In the last two decades, microneedles (MNs) have received significant interest due to their potential for painless transdermal drug delivery (TDD) and minimal skin damage. MNs have found applications in a range of research and development areas in drug delivery. They have been prepared using a variety of materials and fabrication techniques resulting in MN arrays with different dimensions, shapes, and geometries for delivery of a variety of drug molecules. These parameters play crucial roles in determining the drug release profiles from the MNs. Developing mathematical modelling, simulation, and optimisation techniques is vital to achieving the desired MN performances. These will then be helpful for pharmaceutical and biotechnological industries as well as professionals working in the field of regulatory affairs focusing on MN based TDD systems. This is because modelling has a great potential to reduce the financial and time cost of both the MNs’ studies and manufacturing. For example, a number of robust mathematical models for predicting the performance of the MNs in vivo have emerged recently which incorporate the roles of the structural and mechanical properties of the skin. In addressing these points, this review paper aims to highlight the current status of the MN modelling research, in particular, the modelling, simulation and optimisation of the systems for drug delivery. The theoretical basis for the simulation of MN enhanced diffusion is discussed within this paper. Thus, this review paper provides a better understanding of the modelling of the MN mediated drug delivery process. The microneedle (MN) system has received significant interest in the last two decades as an important method for transdermal drug delivery (TDD). The MNs consist of micron size needles that facilitate drug molecules to overcome the stratum corneum (SC), the outermost layer of skin, without triggering the nerve ending within the dermis ,2. Thus,As shown in Mathematical modelling of the MN systems is an area of growing research where the theories governing drug transport and delivery through the skin can be suitably applied for the development and optimisation of MN systems. However, it is obvious that most of the research in the field of MN is focused towards an aspect of experimental or/and fabrication technique. The attention towards the modelling has been ignored generally as researchers tend to focus more on characterisation and fabrication of the MNs. The exact reason for a significantly less efforts on development of modelling, simulation and optimisation tools for MNs is not known. We believe this is due to a lack of sufficient number of parameters for applying the relevant theories as well as the expertise and interests needed for modelling these systems. An analysis of these publications is shown in The possibility of developing modelling tools towards achieving MN optimisation and testing in support of experimental studies is beneficial and attractive. It can save both the costs and time by eliminating the need for many time-consuming and expensive experimental trials. Modelling MN based TDD typically deals with the simulation of three coupled sub-domains i.e., MNs, skin, and bloodstream. As shown in Keeping the above points in mind, this review aims to present the current status of various modelling approaches for MN design and their utilities. This article also highlights the ways in which skin properties can be incorporated in the modelling of MN based TDD. For completeness of the discussion in this paper, we will briefly discuss the key features of the MNs, and the parameters required for carrying out mathematical modelling of these systems. Published case studies on various types of MNs are also highlighted in the latter part of the paper. We believe that this is the first review paper that entirely focuses on reviewing the state of the published work on mathematical modelling, simulation and optimisation of MNs.Some understanding of the MN features is necessary as the first step in the modelling process. This is because reasonable design criteria are necessary to guarantee that a MN array is functioning well during drug delivery  or extraMorphologically, MNs are categorised into five types, namely, hollow, solid, coated, dissolving, and swellable MNs . As discHowever, there are still withstanding issues, such as the manufacturing cost and complexities, toxicity, and the physicochemical properties of the drug molecules that limit the range of applications needing further attention ,52,53,54Of the key MN features, namely, the material of fabrication of the MNs is a critical factor in the preparation and application of different kinds of MNs. Different designs of MNs require specific properties of materials for preparation. These aspects become important for developing mathematical models of these systems as well. Silicon is known to be the material of the first MNs used for drug delivery due to the advent of MEMS technology during the 1990s ,56. SiliThe limitations of metal and inorganic MNs such as expensive production processes and bio-incompatibility have led to the development of polymeric MNs. These polymers can be used to prepare solid , coated Bombxy mori silkworm and were micro-molded into an array of MNs. The silk fibroin MNs has enough mechanical strength to pierce the model skin (porcine skin), and they have been shown to be suitable for encapsulating methylene blue used as a model drug. Other early works also developed MNs from maltose [With the development of dissolving MNs, researchers have started to encapsulate drugs and vaccines in polymers for TDD, which make the biocompatibility of these polymers a top priority for their design ,68. MNs  maltose . Olatunj maltose  prepared maltose ,73. The  maltose .As we analyse the key MN features, it is imperative to consider the designs of these MNs more carefully as they are important for the modelling purposes. There are, in fact, a number of special designs amongst the MNs depending on their applications, for instance, one may consider the pocketed solid MNs for the higher load of drugs  and hollSeveral studies have adopted the tapered MN design for drug delivery. Olatunji et al.  modelledZhang et al.  employedIn an innovative approach to hollow MN design, crown-shaped MNs, which did not have an actual lumen but can be used for fluid extraction, were presented . The quaLim et al.  have comRecently, Chen et al.  have alsFrom the above discussion, it is evident that different MN structures have been fabricated by the researchers for efficient transfer of drugs into the skin. These systems are then considered at various bodies that approve MN based products, e.g., the US Food and Drug Administration . For theIn addressing the same issue of controlling variability of MN characteristics, the design of experiment (DoE) analysis was exploited by Jing et al.  for fabrDuring the modelling of MNs, one needs to convert either the conceptual or the real MN design into a computational domain, which can be used to run numerical simulations or other modelling exercise . It seems that the first work which reported MN modelling was in 1999 when the authors looked at modelling fluid extraction using hollow MNs . These aOne of the main objectives of MN modelling is to obtain the required design parameters of MNs so that the desired/optimum MN performance can be achieved. The outputs of such modelling exercise are typically the penetration depth and drug release rate from MNs. These outputs are related to various parameters of both skin and MN ,93. The The dimensions of the designed MNs need to be converted into input parameters for modelling, so the created computational domain is usable for further modelling and simulation exercises. The generated domain is a substitute for the designed MNs for the computer simulation where different tests can be achieved within that domain. For example, a computational domain of a hollow MN created in the computer program ‘Ansys’ is shown in t’ (Pitch) [The parameters involved to describe the MNs geometry are input to the computer program as well as the alignment of those MNs for a modelling process. For the parameters describing MNs’ geometry and arrangement on the MNs patch, there are five key aspects which have been analysed and discussed: the penetration depth/MNs length, both tip and base diameter of the MNs, center to center spacing between two MNs, numbers of MNs in the array and the distribution of the MNs in an array . Howeve (Pitch) . α descr (Pitch) . In the Skin composes of different layers  and dermis (DE)), which have different mechanical strengths and molecular diffusivities values . ModelliOnce the appropriate skin domain and their properties are chosen, the domain is used for simulation of drug permeation studies, which require information about the related drug molecules. Parameters of the molecules, such as the molecular weight and partition coefficient, can affect the drug diffusion coefficient. To demonstrate how molecular weight affects diffusion rates, Gomaa et al.  selectedA number of computer-based tools have been developed for MN modelling using both in-house programming and commercial software. These tools are usually developed for specific modelling purposes. For instance, when the parameters such as the aspect ratio (α) were needed to be tested for the optimisation of the squared MNs patch, an in-house java programming tool was developed to achieve the purpose . An optiA relatively recent study used a MATLAB-based image processing tool to acquire skin pore profiles from histological images that show the cross-sectional views of MNs treated skin . The comThe diffusion profiles in the computational domain are generally simulated using commercial software that are based on a numerical method such as the finite element method (FEM), e.g., COMSOL ,108, AnsThis section highlights the key work done by various researchers in modelling MN-based TDD. The review is organized according to key aspect of MN based drug delivery addressed in the paper.As discussed above, various MNs have been developed in the past to allow for TDD. The particular MN designs rely on the manufacturing techniques used for fabrication, and their success is totally dependent upon their ability to puncture the skin . HoweverAoyagi et al.  carried As part of numerical modelling of MN systems, one is required to generate the specific numerical meshes of the computational domain over which the governing equations of the specific numerical model can be solved. The following two examples illustrate this point further. Groves  has analBoonma et al.  used finThe dissolving MNs consisting of polymers are relatively soft compared to the solid MNs (polymeric). Hence, the mechanical properties of dissolving MNs are imperative to be measured in order to optimise the formulation of the MNs and the maximum load it can bear. An example model  of dissobending is the force that can bend the skin, Findentation is the force that the MNs start disrupting the SC layer, Fcutting is the force that the MNs start piercing into the skin, Fbuckling is the force causing skin deformation and Ffriction is the frictional force during the MNs penetration. The forces were calculated for conical shaped MN using FEM and results were compared with experimental data. The results of those force components were shown to be not only dependent on the mechanical properties of the skin but also affected by the geometry and alignment of the MNs on the patch. The deformation of the skin caused by those forces could be different from the shape of the MNs, thereby changing the results of the simulation. Simulation results from the FEM agreed well with the experimental results carried out using neonatal porcine skin mounted on Franz diffusion apparatus.Olatunji et al.  analysedRecently, Kim et al.  have devFrom the above discussion, it is evident that most of the modelling for MN insertion in skin have been performed taking into account a single MN rather than the design of the whole MN array. This seems to limit the range of validity of these results because an accurate calibration of the insertion force should take into account both the individual MN design as well as the particular distribution of the individual MNs on the patch , e.g., to investigate the effect of MN interspacing (pitch width) on the insertion force.12 and K21 are the transfer rate between skin reservoir and blood circulation, Ct is the concentration in the skin reservoir and Vt is volume of distribution in the skin reservoir. Although the pharmacokinetic model can display the mass transfer status of all components in the system at both transient and steady states, the parameters such as K12, K21 and Ke are difficult to quantify. Therefore, the blood stream is sometimes assumed to have 100% absorption on drug solutions to avoid the complexity and back diffusion of the drug molecule into the skin is ignored [Once a particular MN design is converted into a modelling domain e.g.,  and suit ignored .TOT) was defined as [ISP is the fractional skin surface area of the intact skin pathway, JISP is the flux through the intact skin pathway, fMCP is the fractional skin surface area of the microchannel pathway, and JMCP is the flux through the microchannel pathway. The cumulative amount-time profiles for skin layer (SC and viable tissue (VT)) were solved using Fick’s law of diffusion. In addition, they considered microchannel closure process which affect the drug transport in vivo. The permeability of the microchannel was interpreted with respect to the fractional skin surface area of the microchannel that remains open. The pore closure process affects the drug diffusion process and Equation (5) was modified by incorporation of new parameter for skin closure given by:Milewski et al.  developefined as :(5)JTOT=OMF stands for the open microchannel surface area fraction. The value of fOMF is unity for the in vitro process and Equation (6) changes to Equation (5). The drug is taken away by the systemic circulation once it crosses the viable skin tissue. A regression analysis was performed on drug pharmacokinetic data which showed a good match with the microchannel closure rate and in vitro permeation data.Here fRajoli et al.  developess is steady state flux, D is the diffusion coefficient, C is the drug concentration in the skin and x is the depth from top surface of the skin. The effective diffusion coefficient, which is related to the properties of the skin and size of the drug molecules, is approximated using Stokes–Einstein equation (Equation (9)) for drug transport through MN-pierced skin [The skin layers perform as obstructions between the drug formulation in MNs and blood stream at different length scales . The difced skin .