YAP phosphorylation within integrin adhesions: Insights from a computational model.

Mechanical and biochemical cues intricately activate Yes-associated protein (YAP), which is pivotal for the cellular responses to these stimuli. Recent findings reveal an unexplored role of YAP in influencing the apoptotic process. It has been shown that, on soft matrices, YAP is recruited to small adhesions, phosphorylated at Y357, and translocated into the nucleus triggering apoptosis. Interestingly, YAP Y357 phosphorylation is significantly reduced in larger mature focal adhesions on stiff matrices. Building upon these novel insights, we have developed a stochastic model to delve deeper into the complex dynamics of YAP phosphorylation within integrin adhesions. Our findings emphasize several key points: firstly, increasing the cytosolic diffusion rate of YAP correlates with higher levels of phosphorylated YAP (pYAP); secondly, increasing the number of binding sites and distributing them across the membrane surface, mimicking smaller adhesions, leads to higher pYAP levels, particularly at lower diffusion rates. Moreover, we show that the binding and release rate of YAP to adhesions as well as adhesion lifetimes significantly influence the size effect of adhesion-induced YAP phosphorylation. The results highlight the complex and dynamic interplay between adhesion lifetime, the rate of pYAP unbinding from adhesions, and dephosphorylation rates, collectively shaping overall pYAP levels. In summary, our work advances the understanding of YAP mechanotransduction and opens avenues for experimental validation.

Insights gained from computational modeling of YAP/TAZ signaling for cellular mechanotransduction.

YAP/TAZ signaling pathway is regulated by a multiplicity of feedback loops, crosstalk with other pathways, and both mechanical and biochemical stimuli. Computational modeling serves as a powerful tool to unravel how these different factors can regulate YAP/TAZ, emphasizing biophysical modeling as an indispensable tool for deciphering mechanotransduction and its regulation of cell fate. We provide a critical review of the current state-of-the-art of computational models focused on YAP/TAZ signaling.

Beyond PDE4 inhibition: A comprehensive review on downstream cAMP signaling in the central nervous system.

Cyclic adenosine monophosphate (cAMP) is a key second messenger that regulates signal transduction pathways pivotal for numerous biological functions. Intracellular cAMP levels are spatiotemporally regulated by their hydrolyzing enzymes called phosphodiesterases (PDEs). It has been shown that increased cAMP levels in the central nervous system (CNS) promote neuroplasticity, neurotransmission, neuronal survival, and myelination while suppressing neuroinflammation. Thus, elevating cAMP levels through PDE inhibition provides a therapeutic approach for multiple CNS disorders, including multiple sclerosis, stroke, spinal cord injury, amyotrophic lateral sclerosis, traumatic brain injury, and Alzheimer's disease. In particular, inhibition of the cAMP-specific PDE4 subfamily is widely studied because of its high expression in the CNS. So far, the clinical translation of full PDE4 inhibitors has been hampered because of dose-limiting side effects. Hence, focusing on signaling cascades downstream activated upon PDE4 inhibition presents a promising strategy, offering novel and pharmacologically safe targets for treating CNS disorders. Yet, the underlying downstream signaling pathways activated upon PDE(4) inhibition remain partially elusive. This review provides a comprehensive overview of the existing knowledge regarding downstream mediators of cAMP signaling induced by PDE4 inhibition or cAMP stimulators. Furthermore, we highlight existing gaps and future perspectives that may incentivize additional downstream research concerning PDE(4) inhibition, thereby providing novel therapeutic approaches for CNS disorders.

YAP mediates apoptosis through failed integrin adhesion reinforcement.

Extracellular matrix (ECM) rigidity is a major effector of cell fate decisions. Whereas cell proliferation on stiff matrices, wherein Yes-associated protein (YAP) plays a pivotal role, is well documented, activation of apoptosis in response to soft matrices is poorly understood. Here, we show that YAP drives the apoptotic decision as well. We find that in cells on soft matrices, YAP is recruited to small adhesions, phosphorylated at the Y357 residue, and translocated into the nucleus, ultimately leading to apoptosis. In contrast, Y357 phosphorylation levels are dramatically low in large adhesions on stiff matrices. Furthermore, mild attenuation of actomyosin contractility allows adhesion growth on soft matrices, leading to reduced Y357 phosphorylation levels and resulting in cell growth. These findings indicate that failed adhesion reinforcement drives rigidity-dependent apoptosis through YAP and that this decision is not determined solely by ECM rigidity but rather by the balance between cellular forces and ECM rigidity.

