct. The second hypothesis, i.e., that the GAPDH plus PGK route delivers less than the 1 ATP per pyruvate that is delivered by the GAPN route, is only correct when GAPDH reaction has a high rate and 1,3-bis-phosphoglycerate (BPG) spontaneously degrades to 3PG at a high rate.

Targeting pathogen metabolism without collateral damage to the host.

The development of drugs that can inactivate disease-causing cells (e.g. cancer cells or parasites) without causing collateral damage to healthy or to host cells is complicated by the fact that many proteins are very similar between organisms. Nevertheless, due to subtle, quantitative differences between the biochemical reaction networks of target cell and host, a drug can limit the flux of the same essential process in one organism more than in another. We identified precise criteria for this 'network-based' drug selectivity, which can serve as an alternative or additive to structural differences. We combined computational and experimental approaches to compare energy metabolism in the causative agent of sleeping sickness, Trypanosoma brucei, with that of human erythrocytes, and identified glucose transport and glyceraldehyde-3-phosphate dehydrogenase as the most selective antiparasitic targets. Computational predictions were validated experimentally in a novel parasite-erythrocytes co-culture system. Glucose-transport inhibitors killed trypanosomes without killing erythrocytes, neurons or liver cells.

The JWS online simulation database.

SUMMARY: JWS Online is a web-based platform for construction, simulation and exchange of models in standard formats. We have extended the platform with a database for curated simulation experiments that can be accessed directly via a URL, allowing one-click reproduction of published results. Users can modify the simulation experiments and export them in standard formats. The Simulation database thus lowers the bar on exploring computational models, helps users create valid simulation descriptions and improves the reproducibility of published simulation experiments. AVAILABILITY AND IMPLEMENTATION: The Simulation Database is available on line at https://jjj.bio.vu.nl/models/experiments/ . CONTACT: jls@sun.ac.za .

FAIRDOMHub: a repository and collaboration environment for sharing systems biology research.

The FAIRDOMHub is a repository for publishing FAIR (Findable, Accessible, Interoperable and Reusable) Data, Operating procedures and Models (https://fairdomhub.org/) for the Systems Biology community. It is a web-accessible repository for storing and sharing systems biology research assets. It enables researchers to organize, share and publish data, models and protocols, interlink them in the context of the systems biology investigations that produced them, and to interrogate them via API interfaces. By using the FAIRDOMHub, researchers can achieve more effective exchange with geographically distributed collaborators during projects, ensure results are sustained and preserved and generate reproducible publications that adhere to the FAIR guiding principles of data stewardship.

Fourth-Generation Progestins Inhibit 3ß-Hydroxysteroid Dehydrogenase Type 2 and Modulate the Biosynthesis of Endogenous Steroids.

Progestins used in contraception and hormone replacement therapy are synthetic compounds designed to mimic the actions of the natural hormone progesterone and are classed into four consecutive generations. The biological actions of progestins are primarily determined by their interactions with steroid receptors, and factors such as metabolism, pharmacokinetics, bioavailability and the regulation of endogenous steroid hormone biosynthesis are often overlooked. Although some studies have investigated the effects of select progestins on a few steroidogenic enzymes, studies comparing the effects of progestins from different generations are lacking. This study therefore explored the putative modulatory effects of progestins on de novo steroid synthesis in the adrenal by comparing the effects of select progestins from the respective generations, on endogenous steroid hormone production by the H295R human adrenocortical carcinoma cell line. Ultra-performance liquid chromatography/tandem mass spectrometry analysis showed that the fourth-generation progestins, nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP), unlike the progestins selected from the first three generations, modulate the biosynthesis of several endogenous steroids. Subsequent assays performed in COS-1 cells expressing human 3ßHSD2, suggest that these progestins modulate the biosynthesis of steroid hormones by inhibiting the activity of 3ßHSD2. The Ki values determined for the inhibition of human 3ßHSD2 by NES (9.5 ± 0.96 nM), NoMAC (29 ± 7.1 nM) and DRSP (232 ± 38 nM) were within the reported concentration ranges for the contraceptive use of these progestins in vivo. Taken together, our results suggest that newer, fourth-generation progestins may exert both positive and negative physiological effects via the modulation of endogenous steroid hormone biosynthesis.