(9)D=kBB is the Boltzmann’s constant, T is the absolute temperature, η is the viscosity of drug molecule and r is the radius of the drug molecules. McAllister et al. used Equation (9) to determine the drug diffusion coefficient in the drug solution within the holes created by the MNs. The authors then used a hindrance factor to work out the effective diffusion coefficient within skin. However, skin has different layers and complicated inner structures which are likely to make the values of effective diffusion coefficient calculated from this approach inaccurate.Here, k1.The diffusion coefficient can be deduced from the partition coefficient considering skin as a multilayer structure. The values of the diffusion coefficient are different for each layer of the skin, which can be calculated separately using Equation (10) .(10)D=−i/j is the partition coefficient between different diffusion compartments , h is the thickness of the skin layer and c0 is the initial drug concentration in the skin layer.Here, K2.To further explore the properties of different layers, the skin is considered as a porous material in which tortuous channels are defined to exist as pathways for the transportation of drug molecules. Equation (9) provides diffusion coefficients for every layer in the skin. While the drug molecules are inside those channels, the hindrance factor H(λ) also needs to be included where λ is the ratio of molecule radius over skin pore radius. Therefore, the size of the molecules decides the significance of the hindrance factor. Equation (11) shows the hindrance factor in cases where λ is less than 0.4 .(11)H and (13) ,122. needs to be measured. Although this method considers the skin as homogeneous material, the diffusion coefficient is still more accurate than that obtained using Equation (6). The theoretical relation between the diffusion coefficient and the time lag is shown in Equation (14) :(14)D=l2 in skin , howeverDue to the special characteristics of skin, three other methods have been introduced in the literature in order to increase the accuracy of the calculations for drug diffusion coefficient as discussed below:v, np, rp and MW are the vehicle drug concentration, skin pore density, pore radius and the molecular weight of the drug, respectively. The validity of the equation has been confirmed by a regression analysis of literature data. The equation has been further validated by performing in vitro experiments on human abdominal skin with three different drug molecules.Recently, Rzhevskiy et al.  have devThe MNs act as the drug delivery vehicle in the system where all the alterable parameters are included. The parameters that are related to the geometry and alignment of MNs have been mentioned in t is defined as the center to center distance between two adjacent MNs. It ensures that the MNs on a patch do not overlap with others and avoid a disordered pattern [The solid MNs can be either coated with drugs while insertion (coat and patch) or used to create pores on the skin where drugs will be applied after the MNs are removed (poke and patch). The poke with patch method has more capacity for drug loading, so the pores created by MNs should be considered as reservoir for drug solutions. The permeability (K) inside the reservoir can be described by .(16)K=fgiven by .(17)g=n pattern .(18)Pt=tn) and longitudinal direction (Ptm) must be specified. After the pattern has been chosen, the optimised α will be deduced so that the relationship between pitch and the parameters in Equation (17) can be found. By applying Fick’s law, the correlation between the permeability and diffusion coefficient of different drug molecules based on those optimum parameters can be calculated which are shown in Equation (18) indicates the MNs on patch are arranged in square pattern. To achieve other patterns such as triangular, diamond or rectangular, the pitch component in transverse direction  and D is the diffusion coefficient of drug in untreated skin. Correlations for dimensionless concentrations with different dimensionless parameters of MNs are as shown in Equations (20) and (21).Uppuluri et al.  analysedGood correlations were observed between the dimensionless concentrations determined theoretically as well as experimentally . Lyashko et al.  developeNumerical simulations have also been performed for solid MNs coated with drug formulations. Römgens et al.  performeHollow MNs have narrow capillaries that can infuse drug solution into the skin. Various numerical simulations have been completed on flow and structural analysis of MNs. Shibata et al.  performeint is the velocity of the moving interface, u0 is initial injection velocity, φ is the drug solution absorption rate per unit volume of tissue, β is the absorption coefficient of drug taken by blood stream and ε is the average porosity of the skin. The results indicate that higher diffusion rate can be achieved by increasing u0 while the porosity and absorption rate of skin are considered as intrinsic variables. In the said study, the moving interface position was measured in an assumed thickness x0 =15 µm skin sample which is shown in The hollow MNs have channels that can continuously deliver drug solution into the skin. However, the annular wall of the MNs limits the contact area of the drug solution to the skin, hence, the parameter f in Equation (16) needs to be redefined which is shown in Equation (22) .(22)f=nen below :(23)uintRecently, Liu et al.  developef is friction force between the piston seal ring and syringe wall. All of the terms included in the equations are defined in In Equation (24), F is the force applied by the pump on the syringe, N is the number of MNs and FMicropumps have been investigated in combination with hollow MNs to carry out the drug delivery and diagnosis. Chen et al.  proposedc is the volume of the dissolving MNs, kD is dissolution rate constant of the drug matrix, ρ is the density of the drug matrix, h is the height of the MNs, Cs is the solubility of the drug matrix in water, C is the drug concentration in the skin, β is the mass fraction of drug in the MNs and tanθ is the original ratio of radius over the height of MNs. Equation (23) indicates the boundary conditions of the simulation are changing with time and highly related to the dissolution rate of the drug formula. Besides, the alignment of the MNs and mass fraction of the drug in the MNs is also adjustable parameters that can affect the diffusion rate.Dissolving MNs are of utmost importance now for TDD as they can improve regulatory compliance and safety for patients. The dissolving MNs have similar mechanisms to solid MNs when using poke with patch method. However, the drug matrix in the reservoir of dissolving MNs is formulated so that it can be released over time. Therefore, the actual amount of drug solution is a time dependent function that is related to the dissolving rate of the drug matrix, i.e., the volume change rate of a conical dissolving MNs is given by .(25)dvcL is elimination rate constant and v(t) is volume of skin layer. The parameters KD and KL were estimated by carrying out the nonlinear regression of experimental data. Cumulative percentage of drug release M(t) is calculated by using the equation:w is pitch width, CSD is drug concentration in solid phase, vc,0 is the initial volume of skin layer, ds is skin depth and m0 is mass of drug in the baseplate. The results showed that drug concentration in the skin increased with increase in drug loading and height of MN. As the pitch width was increased the level of sumatriptan permeation in the skin also reduced.This approach was further investigated by Ronnander et al.  for modeRonnander et al.  reportedBoth Kim et al.  and Ronnn0 is the initial number averaged molecular weight while k is the degradation rate constant. The mass conservation equations of the model were solved for all system components using a finite difference scheme, and the model solutions were validated against experimental data from the literature [In Equation (29), Mterature . This moterature .Watanabe et al.  have expThe correlations indicated that the effect of the change in MN length on the effective insulin delivery can be easily explained by optimal scaling analysis. This enabled Chen et al.  to demoneff can be calculated using Equation (31) [VS is the diffusion coefficient in the VE and Cm is the concentration coated on the MNs.Besides these, new concepts are emerging based on the previous parameters to increase the accuracy of modelling and simulation, i.e., effective thickness is introduced which is calculated from the effective permeability of VE using Fick’s law. It is considering the skin deformation after the MNs insertion in order to give a more realistic value to skin thickness because the MNs reduce the distance between the drug loads to the bloodstream. The effective thickness Hion (31) .(31)HefTo apply all these parameters for simulation, the MNs design needs to be digitalised for computer program to process. Finite element method (FEM) is chosen as the algorism by most of the studies because it can provide detailed diffusion or force distribution profiles ,138 and Furthermore, the simulation of drug delivery using dissolving MNs can be advanced by modelling the drug-polymer interactions within the MN structure. This allows the inclusion of the mechanism by which the drug travels through the MN structure and the distribution of the drugs within the MN. This advances from the previous assumption of the skin-MN interface acting as a bulk constant concentration.Feng et al.  used molMN modelling is still at its early stage but its importance in the development and optimisation of MN systems are growing as evidenced from an increasing number of publications as well as a complex range of issues being modelled by these publications. Given the interests in these models, it is concluded from this review that development of mathematical models of MN systems will enable efficient design and optimisation of these systems, as the system themselves are developed for more complex range of TDD problems. As the theoretical basis of MN-based TDD continues to be developed further, the MNs modelling will be a key in developing the MNs for researches and manufactures.Modelling and optimisations have already been performed on different types of MNs. Different algorithms have been developed for solving the series of equations, thus, providing frameworks which can be adapted to predict performance of future MN arrays. Furthermore, the role of skin has also been incorporated in the modelling to understand the MN-based TDD process in an efficient way. The optimised MNs have been shown to enhance the permeation of drug potentially in many laboratory studies. However, there are yet to be sufficiently accurate and robust MN models for practical, medical or manufacturing applications. Most of the modelling studies have been performed considering the single MN which makes it hard to incorporate the combined effect of the whole MN array. Also, full attention needs to be placed on the material properties of the MNs for maintaining the pharmacokinetics and pharmacodynamics of drug molecule. Some models ignore the skin deformation and changing boundary conditions as there is a continuous change in contact between the microneedle and skin with time. Neglecting these factors while modelling can result in under or overpredictions of the MN performance and, consequently, the result can be unreliable.With more information acquired from MN experiments in vivo and in vitro, the interactions between the MNs and the skin as well as the drug diffusion profiles in the skin can be resolved. This will solve various challenges faced during preclinical and clinical evaluation of novel formulations and modes of delivery. MN modelling are therefore expected to play a greater part in improving the fabrication and application of MN systems in the future.