Blood Monocyte Phenotype Is A Marker of Cardiovascular Risk in Type 2 Diabetes.

BACKGROUND: Diabetes is a major risk factor for atherosclerotic cardiovascular diseases with a 2-fold higher risk of cardiovascular events in people with diabetes compared with those without. Circulating monocytes are inflammatory effector cells involved in both type 2 diabetes (T2D) and atherogenesis. METHODS: We investigated the relationship between circulating monocytes and cardiovascular risk progression in people with T2D, using phenotypic, transcriptomic, and metabolomic analyses. cardiovascular risk progression was estimated with coronary artery calcium score in a cohort of 672 people with T2D. RESULTS: Coronary artery calcium score was positively correlated with blood monocyte count and frequency of the classical monocyte subtype. Unsupervised k-means clustering based on monocyte subtype profiles revealed 3 main endotypes of people with T2D at varying risk of cardiovascular events. These observations were confirmed in a validation cohort of 279 T2D participants. The predictive association between monocyte count and major adverse cardiovascular events was validated through an independent prospective cohort of 757 patients with T2D. Integration of monocyte transcriptome analyses and plasma metabolomes showed a disruption of mitochondrial pathways (tricarboxylic acid cycle, oxidative phosphorylation pathway) that underlined a proatherogenic phenotype. CONCLUSIONS: In this study, we provide evidence that frequency and monocyte phenotypic profile are closely linked to cardiovascular risk in patients with T2D. The assessment of monocyte frequency and count is a valuable predictive marker for risk of cardiovascular events in patients with T2D. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04353869.

Force-dependent focal adhesion assembly and disassembly: A computational study.

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

Computational evidence for multi-layer crosstalk between the cadherin-11 and PDGFR pathways.

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell-cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR-α binding to extracellular cadherin-11 regions increases the PDGFR-α activity, whereas the interaction between PDGFR-ß and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between ß-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR-α and -ß. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.

Development of Mesoporous Silica Nanoparticle-Based Films with Tunable Arginine-Glycine-Aspartate Peptide Global Density and Clustering Levels to Study Stem Cell Adhesion and Differentiation.

Stem cell adhesion is mediated via the binding of integrin receptors to adhesion motifs present in the extracellular matrix (ECM). The spatial organization of adhesion ligands plays an important role in stem cell integrin-mediated adhesion. In this study, we developed a series of biointerfaces using arginine-glycine-aspartate (RGD)-functionalized mesoporous silica nanoparticles (MSN-RGD) to study the effect of RGD adhesion ligand global density (ligand coverage over the surface), spacing, and RGD clustering levels on stem cell adhesion and differentiation. To prepare the biointerface, MSNs were chemically functionalized with RGD peptides via an antifouling poly(ethylene glycol) (PEG) linker. The RGD surface functionalization ratio could be controlled to create MSNs with high and low RGD ligand clustering levels. MSN films with varying RGD global densities could be created by blending different ratios of MSN-RGD and non-RGD-functionalized MSNs together. A computational simulation study was performed to analyze nanoparticle distribution and RGD spacing on the resulting surfaces to determine experimental conditions. Enhanced cell adhesion and spreading were observed when RGD global density increased from 1.06 to 5.32 nmol cm-2 using highly clustered RGD-MSN-based films. Higher RGD ligand clustering levels led to larger cell spreading and increased formation of focal adhesions. Moreover, a higher RGD ligand clustering level promoted the expression of alkaline phosphatase in hMSCs. Overall, these findings indicate that both RGD global density and clustering levels are crucial variables in regulating stem cell behaviors. This study provides important information about ligand-integrin interactions, which could be implemented into biomaterial design to achieve optimal performance of adhesive functional peptides.