The ubiquitin protease pathway plays important role in human bone marrow-derived mesenchymal stem cell (hBMSC) differentiation, including osteogenesis. However, the function of deubiquitinating enzymes in osteogenic differentiation of hBMSCs remains poorly understood. In this study, we aimed to investigate the role of ubiquitin-specific protease 53 (USP53) in the osteogenic differentiation of hBMSCs. Based on re-analysis of the Gene Expression Omnibus database, USP53 was selected as a positive regulator of osteogenic differentiation in hBMSCs. Overexpression of USP53 by lentivirus enhanced osteogenesis in hBMSCs, whereas knockdown of USP53 by lentivirus inhibited osteogenesis in hBMSCs. In addition, USP53 overexpression increased the level of active β-catenin and enhanced the osteogenic differentiation of hBMSCs. This effect was reversed by the Wnt/β-catenin inhibitor DKK1. Mass spectrometry showed that USP53 interacted with F-box only protein 31 (FBXO31) to promote proteasomal degradation of β-catenin. Inhibition of the osteogenic differentiation of hBMSCs by FBXO31 was partially rescued by USP53 overexpression. Animal studies showed that hBMSCs with USP53 overexpression significantly promoted bone regeneration in mice with calvarial defects. These results suggested that USP53 may be a target for gene therapy for bone regeneration. The imbalance of these processes leads to low bone density and deterioration of the bone matrix, which increases the risk of skeletal disorders, such as osteoporosis4. Osteoporosis causes serious socioeconomic problems6. Several therapeutics have been developed to maintain bone homeostasis8, including bone-forming and antiresorptive compounds. However, these compounds have serious side effects, including peptic ulcers, atypical femur fractures, osteonecrosis of the jaws, and increased risk of bone tumor10. Therefore, novel therapeutic agents are urgently needed.Bone homeostasis is regulated by the balance of osteogenesis (bone formation) and osteoclastogenesis (bone resorption)11. Up to 80% of all intracellular proteins are degraded through the UPP. The reaction occurs through the hierarchal transfer of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), resulting in target protein degradation11. Deubiquitinating enzymes (DUBs) then remove ubiquitin chains from a target protein, controlling the function and stability of the protein13.The ubiquitin-proteasome pathway (UPP) is an ATP-dependent, reversible pathway that degrades target proteins14. Runt-related transcription factor 2 (RUNX2), a master transcription factor in osteogenesis, interacts with smad ubiquitination regulatory factor 1 (Smurf1) and Smurf2, E3 ligases, leading to the degradation of RUNX215. As an E3 ligase, WW domain-containing E3 ubiquitin protein ligase 1 negatively regulates osteoblast differentiation targeting RUNX216. The E3 ligase, Itch, also negatively regulates osteoblast function17. Accumulating evidence indicates that E3 ligases play important roles in the regulation of osteogenesis in hBMSCs19. However, few studies have addressed the functions of DUBs in the osteogenic differentiation of hBMSCs21.UPPs have important roles in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs)22. Mice lacking Usp53 exhibit progressive hearing loss22; additionally, USP53 is mutated in novel syndromic forms of cholestatic liver disease in humans23. However, the functions of USP53 are unclear.Ubiquitin-specific peptidase 53 (USP53), a member of the deubiquitinating enzyme family, contains a catalytically inactive ubiquitin-specific protease domain and is expressed in cochlear hair cellsAccordingly, in this study, we identified USP53 as a differentially expressed gene (DEG) in re-analysis of the Gene Expression Omnibus (GEO) database and investigated the roles of USP53 in osteogenic differentiation of hBMSCs. We hypothesized that USP53 may positively regulate the osteogenic differentiation of hBMSCs in vitro and that local delivery of AAV2-USP53 to calvarial defects would thus enhance bone formation. Our findings provide important insights into the potential applications of AAV2-USP53-activated hBMSCs in gene therapy for bone regeneration.24, commonly upregulated DUBs in hBMSC osteogenesis were analyzed (Supplementary Table Based on GEO datasets and previous studiesWe then investigated the roles of USP53 in the osteogenic differentiation of hBMSCs using short hairpin RNA (shRNA) targeting USP53. Approximately 90% knockdown efficiency was achieved , bone morphogenetic protein (BMP), parathyroid hormone-related protein (PTHrP), and insulin-like growth factor (IGF) signaling plays important roles in regulating RUNX2 expression. Thus, western blot analysis was performed to evaluate the expression of Wnt/β-catenin signal pathway mediators in hBMSCs with overexpression or knockdown of USP53. The levels of unphosphorylated β-catenin were higher in hBMSCs overexpressing USP53 than in the control Fig. . ConversTo investigate whether USP53 promoted the osteogenesis of hBMSCs through the Wnt/β-catenin signaling pathway, we examined the effects of a Wnt/β-catenin activator (Wnt3a) and inhibitor (DKK1) in hBMSCs with knockdown or overexpression of USP53. Inhibition of the osteogenic differentiation capacity in the shUSP53 group was rescued in response to Wnt3a, an activator of the Wnt pathway Fig. . ConversImmunoprecipitation (IP) and LC–MS/MS were performed to identify proteins that interacted with USP53 in the osteogenesis of hBMSCs  were created and then covered with fibrin glue mixed with AAV2-control-infected hBMSCs or AAV2-USP53-infected hBMSCs Fig. . Eight wNext, to investigate new bone formation, we performed M&T staining. The results showed that newly formed bone in the AAV2-USP53-infected hBMSC group was increased compared with that in the AAV2-control-infected hBMSC group Fig. . Evaluat30 was analyzed. The results showed that USP53 expression in BMSCs from patients with osteoporosis was downregulated compared with that in normal controls 14. Since USPs regulate cell cycle activity that is involved in post-translational modification, it is crucial in various diseases. Recent studies reported that USPs play an important role in cancer progression. In case of USP2a, it is overexpressed around 50% in human prostate cancers and affects the invasion ability of prostate cells31. USP22 is also upregulated in many cancer cells and activates the proliferation, migration, and invasion of gastric cancer, colorectal cancer, and breast cancer34. Another study reported that USP4 is overexpressed in hepatocellular carcinoma (HCC) tissues. And, USP4 increases progression of HCC through stabilization of cyclophilin A and deubiquitination35. Recently, USP34 has been reported as a positive regulator in osteogenic differentiation of hBMSCs36. Additionally, in the study about USP53, it was overexpressed in cervical cancer tissues and the inhibition of USP53 reduced cancer cell arrest37. But, none of the studies has reported about deubiquitinase enzyme which is related to osteoporosis. Here, we found that USP53 positively regulated the osteogenesis of hBMSCs in vitro and in vivo. Additionally, we confirmed that USP53 overexpression promoted osteogenesis in hBMSCs by activating the Wnt/β-catenin signaling pathway in vitro and that USP53 bound with FBXO31 during the osteogenic differentiation of hBMSCs. Mechanistically, FBXO31 negatively regulated the osteogenic differentiation of hBMSCs by acting as an E3 ubiquitin ligase and degrading β-catenin. Finally, USP53 expression was decreased in patients with osteoporosis and in a mouse model of estrogen deficiency-induced osteoporosis. Our findings provided further insights into the roles of DUBs in the pathogenesis of skeletal diseases and will be valuable for the development of novel therapeutic strategies for bone regeneration in tissue engineering.Understanding the molecular mechanisms that regulate bone homeostasis is essential for the development of improved therapeutics to treat human skeletal disorders. UPPs are required for the osteogenic differentiation of hBMSCs38. Because the F-box motif is essential for the interactions within the SCF complex, this motif mediates the degradation of target proteins39. FBXO31 is a member of the FBP family and interacts with the SCF complex to form a functional SCF-FBXO31 E3 ubiquitin ligase, which regulates the ubiquitination of target proteins, such as mouse double minute 2, cyclin D1, forkhead box 1, chromatin licensing and DNA replication factor 1, Slug, mitogen-activated protein kinase kinase 6, and Par6c40. FBXO31 has been identified as a putative tumor-suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. Its inactivation, due to heterozygosity loss, is associated with several cancers41. Additionally, FBXO31 has been reported to interact with the Skp1 protein26, and Cullin-1 and Roc-1 interact with myc-FBXO31/HA-Skp126. Therefore, we hypothesized that FBXO31 may regulate the osteogenic differentiation of hBMSCs by modulating osteogenesis-related genes. Because the overexpression of USP53 by lentivirus results in enhanced osteogenic differentiation of hBMSCs through the Wnt/β-catenin signal pathway, β-catenin may be a target of the SCF-FBXO31 E3 ubiquitin ligase43. Here, we confirmed that SCF-FBXO31 bound with β-catenin and degraded β-catenin in hBMSCs. Moreover, FBXO31 mutant did not promoted the proteasomal degradation of β-catenin. Thus, FBXO31-SCF complex interaction was removed in FBXO31 mutant. Moreover, siFBXO31 enhanced the expression of active β-catenin and promoted the osteogenesis of hBMSCs. Conversely, overexpression of FBXO31 decreased active β-catenin and RUNX2 levels, thereby suppressing hBMSC osteogenesis. These results suggested that FBXO31 acted as an E3 ubiquitin ligase to degrade β-catenin, which then negatively regulated hBMSC osteogenesis. Accordingly, the FBXO31/β-catenin/USP53 axis may positively regulate the osteogenic differentiation of hBMSCs  are core components of E3 ubiquitin ligases and determine substrate specificity for degradation by protein targetsSCs Fig. .46. The most commonly used AAV serotype in bone biology is AAV2, which is a potential carrier for target gene delivery in the bone tissue50. Recently, AAV9 was classified as an attractive vector for therapeutic gene delivery in bone tissue52. In this study, we investigated the effects of USP53 overexpressed in hBMSCs by delivering AAV2 in a mouse calvarial defect model. These mice showed better regeneration, as characterized by increased BV/TV, bone surface density, and mineral apposition rates in comparison with control mice. However, marked bone regeneration was not observed in AAV2-USP53-infected hBMSCs. These results may be related to the complete removal of the periosteum, which has bone regeneration capacity.To date, AAV is considered the most promising vector for gene therapy. AAV is a nonenveloped parvovirus with a single-stranded DNA genome 4.7 kb in length. This genome encodes four nonstrucutural replication proteins  and three structural capsid proteins 30. Notably, USP53 expression in patients with osteoporosis was downregulated compared with that in normal controls. Similar results were observed in OVX mice. Thus, these findings indicated that USP53 may play important roles in the pathogenesis of bone disorders such as osteoporosis.We also analyzed the USP53 expression in hBMSCs of elderly patients (79–94 years old) with osteoporosis using a GEO dataset (GSE35959)Prx1-Cre;Usp53f/f, Sp7-Cre;Usp53f/f, or Ocn-Cre;Usp53f/f mice.In summary, USP53 acted as a positive regulator of hBMSC osteogenic differentiation through Wnt/β-catenin signaling. The FBP, FBXO31, a component of the SCF complex, was shown to modulate the osteogenic differentiation of hBMSCs by acting as an E3 ubiquitin ligase to mediate the degradation of β-catenin. Intriguingly, USP53 was also shown to bind with the SCF ubiquitin ligase complex to reduce the polyubiquitination of β-catenin with FBXO31 dependently. Notably, transplantation of AAV2-USP53-infected hBMSCs improved bone regeneration in a mouse calvarial defect model. Taken together, our findings provide insights into the development of novel therapeutic strategies for the treatment of bone diseases. However, our findings are limited because we did not examine whether USP53 mutation/deletion was present in patients with osteoporosis. Further investigation is needed to determine the roles of USP53 in osteoblast lineages using 53. Wnt3a  was used at a concentration of 10 ng/mL in hBMSCs55.This study was approved by the Institutional Review Board of Yonsei University College of Medicine (IRB no. 4-2017-0232). Human bone marrow aspirates were obtained from the point 3 cm posterior to the anterior superior iliac spine of 10 healthy adult donors with a mean age of 35 years (range: 30–40 years). hBMSCs were maintained in low-glucose Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and 1% antibiotic-antimycotic solution (Gibco). Human embryonic kidney 293 (HEK293) and 293FT cells were obtained from American Type Culture Collection . These cells were maintained in high-glucose DMEM  containing 10% FBS and 1% antibiotic-antimycotic solution (Gibco). DKK1 (R&D Systems) was used at a concentration of 0.1 mg/mL in hBMSCshttps://www.ncbi.nlm.nih.gov/geo/) based on GSE1804324. A web-based tool, GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the DEGs by comparing up-regulated DUBs during hBMSC osteogenesis at different time points . The cut-off value was set as P < 0.05, log (fold change) > 2.0. The mRNA expression profiles of hBMSCs from five patients with osteoporosis (79–94 years old) were found in GEO datasets based on GSE3595930 that contained a total of 9 samples . The cut-off value was set as P < 0.05, log (fold change) < 0.5.The mRNA expression profiles of hBMSCs in osteogenic differentiation medium were found in GEO datasets 53. The osteogenic medium was replenished every 3 days. Alizarin red S and alkaline phosphatase (ALP) staining procedures were performed as previously described44.hBMSCs were seeded onto 12-well culture plates at a density of 8 × 10Total RNA was isolated from cells using an RNeasy kit . RNA (2 μg) was then reverse transcribed using an Omniscript kit . qRT-PCR was performed using a StepOnePlus Real-Time PCR System  and 2× qPCRBIO SyGreen Mix  according to the manufacturers’ guidelines. Primer sequences are listed in Supplementary Table Total proteins were prepared from cells using passive lysis buffer , and 30 μg of each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After blocking with tris-buffered saline with Tween 20 containing 5% skim milk, blots were incubated overnight at 4 °C with primary antibodies. All membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. Finally, the western blots were imaged using LAS 4000 (Fujifilm). Primary antibodies are listed in Supplementary Table FLAG-HA-USP53 was gifted by Wade Harper (Addgene plasmid #22606). FLAG-β-catenin was a gift from Eric Fearon (Addgene plasmid #16828). pCMV-Myc (Clontech plasmid #631604) was purchased from Clontech . myc-F-box only protein 31 (FBXO31) and myc-FBXO31ΔF were kind gifts from Prof. David F. Callen . The FBOX31 clone (untagged) was provided by the Korea Human Gene Bank, Medical Genomics Research Center . Green fluorescent protein (GFP)-RUNX2, myc-Ub, HA-Ub, and HA-Ub(K48) were kindly provided by Prof. Jae-Hyuck Shim . Lentiviral control vector  and lentiviral vector-USP53  were purchased from Applied Biological Materials . Scramble negative control small interfering RNA , USP53 siRNA (siRNA no. 54532), and FBXO31 siRNA (siRNA no. 79791) were purchased from Bioneer .5 cells/well and transfected with purified lentiviruses for 24 h. Puromycin  was used for selection, and stable lentivirus-infected hBMSCs were used in experiments. Information on the lentiviral vectors is provided in Supplementary Table 293FT cells were transfected with USP53-overexpressing lentivirus or USP53 shRNA along with packaging plasmid (delta 8.9) and envelop plasmid (VSV-G) using Lipofectamine LTX & Plus Reagent  according to the manufacturer’s instructions. DMEM-LG supplemented with 10% FBS was changed 6 h after transfection. The supernatants containing virus were collected 24 and 48 h after initial transfection, filtered with a 0.45-μm syringe filter (Millipore), separated into aliquots, and stored at -80 °C until use. hBMSCs were seeded onto 6-well plates at a density of 1 × 109 genome copies [GC]/mL, AAV2-control, AAVP022) and AAV-USP53 with GFP  were purchased from Applied Biological Materials. For AAV infection, hBMSCs were seeded onto 12-well plates at a density of 8 × 104 cells/well. After 24 h, hBMSCs were incubated with 40 μL AAV2-control and AAV2-USP53 using X-tremeGENETM9 DNA transfection reagent (Sigma). AAVs infected with hBMSCs were grown to 80% confluence and then replated at a density of 1 × 105 cells in a 10-cm2 dish.AAV blank control with GFP  for 6 h, and whole cells were lysed using nondenaturing lysis buffer . The lysates were incubated with anti-myc or HA antibodies overnight at 4 °C. Then, lysates were conjugated with 40 μL pre-washed protein A/G agarose beads  for 4 h at 4 °C with gentle rotation. Immunoprecipitates were eluted with 2× lane marker-reducing sample buffer (Thermo Fisher Scientific). Eluates were separated using SDS-PAGE.To investigate USP53-binding proteins in hBMSCs, the cells were transfected with lentiviral control vector or lentiviral vector-USP53 and cultured in osteogenic induction medium for 4 days. The cells were then treated with MG132 (10 μM) for 6 h, and hBMSCs were harvested using passive lysis buffer (Promega). Total protein (approximately 500 μg) was incubated with an antibody against GFP  and IgG control  at 4 °C overnight. SDS-PAGE was performed, following which the gels were stained with an InstantBlue staining kit . Differential bands were collected for LC-MS/MS, which was performed using a LTQ orbitrap mass spectrometer .5 cells/well, and cells were transfected with 1 μg pCMV-myc, myc-FBXO31, myc-FBXO31ΔF plus TOP-flash (wild-type T cell factor binding site), or FOP-flash (mutant T cell factor binding site) using Lipofectamine LTX & Plus Reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h, the cells were maintained for 5 days in osteogenic induction medium. The cells were analyzed using a simple dual-luciferase assay (Promega).hBMSCs were seeded onto 6-well plates at a density of 1 × 105 cells/well onto 6-well plates and transfected with Flag-β-catenin and control or Flag-USP53. Cells were cultured in the presence of Wnt3a. After 48 h, 100 μg/mL cycloheximide  was added. Cells were harvested at the indicated time points.hBMSCs were seeded at a density of 1 × 105) with AAV2-control group, and (3) hBMSCs (5 × 105) with AAV2-USP53. Fibrin gel  was mixed with hBMSCs and implanted into defects. Finally, the defect site was closed with vicryl plus (Ethicon 3-0) and nylon . Metacam (1 mg/kg) was used as an analgesic. Mice were sacrificed 8 weeks after surgery, and the skulls were harvested.The Committee on the Ethics of Animal Experiments of Yonsei University College of Medicine approved all animal experiments and protocols (permit no. IACUC-2019-0168). Fifteen male Institute of Cancer Research (ICR) mice (8 weeks old) were anesthetized with zoletil  and rompun  by intraperitoneal injection. After shaving the skull, an alcohol swab was used before hair trimming. The skin was retracted. Critical-sized calvarial bone defects (4 mm inside diameter/5 mm outside diameter) were created using a trephine burr (Shudent). The animals were divided into three groups as follows: (1) fibrin gel only, (2) hBMSCs  for 3 days, following which μCT was performed using a Skyscan 1173 High Energy Micro-CT scanner  with a 40–130 kV, 8 W X-ray source. For histological analysis, skulls or femurs were decalcified with 5% formic acid (Sigma) or 0.5 M EDTA for 1 week. Fixed specimens were then embedded in paraffin blocks and cut into 4-μm-thick sections. Sections were deparaffinized in xylene and serially rehydrated in ethanol. Sections were sequentially stained with Masson trichrome (M&T) staining and immunohistochemistry (IHC). Primary antibodies are listed in Supplementary Table The Committee on the Ethics of Animal Experiments of Yonsei University College of Medicine approved all animal experiments and protocols (permit no. IACUC-2020-0045). Female sham-operated and ovariectomized (OVX) mice  were purchased from Japan SLC . After 8 weeks, the sham and OVX mice were sacrificed, and serum was collected by cardiac puncture. Mouse RatLaps  was used according to the manufacturer’s recommendations to measure serum level of CTX-1.P values less than 0.05 were considered statistically significant. All data are presented as means ± standard deviations (SDs). No animals or samples were excluded from the analysis. We first performed Shapiro-Wilk normality tests for normal distributions of the groups. If normality tests were passed, two-tailed, unpaired Student’s t-tests were used for the comparisons between two groups. For comparisons of three or four groups, we used one-way analysis of variance (ANOVA) if normality tests were passed, followed by Tukey’s multiple comparison tests for all pairs of groups. GraphPad PRISM software  was used for statistical analysis.For each experiment, samples were analyzed at least in triplicate. Results with Supplementary Table S1–S8Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5