Optimization of Media Change Intervals through Hydrogels Using Mathematical Models.

Three-dimensional cell culture in engineered hydrogels is increasingly used in tissue engineering and regenerative medicine. The transfer of nutrients, gases, and waste materials through these hydrogels is of utmost importance for cell viability and response, yet the translation of diffusion coefficients into practical guidelines is not well established. Here, we combined mathematical modeling, fluorescent recovery after photobleaching, and hydrogel diffusion experiments on cell culture inserts to provide a multiscale practical approach for diffusion. We observed a dampening effect of the hydrogel that slowed the response to concentration changes and the creation of a diffusion gradient in the hydrogel by media refreshment. Our designed model combined with measurements provides a practical point of reference for diffusion coefficients in real-world culture conditions, enabling more informed choices on hydrogel culture conditions. This model can be improved in the future to simulate more complicated intrinsic hydrogel properties and study the effects of secondary interactions on the diffusion of analytes through the hydrogel.

Prevalence and Description of the Skin Reactions Associated with Adhesives in Diabetes Technology Devices in an Adult Population: Results of the CUTADIAB Study.

Objective: The use of continuous glucose monitoring (CGM) systems and continuous subcutaneous insulin infusion (CSII) devices adhering to the skin can lead to skin reactions. The objective was to determine the prevalence and consequences of skin reactions at CGM or CSII sites in a large unbiased population. Research Design and Methods: This is a cross-sectional multicenter study. All adult patients with diabetes seen in consultation over a period of 7 months and using or having used a system with skin adhesives (in the last 10 years) were included and filled out a self-assessment questionnaire. Results: Among 851 patients, skin reaction was reported in 28% with CGM and 29% with CSII. Patients reporting reactions were more frequently women using CGM and CSII, and CGM users had type 1 more often than type 2 diabetes (P < 0.001). Manifestations were similar for reactions to CGM and CSII: redness and pruritus in 70%-75% of patients with reactions, pain in 20%-25%, and vesicles and desquamation in 12%-15%. Manifestations occurred within the first 24 h of first use in 22%-24% of patients with reactions to CGM and CSII, but after more than 6 months in 38% and 47% of patients with reactions to CGM and CSII, respectively. Device use was definitively stopped in 12% of patients with reactions to CGM (3.2% of all users) and 7% with reactions to CSII (2.1% of all users). Conclusions: Skin reactions were common, with similar presentations in CGM and CSII users. Manifestations suggested skin irritation rather than allergies. These reactions rarely led to the definitive discontinuation of the use of the device.

Intact Fish Skin Graft vs. Standard of Care in Patients with Neuroischaemic Diabetic Foot Ulcers (KereFish Study): An International, Multicentre, Double-Blind, Randomised, Controlled Trial Study Design and Rationale.

BACKGROUND: Cell and/or tissue-based wound care products have slowly advanced in the treatment of non-healing ulcers, however, few studies have evaluated the effectiveness of these devices in the management of severe diabetic foot ulcers. METHOD: This study (KereFish) is part of a multi-national, multi-centre, randomised, controlled clinical investigation (Odin) with patients suffering from deep diabetic wounds, allowing peripheral artery disease as evaluated by an ankle brachial index equal or higher than 0.6. The study has parallel treatment groups: Group 1 treatment with Kerecis® Omega3 Wound™ versus Group 2 treatment with standard of care. The primary objective is to test the hypothesis that a larger number of severe diabetic ulcers and amputation wounds, including those with moderate arterial disease, will heal in 16 weeks when treated with Kerecis® Omega3 Wound™ than with standard of care. CONCLUSION: This study has received the ethics committee approval of each participating country. Inclusion of participants began in March 2020 and ended in July 2022. The first results will be presented in March 2023. The study is registered in ClinicalTrials.gov as Identifier: NCT04537520.

Computational investigation of the dynamic control of cAMP signaling by PDE4 isoform types.