Unequal financial outcomes often originate from unequal chances. Yet, compared to outcomes, little is known about how individuals perceive unequal distributions of chances. We investigate empirically the role of different sources of unequal chances in shaping inequality perceptions. Importantly, we do so from an ex ante perspective—i.e., before the chances are realized—which has rarely been explored. In an online survey, we asked uninvolved respondents to evaluate ex ante the fairness of unequal allocations of chances. We varied the source of inequality of chances, using a comprehensive range of factors which resemble several real world situations. Respondents also evaluated how much control individuals hold over the distribution of chances. Results show that different sources generate different ex ante perception of fairness. That is, unequal chances based on socioeconomic and biological factors, such as gender, family income and ethnicity, are evaluated to be unfair relative to the same chances based on effort, knowledge, and benevolence. Results also show that, for most individuals, there is a positive correlation between perceived control of a factor and fairness of unequal chances based on that factor. Luck appears to be an exception to this correlation, ranking as high in fairness as effort, knowledge, and benevolence, but similarly low in individual control as ethnicity, family income, and gender. They attempt to “level the playing field” as the saying goes. Such policies can be observed across the globe. Brazil invests additional funds in the education of low-income children, through its Bolsa Família cash-transfer program. Norway as well as other European countries require a gender quota on corporate boards. Universities in India, South Africa, and the United States increase the admission chances of disadvantaged groups through Affirmative Action. Nevertheless, our understanding of how individuals perceive the unequal chances that these policies address is minimal. In fact, there is a noticeable empirical gap in the existing literature on distributive fairness ideals: we have limited knowledge about which types of unequal chances are considered to be fair from the ex ante standpoint—i.e., before chances are realized and incomes are known. We employ ourselves to bridge this gap.Unequal chances abound in the pursuit of financial rewards; erecting the landscape of the financial outcomes likely to be achieved by different groups of individuals. A striking recent study6, we examine the fairness perceptions of an impartial or uninvolved spectator. While non-monetarily incentivized, the perspective of the spectator is of value because it is expected to be devoid of self-interest3. That is, the spectator’s judgment should be impartial, providing us with a normative account of what is considered fair in the ex ante domain.Using a survey, we investigate the fairness perceptions of different factors used to distribute unequal chances, from the ex ante perspective. As many studies before7 also makes conclusions about “sheer luck” and “performance” by studying height and a mix of effort and knowledge, but those are proxies of unknown quality. Moreover, we add identification numbers (ID) to our factors because experimenters sometimes allocate students to treatments based on IDs in laboratory experiments to mimic random assignment, so that it is practical to understand the fairness of such a chance assignment. Second, from a theoretical viewpoint, liberal egalitarianism, which we discuss below, as well as a few other concepts could affect fairness perceptions of some of our factors. Those include repugnance8, psychological cost9, and indirect reciprocity10, which we discuss in the Methods section.We use a large set of factors, which include factors often serving as bases for unequal chances in society such as effort, knowledge, luck, gender, ethnicity, and family income. Those factors are more comprehensive than those that the literature on fairness has employed, enabling a more fine-grained exploration of differences among a range of situations that can generate unequal chances. The motivation for employing this wider range is twofold. First, our primary motivation is empirical, as whether differences exist in fairness perceptions is unknown for almost all chance factors studied here, and several of those unstudied factors such as ethnicity and gender are valuable for public policy. The only study investigating chance factors from an ex ante viewpoint15. Furthermore, whether this relationship extends empirically to chance inequality and ex-ante fairness judgements has not been explored. Lastly, we touch upon Affirmative Action, which raises the odds for disadvantaged groups over advantaged groups. We do this by testing whether it is fairer to provide higher chances to groups that are generally disadvantaged than to groups that are generally advantaged. Our hope is that understanding how individuals perceive unequal chances originating from different factors can provide insights to policymakers in terms of which unequal chances are perceived as justifiable and which may call for rectification.Moreover, we examine the perception of individual control over these factors and the relationship between control and ex ante fairness. This is important because economic and philosophy research on the liberal egalitarian fairness ideal has emphasized the positive relationship between factors perceived as within one’s control and fairness perception of different types of outcome inequality and the lasting theoretical difficulties in pinpointing exactly which factors are under one’s controlex post fairness concerns the distribution of incomes between individuals once these incomes are known16, ex ante fairness applies to the distribution of chances that are to generate incomes17. The literature in psychology and management21 also studies the factor generating outcomes separately from the outcomes themselves and uncovers crucial elements that influence individuals’ appraisal of factors, such as respect, an opportunity to be involved or voice their concerns, and the absence of biases.The economic literature investigating inequality and redistribution decisions draws a line between ex post and ex ante fairness. While 26. Studies have also examined ex-post fairness from the non-monetarily incentivized spectator viewpoint, showing that many individuals also prefer to equalize earnings of equally-deserving participants27 and judge such unequal situations to be unfair when asked in a hypothetical manner5.The empirical literature on redistribution focuses mostly on ex post fairness, once incomes are already known. Studies show that a substantial proportion of individuals display concern for equality of incomes, exhibiting a willingness to decrease their own income to improve the conditions of others through redistribution29 presented participants with unequal chances to win a lottery, and the opportunity to redistribute chances before the realization of that lottery (note that other types of experiments exist in the same vein30). In those experiments, many individuals indeed show concern for equality when redistributing chances, even if it reduces their own chances of winning. Similarly, experiments show that individuals do consider the distribution of initial chances in their redistribution decisions even after the realization of these chances, both when they are themselves involved33 or when spectators33. Finally, theoretical work on social preferences for obtaining a fair chance has been the focus of a number of papers in the past twenty years37.There is also empirical evidence that individuals care about ex ante inequality, although the topic is less studied. For instance, recent economic experiments38. Typically, a sizeable proportion of individuals are more accepting of inequality caused by effort and knowledge than luck, both when involved38 and when spectators27. The prominent explanation for this type of behavior is the view that individuals should be held accountable for factors under their control, such as effort and knowledge, but not for others which they cannot influence, such as luck. This is the position taken by the liberal egalitarian fairness ideal12 and the accountability principle6. Several earlier equality concepts in philosophy also hold individuals responsible for their choices, the difference often being to which extent one is responsible for certain factors and not for others, a notoriously difficult matter to determine theoretically15.Studies investigate the fairness perception and the distribution decisions of incomes that have been generated through different factors, but they do so almost exclusively from an ex post perspective. For example, they examine whether the patterns of fairness perceptions and income distribution decisions differ if income inequality is caused by different factors, such as effort, knowledge, and luck39.In contrast, how different factors behind the distribution of chances affect the ex ante fairness perception of these chances has scarcely been explored by empirical studies. We believe that this missing step is socially valuable because many policies—such as the Affirmative Action described in the first paragraph—intend not to correct incomes once unequal chances have been realized, but to correct different types of unequal chances before they are realized. Furthermore, whether the positive relationship between perceived control and fairness of inequality documented ex post is typical from an ex ante perspective has not been researched much. Studying ex ante perceptions is essential because our knowledge regarding ex post fairness views does not necessarily inform us about ex ante fairness views. For instance, theoretically, a society’s fairness evaluation of a factor based on luck may differ ex ante and ex post: providing equal chances of obtaining either a high or low income to everyone might be judged as fair at the outset, only to be considered unfair once unequal incomes have been randomly allocated7. Using those measures of luck and performance, the study finds that, ex ante, individuals are more prone to support future redistribution when luck influences chances than when performance does, which provides some evidence that liberal egalitarianism is present ex ante. We add to this research by investigating fairness perceptions of a wide range of factors underlying unequal distribution of chances, which are mostly chosen to resemble real-world situations of inequality. Interestingly, we measure fairness perceptions of height and luck, and find large differences in fairness ratings between the two, whereas luck rates similarly in fairness as effort and knowledge. Moreover, we measure individuals’ perceptions of control over the different factors. We note that the previous study also included questions regarding fairness perceptions of factors, but did not find significant differences between the reported fairness of the height and the effort and knowledge factors. However, as the author reports, its use of a between-subject design might generate too much noise to distinguish fairness differences between the factors. Our within-subject design is likely to alleviate this issue.The only study that has empirically investigated the role of different factors in ex ante fairness views aims to compare how distributions of chances based on “sheer luck” vs. “performance” impact individuals’ ex ante choice of future redistribution by employing chances based on height vs. a combination of effort and knowledge in a quiz7. We instead use survey questions about fairness perceptions in a hypothetical scenario, as others have done40. Note that economic experiments in other domains also sometimes employ non-incentivized measures of fairness perceptions, e.g., in labor experiments42.Finally, we note the following point about our measures of fairness. In economic studies of distributive moral ideals, it is theoretically assumed that individuals hold fairness preferences or ideals that translate into income redistribution decisions, and the common empirical way of studying fairness is then through revealed preferences: we measure redistribution decisions and then infer preferences from those decisions43 of the Behavioral and Experimental Economics Laboratory (BEELab) at the School of Business and Economics of Maastricht University (The Netherlands) to take part in an online Qualtrics survey. We obtained informed consent from participants to take part in the study. They completed the survey in April and May 2017 and received 5 EUR for their participation. In terms of demographic characteristics, 41% are men and the mean age is 21.5 years. The study was conducted following the peer-approved factors