Cyclic adenosine monophosphate (cAMP) is a generic signaling molecule that, through precise control of its signaling dynamics, exerts distinct cellular effects. Consequently, aberrant cAMP signaling can have detrimental effects. Phosphodiesterase 4 (PDE4) enzymes profoundly control cAMP signaling and comprise different isoform types wherein enzymatic activity is modulated by differential feedback mechanisms. Because these feedback dynamics are non-linear and occur coincidentally, their effects are difficult to examine experimentally but can be well simulated computationally. Through understanding the role of PDE4 isoform types in regulating cAMP signaling, PDE4-targeted therapeutic strategies can be better specified. Here, we established a computational model to study how feedback mechanisms on different PDE4 isoform types lead to dynamic, isoform-specific control of cAMP signaling. Ordinary differential equations describing cAMP dynamics were implemented in the VirtualCell environment. Simulations indicated that long PDE4 isoforms exert the most profound control on oscillatory cAMP signaling, as opposed to the PDE4-mediated control of single cAMP input pulses. Moreover, elevating cAMP levels or decreasing PDE4 levels revealed different effects on downstream signaling. Together these results underline that cAMP signaling is distinctly regulated by different PDE4 isoform types and that this isoform specificity should be considered in both computational and experimental follow-up studies to better define PDE4 enzymes as therapeutic targets in diseases in which cAMP signaling is aberrant.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Implementing Computational Modeling in Tissue Engineering: Where Disciplines Meet.

In recent years, the mathematical and computational sciences have developed novel methodologies and insights that can aid in designing advanced bioreactors, microfluidic setups or organ-on-chip devices, in optimizing culture conditions, or predicting long-term behavior of engineered tissues in vivo. In this review, we introduce the concept of computational models and how they can be integrated in an interdisciplinary workflow for Tissue Engineering and Regenerative Medicine (TERM). We specifically aim this review of general concepts and examples at experimental scientists with little or no computational modeling experience. We also describe the contribution of computational models in understanding TERM processes and in advancing the TERM field by providing novel insights. Impact Statement Although in recent years the use of mathematical and computational sciences has increased in the Tissue Engineering and Regenerative Medicine (TERM) field, we believe that a further integration of experimental and computational approaches has a huge potential for advancing the field due to the ability of models to explain and predict experimental results and efficiently optimize TERM product and process designs. By providing an overview of existing computational models, how they have contributed to the field, as well as a future perspective, this review represents an important step to help realize TERM's ultimate goal: a cure instead of care.

Sex-Specific Computational Models of Kidney Function in Patients With Diabetes.

The kidney plays an essential role in homeostasis, accomplished through the regulation of pH, electrolytes and fluids, by the building blocks of the kidney, the nephrons. One of the important markers of the proper functioning of a kidney is the glomerular filtration rate. Diabetes is characterized by an enlargement of the glomerular and tubular size of the kidney, affecting the afferent and efferent arteriole resistance and hemodynamics, ultimately leading to chronic kidney disease. We postulate that the diabetes-induced changes in kidney may exhibit significant sex differences as the distribution of renal transporters along the nephron may be markedly different between women and men, as recently shown in rodents. The goals of this study are to (i) analyze how kidney function is altered in male and female patients with diabetes, and (ii) assess the renal effects, in women and men, of an anti-hyperglycemic therapy that inhibits the sodium-glucose cotransporter 2 (SGLT2) in the proximal convoluted tubules. To accomplish these goals, we have developed computational models of kidney function, separate for male and female patients with diabetes. The simulation results indicate that diabetes enhances Na+ transport, especially along the proximal tubules and thick ascending limbs, to similar extents in male and female patients, which can be explained by the diabetes-induced increase in glomerular filtration rate. Additionally, we conducted simulations to study the effects of diabetes and SGLT2 inhibition on solute and water transport along the nephrons. Model simulations also suggest that SGLT2 inhibition raises luminal [Cl-] at the macula densa, twice as much in males as in females, and could indicate activation of the tubuloglomerular feedback signal. By inducing osmotic diuresis in the proximal tubules, SGLT2 inhibition reduces paracellular transport, eventually leading to diuresis and natriuresis. Those effects on urinary excretion are blunted in women, in part due to their higher distal transport capacity.