established by Maastricht University’s Behavioral and Experimental Economics Laboratory (BEELab). Our study was approved by the Maastricht University’s Behavioral and Experimental Economics Laboratory at a public ethics review and project proposal meeting that is mandatory for all scholars who wish to use the BEElab facilities. All methods were performed in accordance with the relevant guidelines and regulations of the BEELab and Maastricht University for studies with human participants.We recruited 82 undergraduate students from the online recruitment system for economic experiments (ORSEE)Participants considered a hypothetical scenario in which unequal chances to win a prize of 10 EUR are allocated between two other individuals. Participants could not win the prize themselves, and, for simplicity, we framed chances as lottery tickets. Chances are always allocated in the following manner: one individual has a Consider a situation in which lottery tickets are distributed between two persons. Generally we ask you to assume that these two persons have a similar social, economic and educational background. In some cases this will not be so and you will be explicitly informed about it. Each ticket gives achance to win the lottery. There can be only one winner in the lottery, who receives 10 EUR as a prize. The ticket allocation is as follows: one person gets 9 tickets and thus has achance of winning the lottery prize, whereas the other person gets 1 ticket and thus has achance of winning the lottery prize. The allocation of tickets is based on various factors.31.We then presented procedures varying 11 different factors in random order. Although all procedures presented led to the same consequences . The order of blocks was randomized. Except for a few pre-selected factors, we are solely interested in the “Regular” Block because the factors in this block described cases that generally occur more often in real life, such as advantaging men, light-skinned individuals, or individuals from a high-income family. All factors are presented in the “Reverse” block simply to avoid disclosing the factors we wanted to focus on in this block. Those pre-selected factors of interest in the “Reverse” block are chosen to evaluate whether asymmetries in fairness might exist for them, which we explain below.not help a person in need. In this specific case, higher chances are allocated to the benevolent person in the “Regular” Block, while higher chances are allocated to the non-benevolent person in the “Reverse” Block. For knowledge, we used a general knowledge quiz, where the person with a higher number of correct answers would receive higher chances in the “Regular” Block and lower chances in the “Reverse” Block. For effort we employed a slider task44, a common effort task in experimental economics, which is explained in the example below. Again, the person exerting more effort would receive higher chances in the “Regular” Block and lower chances in the “Reverse” Block.Table We make a few observations about this effort task. First, we think that the effort scenario that we present makes it clear that the two individuals in the scenario are aware that their effort determines their chances of winning the prize, although it does not explicitly specify the beliefs of individuals in the scenario. Control ratings by respondents in the Results section are in line with this, suggesting participants understand that chances depend on individuals’ effort. Second, we cannot rule out that performance in the slider task is fully unrelated to ability, but it is a gruesome manual task that relies on the willingness to put in effort. Third, since it is one of the most common tasks used in economic experiments, participants in our laboratory pool are likely to be familiar with this task by having experienced it themselves. Even for those who have not, the task and its mechanical nature are relatively easy to comprehend in the description that we provide.Then, for luck we adopted a 6-side die roll where one person would roll a number equal or smaller than 3 and receive higher chances, whereas the other would receive lower chances. For the ID factor, we used greater and smaller student ID numbers . For height we contrasted taller and shorter individuals, while for weight we contrasted heavy and light persons and for gender we employed women versus men. For nationality, we made use of Germans versus non-Germans . For ethnicity, we contrasted light-skinned to dark-skinned individuals. Finally, for family income we considered individuals coming from higher or lower income families.Very Unfair to Very Fair and from No Control to Full Control. At the end of the study, participants provided demographic information, such as age, gender and parental income as well as other characteristics of themselves such as height and weight. The full instructions are provided in the Supplementary materials. As in many studies on moral ideals before us27, our participants’ answers do not affect their own financial welfare. We rely on participants’ intrinsic motivation to pay attention. Still, we made sure that our study provides adequate conditions for them to pay attention: all instructions are carefully explained, the study is designed to be short  score low on fairness.P-values are corrected for multiple comparisons using the Holm-Bonferroni method45 and are indicated by the number of stars. For completeness, fairness ratings of the factors in the Reverse Block can be found in the Supplementary materials.Table This result indicates that not all chance inequalities are considered equally fair from the ex ante viewpoint, which is in line with what other studies have found for the ex post perspective. Indeed, despite the fact that all factors led to an unequal distribution of chances between individuals, some factors were considered fairer than others. Unlike for the ex post perspective, however, factors giving higher chances to those who exert effort or are knowledgeable are not significantly fairer than luck, which simply randomly allocates chances. Factors based on socioeconomic and biological criteria are the ones which really differ from luck, by being substantially less fair.Although we do not aim to formalize groups in terms of fairness, we can assay their presence by conducting a Principal Component Analysis (PCA). It detects three main components . This is line with the advantage of using spectator participants: their judgement is as impartial as possible as they have no stake in the situation.We also examine whether a participant’s characteristic could influence their fairness ratings when facing a situation that allocates chances based on that specific characteristic, e.g., whether a man rates unequal chances advantaging a man any differently than a woman does. To do this, we conduct a non-parametric Wilcoxon-Mann-Whitney test (or Wilcoxon rank-sum test) to test whether this is the case for each of the following 7 self-reported characteristics: gender, height , weight , skin color, general knowledge , laziness , and parental income. We find no statistically significant effect for each of the 7 tests on fairness ratings , as hypothesized, the ex ante fairness rating differs depending on the individual who is favored with higher chances. The exception is gender, for which, unlike what we hypothesized, we did not find a significant asymmetry. Table We make two observations. First, finding no asymmetries in the case of gender might be due to the specific setting that we study—a Dutch business school with a large share of women among its student body, which for students is likely not highly unequal in terms of gender. Second, although we do find asymmetries in fairness, factors that favor disadvantaged groups are still generally considered unfair compared to factors that advantage those who are benevolent, exert effort, or are knowledgeable. This result reflects to some extent the mixed support affirmative action can receive. Even though individuals recognize that giving more chances to the disadvantaged is fairer, such factors are still considered relatively unfair. Alternatively, the low relative fairness of the affirmative action factors that we use might be due to the somewhat egalitarian environment of the students. Another reason for this result is that these factors create a large amount of inequality by distributing very unequal chances instead of slightly advantaging the generally-disadvantaged group.Result 3—Control and fairness:A majority of individuals consider factors that one has control over to be fairer. Interestingly, luck stands out as the exception: it ranks high in fairness but low in control.Table pTo provide a picture of the correlation at the group level, we can employ an individual-level random effects parametric Tobit regression with lower-bound censoring. Fairness is the dependent variable and control is the explanatory variable. The resulting control coefficient of 0.985 .We also examine whether a participant’s characteristic could influence his or her control ratings when facing a situation that allocates chances based on that characteristic. We conduct a non-parametric Wilcoxon-Mann-Whitney test to test whether this is the case for each of the following 7 self-reported characteristics: gender, height , weight , skin color, general knowledge , laziness , and parental income. We find no statistically significant effect for each of the 7 tests on control ratings . If this was the case, one could expect individuals to be more sensitive to scenarios in our study in which their own attributes or characteristics served to create chance inequality. However, results show that individuals’ characteristics  are not significantly related to their fairness ratings or to their control ratings in situations where those characteristics determine chance inequality. In addition, despite keeping information on factors constant in the Regular and Reverse blocks , we observe variability in the fairness ratings of factors between the two blocks. Finally, we note that the differences and ordering in control ratings between factors are not exactly the same as for fairness ratings . Still, it would be interesting to further explore whether and how sensitivity to information can impact fairness judgments in future research.47, while 26% stated in 2019 that race should be a factor in university admission48. Even in our setting of a simple lottery among university students, it is indeed fairer to favor certain disadvantaged groups, such as individuals with dark skin. We observe, however, that increasing the chances of certain groups is still considered unfair on average in this context relative to advantaging those who exert more effort or are more knowledgeable. We do not find that it is fairer to give higher chances to one gender over the other, although we note that this might be the result of using students in a relatively gender-balanced environment. Overall, asymmetries in perceived fairness provide a potential source of expansion for current research.The asymmetry observed in the fairness of certain factors is consistent with at least a substantial number of individuals supporting Affirmative Action for disadvantaged groups, such as ethnic minorities. For instance, 63% of Americans reported in 2014 that they thought that affirmative action programs increasing black and minority university students are a “good thing”4. Interestingly, unlike