The Galapagos Chip Platform for High-Throughput Screening of Cell Adhesive Chemical Micropatterns.

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and -physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell-material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called "Galapagos chip" is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell-ECM interaction.

Self-Oxygenation of Tissues Orchestrates Full-Thickness Vascularization of Living Implants.

Bioengineering of tissues and organs has the potential to generate functional replacement organs. However, achieving the full-thickness vascularization that is required for long-term survival of living implants has remained a grand challenge, especially for clinically sized implants. During the pre-vascular phase, implanted engineered tissues are forced to metabolically rely on the diffusion of nutrients from adjacent host-tissue, which for larger living implants results in anoxia, cell death, and ultimately implant failure. Here it is reported that this challenge can be addressed by engineering self-oxygenating tissues, which is achieved via the incorporation of hydrophobic oxygen-generating micromaterials into engineered tissues. Self-oxygenation of tissues transforms anoxic stresses into hypoxic stimulation in a homogenous and tissue size-independent manner. The in situ elevation of oxygen tension enables the sustained production of high quantities of angiogenic factors by implanted cells, which are offered a metabolically protected pro-angiogenic microenvironment. Numerical simulations predict that self-oxygenation of living tissues will effectively orchestrate rapid full-thickness vascularization of implanted tissues, which is empirically confirmed via in vivo experimentation. Self-oxygenation of tissues thus represents a novel, effective, and widely applicable strategy to enable the vascularization living implants, which is expected to advance organ transplantation and regenerative medicine applications.

A multi-domain shear-stress dependent diffusive model of cell transport-aided dialysis: analysis and simulation.

Kidney dialysis is the most widespread treatment method for end-stage renal disease, a debilitating health condition common in industrialized societies. While ubiquitous, kidney dialysis suffers from an inability to remove larger toxins, resulting in a gradual buildup of these toxins in dialysis patients, ultimately leading to further health complications. To improve dialysis, hollow fibers incorporating a cell-monolayer with cultured kidney cells have been proposed; however, the design of such a fiber is nontrivial. In particular, the effects of fluid wall-shear stress have an important influence on the ability of the cell layer to transport toxins. In the present work, we introduce a model for cell-transport aided dialysis, incorporating the effects of the shear stress. We analyze the model mathematically and establish its well-posedness. We then present a series of numerical results, which suggest that a hollow-fiber design with a wavy profile may increase the efficiency of the dialysis treatment. We investigate numerically the shape of the wavy channel to maximize the toxin clearance. These results demonstrate the potential for the use of computational models in the study and advancement of renal therapies.

Mechanistic Computational Models of Epithelial Cell Transporters-the Adorned Heroes of Pharmacokinetics.

Epithelial membrane transporter kinetics portray an irrefutable role in solute transport in and out of cells. Mechanistic models are used to investigate the transport of solutes at the organ, tissue, cell or membrane scale. Here, we review the recent advancements in using computational models to investigate epithelial transport kinetics on the cell membrane. Various methods have been employed to develop transport phenomena models of solute flux across the epithelial cell membrane. Interestingly, we noted that many models used lumped parameters, such as the Michaelis-Menten kinetics, to simplify the transporter-mediated reaction term. Unfortunately, this assumption neglects transporter numbers or the fact that transport across the membrane may be affected by external cues. In contrast, more recent mechanistic transporter kinetics models account for the transporter number. By creating models closer to reality researchers can investigate the downstream effects of physical or chemical disturbances on the system. Evidently, there is a need to increase the complexity of mechanistic models investigating the solute flux across a membrane to gain more knowledge of transporter-solute interactions by assigning individual parameter values to the transporter kinetics and capturing their dependence on each other. This change results in better pharmacokinetic predictions in larger scale platforms. More reliable and efficient model predictions can be made by creating mechanistic computational models coupled with dedicated in vitro experiments. It is also vital to foster collaborative efforts among transporter kinetics researchers in the modeling, material science and biological fields.