ex post, liberal egalitarianism fails to hold ex ante in the case of luck, which is considered both fair and outside of one’s control. On the one hand, the factor randomly allocating higher chances is considered as fair as the factors advantaging those who exert effort or are knowledgeable. On the other hand, whereas factors based on effort or knowledge are judged to be under individuals’ control, luck is obviously considered to be under no control, as are factors based on biological or socioeconomic factors.Furthermore, factors allocating chances in a fairer manner rate higher in individual control. This is broadly consistent with the liberal-egalitarian fairness ideal that a large share of individuals have been shown to hold in terms of ex post fairness38 highlight that it is unfair ex post? Theoretical work suggests that individuals can be time-inconsistent when making ex ante and ex post fairness judgements49. For instance, they can first support policies that aim at ex ante chance equality, but lead to unequal incomes, and, once these unequal incomes are realized, they then support policies that aim at ex post income equality. This problem can arise when, at the moment of making an ex ante choice, individuals do not take into account possible ex post inequality. A recent working paper also reports behavior consistent with this phenomenon: individuals often make an ex ante fair choice before outcomes are realized, only to then switch to an ex post fair choice once outcomes are known50. A qualitative comparison between our fairness results in the ex ante domain and fairness results of other studies in the ex post domain38 also suggests that individuals may make different ex ante and ex post fairness judgements. However, our study cannot provide a direct test of this time-inconsistency explanation because it does not measure individuals’ perception of both ex ante and ex post fairness, and differences in designs and contexts with previous studies could also have contributed to the observed differences. It would be interesting to investigate not only if fairness ratings differ ex ante and ex post, but to explore if the extent of these differences depend on the source of inequality. An extrapolation of our results would suggest that while differences may be observed for luck, other factors such as effort and knowledge might be considered equally fair from an ex ante and ex point view.How is it possible for the random allocation to be fair from an ex ante perspective when previous workIn addition, we note that it can be puzzling that individuals evaluate chances allocated based on luck as fairer than chances allocated based on other factors that could be easily rationalized as coming from luck, such as height. We note that luck being perceived as fair ex ante and unfair ex post may provide an explanation for this phenomenon. That is, on the one hand, an allocation of chances based on height may be seen through ex post lenses, as the luck that could determine height has already been realized. On the other hand, an allocation of chances based on luck may be seen as still not realized—i.e., as part of a compound lottery—and therefore be perceived through ex ante lenses. Another possibility is that the exception of luck might have arisen through social conventions. That is, although die rolls cannot be controlled, they could be perceived as fair because individuals often voluntarily opt to use them to settle indivisible claims, whereas individuals rarely settle claims based on height.7. That paper7 aimed to compare how distributions of chances based on “sheer luck” vs. “performance” impact individuals’ ex ante choice of future redistribution by using chances based on height vs. a combination of effort and knowledge in a quiz. While we do not study redistribution decisions, our results suggest that there are large differences in fairness perceptions between height and luck, whereas luck rates similarly in fairness as effort and knowledge. Future research will need to address whether this difference between height and luck also translates into a large difference in ex ante redistribution decisions.Importantly, our results on fairness contrast in some respects with the only previous study that has empirically investigated the role of different factors in ex ante fairness viewsOur findings carry two potential implications for policymakers. First, the causes of unequal distributions of chances need to be carefully considered if one wants to make chance-equalizing policies that correspond to the fairness perceptions of individuals. Second, considered with the results of other studies, our study suggests that individuals exhibit time-inconsistency regarding the fairness of different redistribution policies. That is, they may support policies that grant equal chances, but create income inequalities, only to later switch their support to policies that aim at equal incomes. A way to avoid this reversal is perhaps to conscientiously consider the ex post inequalities that equal chances may generate, whenever there are public debates on equal-chance versus equal-income policies.3. It would be interesting for future research to explore how ex ante judgments of involved agents deviate from those of spectators. Despite the fact that spectators, just like involved agents, tend to take ex ante inequality into consideration33 and are also sensitive to the different sources causing inequality in outcomes27, previous studies have also pinpointed differences in the behavior of stakeholders and third-party individuals in general. To start with, spectators tend to fully achieve their distributive fairness ideals more often (due to this achievement being monetarily cheaper) than stakeholders6. Furthermore, when individuals are themselves affected by an unfair allocation of outcomes, they tend to punish fairness violations more strongly52 and show more intense emotional reactions to unfairness53 than third-party individuals do. Neural mechanisms in response to unfairness have also been reported to differ depending on whether individuals are directly affected by a fairness violation or a third-party observer in the situation55. Although these differences are restricted to the ex post domain, it is plausible that some differences will emerge between stakeholders and spectators in the ex ante domain, e.g., stakeholders are likely to act in more selfish ways. However, it is unclear whether there will be differences between stakeholders and spectators in how they evaluate the relative fairness of different factors generating unequal chances.We also note that in our study we opted to investigate the judgments of uninvolved spectators, whose judgment should be impartial56 and it has been shown that ex post fairness views can differ substantially across cultures57. We can therefore also expect that ex ante fairness is affected by culture.An additional interesting question for future research concerns the extent to which differences between cultures arise regarding ex ante fairness evaluation of chance inequality. It is well known in economics that different cultures exhibit different social preferences12, our conjecture is that the positive correlation between fairness and control would not change substantially if we were to reverse the order of presentation between fairness and control. Nevertheless, we cannot fully rule out that asking participants first for their fairness or control ratings would not influence their assessments.We point out that a limitation of our study is that participants always rated the source of inequality first in terms of fairness and then in terms of individual control. We chose this ordering because we are primarily interested in ex ante fairness evaluations, and only interested in control ratings to the extent that they relate to fairness. Considering the strength of the relationship between fairness and individual control and the fact that it is at the core of liberal egalitarianism58, and that individuals are less supportive of income redistribution when it is unclear whether outcome inequality comes from effort or luck than when it clearly arises from luck59. It would be interesting to measure the contribution of these misconceptions to the opposition to and support for public policies that aim to establish ex ante equality, such as Affirmative Action.Finally, we realize that while in our study participants were clearly aware of the reason behind inequality in chances, this is often not the case in real life. Individuals’ misconceptions of the reasons why someone might have higher or lower chances might considerably affect their fairness judgments, and consequently their support for redistributive policies. Recent studies have found that ambiguity in the perception of others’ responsibility in their life outcomes leads to lower allocation of resources to themSupplementary Information.


Examine the effect of aerobic exercise (EX) combined with standard medical care (SC) (EX + SC) compared to SC alone on cardiometabolic health and quality of life in relation to surgical outcomes.n = 7, 1 male, 39.0 ± 5.3 years, body mass index 46.4 ± 3.0 kg/m2; low calorie diet) or EX + SC . Body mass, waist circumference, cardiorespiratory fitness (VO2peak), high sensitivity C-reactive protein (hs-CRP), cytokeratin 18 (CK18), weight related quality of life (QoL), and a 120 min mixed meal tolerance test (MMTT) was performed to assess arterial stiffness via augmentation index normalized to a heart rate of 75 beats per minute (AIx@75), whole-body insulin sensitivity, and glucose total area under the curve (tAUC) pre- and post-intervention (∼2 days prior to surgery). Length of hospital stay (admission to discharge) was recorded.Patients receiving bariatric surgery were match-paired to 30 days of pre-operative SC  compared to SC, but no change in body weight or waist circumference was observed in either group. EX + SC had a greater effect for increased VO2peak  and decreased hs-CRP  compared to SC. EX + SC reduced circulating CK18  and improved QoL (P = 0.02) compared to SC. Although EX + SC had no statistical effect on arterial stiffness compared to SC, we observed a modest effect size for AIx@75 tAUC . EX + SC had a significantly shorter length of hospital stay  than SC, and a shorter length of hospital stay was associated with decreased sugar intake . Decreased AIx@75 tAUC significantly correlated with improved whole-body insulin sensitivity  and glucose tAUC .EX + SC had a greater effect for decreased intake of total calories (EX with SC for 30 days prior to bariatric surgery may be important for cardiometabolic health, quality of life, and surgical outcomes in the bariatric patient. A pleth2peak . This su2peak  but not 2peak . To date2) were included for participation in the study if they were 18–70 years old, undergoing their first RYGB or SG procedure, not pregnant or lactating, or not taking medications known to alter body weight. Participants were excluded if they were physically active (>60 min/wk of exercise), diagnosed with insulin dependent diabetes, had a history of cardiovascular disease, or diagnosed with cancer in the past 5 years. Physicals and physician clearance for participation in the study was completed by one investigator (P.T.H.). All outcomes were assessed pre-intervention and post-intervention . After pre-intervention testing participants were match paired to 30 days of pre-operative EX + SC or SC based on body mass index (BMI), sex, race, and surgery type (RYGB or SG). One investigator (P.T.H.) completed all RYGB or SG surgical procedures and was blinded to the patient’s treatment group. Length of hospital stay was defined as the time of hospital admission to discharge, and surgical residents were responsible for hospital discharge and had no knowledge of the patient’s participation in the present study. Operating time was defined as time of incision to close. These data, along with demographic characteristics were previously published . Participants self-selected a speed and grade was increased 2.5% every 2 min until volitional exhaustion. The highest heart rate value achieved during the VO2peak test was recorded as heart rate peak (HRpeak) and used for the exercise prescription of EX + SC.Body weight was measured to the nearest 0.01 kg on a digital scale and height was measured with a stadiometer to assess BMI. Fat mass and fat free mass (FFM) were measured using air displacement plethysmography . Waist circumference was measured 2 cm above the umbilicus using a flexible tape measure. VO∗diastolic) + systolic)/3] was calculated. An intravenous catheter was then placed in an antecubital vein. Fasting blood was collected to measure glucose, insulin, triglycerides (TG), high-density lipoproteins (HDL), low-density lipoproteins (LDL), total cholesterol, high sensitivity CRP (hs-CRP), and cytokeratin 18 (CK18), a marker of hepatocyte apoptosis and liver health. A mixed meal tolerance test (MMTT) was then administered in which participants consumed 4 fl. oz. of an Ensure Plus  shake , and circulating glucose and insulin was also determined every 30 min up to 120 min after consumption of the mixed meal. The Matsuda Index was used to estimate whole-body insulin sensitivity  with Dinamap , which was averaged over three measurements for data analysis. Mean arterial pressure (MAP) = [ at 0, 60, and 120 min of the MMTT. Pulse wave analysis measures were recorded at each time point in a temperature-controlled room while patients rested quietly in the supine position. AIx@75 total area under the curve (tAUC) was determined using the trapezoid method , augmentation pressure (AP), and augmentation index corrected to a heart rate of 75 bpm using the manufacturer’s software (AIx@75) was measured by applanation tonometry using the SphygmoCord method .The Laval questionnaire was used to assess weight related QoL as it has been validated in participants with morbid obesity and is sensitive to treatment-induced changes . The queAccelerometers  were used to assess non-exercise physical activity for 1 week prior to the mixed meal tolerance test (MMTT) at pre- and post-intervention testing. The average wear time per day and the total number of days the device was worn at each time point is reported. Freedson VM3 (’11) is an energy expenditure algorithm that was used to determine the frequency of sedentary bouts per day as well as percent of wear time spent sedentary, in light physical activity, or in moderate to vigorous physical activity (MVPA) per day . IndividParticipants met with registered dieticians, attended an education session with a registered nurse, and cleared for bariatric surgery by a psychologist and the surgical team prior to study enrollment. Patients attended a pre-operative visit approximately 40 days prior to bariatric surgery. For 2 weeks prior to surgery, patients were instructed by registered dieticians to consume a meal replacement shake for breakfast and lunch, snacks of raw vegetables, a dinner composed of 4 oz. of lean protein and steamed vegetables, and sugar-free beverages.peak for 30 days. Exercise adherence was monitored by the research team using an exercise diary and an A300 Polar fitness and activity tracker . The Polar fitness monitor tracked the duration, heart rate, and calories expended per exercise session. The research team also conducted weekly check-ins by texting, emails, and/or phone calls with study participants to promote adherence.Participants in EX + SC also completed at home walking for 30 min/day, 5 days/week, 65–85% HRad libitum food intake. Participants were provided with reference guides that displayed serving sizes of beverages and food commonly consumed. They were also given detailed instructions for recording food and beverages consumed over the intervention. Intake was analyzed with the Food Processor Nutrition Analysis Software .Three-day food logs, including two weekdays and one weekend day, were used to assess Aprotinin was added to the EDTA vacutainer tube prior to blood collection for insulin. Blood was collected in EDTA and SST vacutainer tubes as well as lithium heparin microtainer bullet tubes and immediately placed on ice. Lithium heparin microtainer bullet tubes were then immediately centrifuged at 13,300 rpm for 60 s, and the YSI 2300 StatPlus Glucose Analyzer system  was used to determine plasma glucose. SST tubes rested for 30 min prior to processing. All EDTA and SST vacutainers were centrifuged for 10 min at 4°C and 3,000 rpm, and samples was aliquoted to cryotubes and stored at −80°C until later analysis. Samples were batch analyzed in duplicate to minimize inter-assay variability. The University of Virginia’s Health System Medical Laboratories determined HDL and total cholesterol as well as TG by colorimetric assays from serum collected in SST tubes. LDL cholesterol was calculated from the Friedewald equation . Plasma t-tests. A non-parametric Wilcoxon Signed Rank test was performed to determine differences from pre- to post-intervention in weight related QoL, and Mann–Whitney U Test evaluated differences between groups in the score change from pre- to post-intervention. All other outcomes were analyzed using a 2 × 2 repeated measure analysis of variance (ANOVA). Cohen’s d effect sizes (ES) were also calculated on the interaction of treatments for physiological outcomes, and relevance was interpreted as small (d = 0.2), medium (d = 0.5), or large (d = 0.8). Pearson’s correlation was used to assess associations. Body composition, VO2peak, whole-body insulin sensitivity, and metabolic flexibility was previously reported   and insureported , but then = 7) and EX + SC (n = 7) in age (SC 39.0 ± 5.3 vs. EX + SC 45.6 ± 4.8 years), sex , race/ethnicity , or type of surgical procedure  . Although body weight (SC −0.6 ± 1.6 vs. EX + SC −0.5 ± 0.7 kg) and waist circumference (SC −1.1 ± 0.6 vs. EX + SC −0.8 ± 1.4 cm) was unchanged with SC and EX + SC, FFM decreased with both interventions (P = 0.05) and SC (−1.5 ± 0.5kg) had a greater decrease in FFM than EX + SC (−0.6 ± 0.8kg) . There was a medium effect size  for an increase in fat mass with SC (0.9 ± 1.0kg) compared to a slight reduction with EX + SC (−0.3 ± 0.8kg). EX + SC had a large effect size for increased VO2peak relative to body weight (mL/kg/min)  compared to SC, albeit not statistically significant.There was no difference between SC (P = 0.003), saturated fat , trans fat , cholesterol , and fiber  over the study  compared to SC. EX + SC also had a greater effect for decreased intake of CHO , sugar , and unsaturated fat  compared to increased intake with SC  which equated to 248.8 ± 24.0 kcal/session. There was no difference in days of accelerometer wear between SC and EX + SC at baseline  nor was there a difference between groups after treatment . Both treatments had the same frequency of sedentary bouts per day at baseline  and after treatment . Non-exercise sedentary time appeared to increase with EX + SC from baseline (SC 30.1 ± 5.2 vs. EX + SC 38.5 ± 2.3%) to post-intervention . This observation remained for percent of total time spent sedentary (including exercise within the day) from baseline (SC 30.1 ± 5.2 vs. EX + SC 38.5 ± 2.3%) to post-intervention . SC and EX + SC had no changes in percent of non-exercise time spent in light physical activity or MVPA (data not shown). However, EX + SC (baseline 6.9 ± 0.8 vs. post-intervention 11.2 ± 1.1%) spent significantly more of their total time in MVPA compared to SC (baseline 4.4 ± 0.8 vs. post-intervention 3.9 ± 0.9%) during the study .EX + SC participants completed 18.4 ± 3.0 exercise sessions for 37.3 ± 2.6 min/session at moderate to vigorous intensity  and MAP  compared to EX + SC  and 120 min  AIx@75 as well as AIx@75 tAUC  compared to SC (P = 0.05), yet SC had a greater effect than EX + SC on fasted PP  as well as fasted , 60 min , and 120 min  AP  AP .P = 0.26; ES = 0.63) compared to EX + SC  compared to SC, and EX + SC decreased CK18 compared to an increase with SC   .P = 0.20, ES = 0.33). EX + SC increased metabolic flexibility compared to a lowered effect with SC .EX + SC had a small effect size for increased whole-body insulin sensitivity compared with SC , emotions (P = 0.03), and social interaction (P = 0.05) as well as total score (P = 0.03) (P = 0.02) as well as total score compared to SC (P = 0.02) (EX + SC significantly improved domains of QoL including symptoms ( = 0.03) . EX + SC = 0.02) .P = 0.05, ES = 1.38). There was no difference between treatments in length of surgery .EX + SC had a shorter length of hospital stay compared to SC  and metabolic flexibility  as well as reduced glucose tAUC . The decrease in AIx@75 tAUC  and increase in whole-body insulin sensitivity  was associated with a reduction in TG. The decrease in caloric intake was associated with an increase in metabolic flexibility  and increased percent of total time in MVPA . The decrease in sugar intake after treatment was correlated with increased whole-body insulin sensitivity  and metabolic flexibility  as well as a shorter length of hospital stay . The increase in number of sedentary bouts was linked to increased fasting glucose .A decrease in AIx@75 tAUC correlated with increased whole-body insulin sensitivity  had no change in similar cardiometabolic risk factors. Yet, high amount/vigorous intensity exercise  lowered blood pressure and raised HDL in overweight and obese adults , suggestn = 14), and therefore findings may not be generalizable to the bariatric population as a whole. For instance, race/ethnicity (The present study has limitations that require acknowledgment. This study had a small sample size (thnicity , sex (Duthnicity , socioecthnicity , educatithnicity , and agethnicity  are all thnicity , we werethnicity  and a reIn conclusion, EX + SC had favorable cardiometabolic health effects as well as improved weight related QoL compared to SC in patients with obesity prior to bariatric surgery. Exercise had a greater effect for increased adherence to pre-operative dietary recommendations, and the decrease in sugar intake pre-operatively was associated with a shorter length of hospital stay. Collectively, these findings suggest that pre-operative EX + SC may be important for cardiometabolic health, quality of life, and surgical outcomes in the bariatric patient.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by University of Virginia’s Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.SM conceptualized the research. NG, NE, and ER were responsible for subject recruitment and retention. NG, NE, MK, and PH were primarily responsible for data collection and management. NG, SK, AW, PH, and SM analyzed and interpreted the data. NG and SM were primarily responsible for writing the manuscript. NG, NE, MK, ER, SK, AW, PH, and SM reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.