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Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease

Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease fcell-08-00096 February 20, 2020 Time: 15:20 # 1 MINI REVIEW published: 21 February 2020 doi: 10.3389/fcell.2020.00096 Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease 1 2 3 3 4 Peta Bradbury , Hanjie Wu , Jung Un Choi , Alan E. Rowan , Hongyu Zhang , 5 3 2 Kate Poole , Jan Lauko and Joshua Chou * 1 2 Respiratory Technology, Woolcock Institute of Medical Research, Sydney, NSW, Australia, School of Biomedical Engineering, Faculty of Engineering and Information Technology, University of Technology Sydney, Sydney, NSW, Australia, 3 4 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia, State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing, China, EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia A lack of gravity experienced during space flight has been shown to have profound effects on human physiology including muscle atrophy, reductions in bone density and immune function, and endocrine disorders. At present, these physiological changes present major obstacles to long-term space missions. What is not clear is which pathophysiological disruptions reflect changes at the cellular level versus changes that occur due to the impact of weightlessness on the entire body. This review Edited by: focuses on current research investigating the impact of microgravity at the cellular level Michael Smutny, including cellular morphology, proliferation, and adhesion. As direct research in space University of Warwick, is currently cost prohibitive, we describe here the use of microgravity simulators for United Kingdom studies at the cellular level. Such instruments provide valuable tools for cost-effective Reviewed by: Sarah Boyle, research to better discern the impact of weightlessness on cellular function. Despite Centre for Cancer Biology (CCB), recent advances in understanding the relationship between extracellular forces and Australia Ludmila Buravkova, cell behavior, very little is understood about cellular biology and mechanotransduction Institute of Biomedical Problems under microgravity conditions. This review will examine recent insights into the impact of (RAS), Russia simulated microgravity on cell biology and how this technology may provide new insight *Correspondence: into advancing our understanding of mechanically driven biology and disease. Joshua Chou Joshua.chou@uts.edu.au Keywords: microgravity, mechanobiology, mechanotransduction, cytoskeletal, mechanosensing Specialty section: This article was submitted to INTRODUCTION Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Humans are subjected to persistent gravitational force and the importance of gravity for Biology maintaining physiological function has been revealed by the detrimental impacts of space travel on human health. During space flight, astronauts are exposed to a prolonged state of microgravity Received: 30 September 2019 Accepted: 04 February 2020 and develop a myriad of physiological disruptions including a loss of muscle mass and bone Published: 21 February 2020 density, impaired vision, decreased kidney function, diminished neurological responses, and a Citation: compromised immune system (White and Averner, 2001; Horneck et al., 2003; Crucian et al., 2014; Bradbury P, Wu H, Choi JU, White et al., 2016). This review will discuss recent data that highlight the impact of microgravity Rowan AE, Zhang H, Poole K, at the cellular level. Additionally, this review addresses how such research can be conducted on Lauko J and Chou J (2020) Modeling earth by simulating the microgravity state. These studies are not only important for understanding the Impact of Microgravity how humans are affected by microgravity but have the potential to elucidate the role of mechanical at the Cellular Level: Implications stimuli on cellular function and the development of mechanically driven disease states. for Human Disease. Mechanobiology is the study of how cells are influenced by their physical environment. Front. Cell Dev. Biol. 8:96. doi: 10.3389/fcell.2020.00096 This emerging field of research provides an important perspective on understanding Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 2 Bradbury et al. Modeling the Impact of Microgravity many aspects of cellular function and dysfunction. Cells 10 g (or as close to this value as possible) but cannot achieve can convert mechanical inputs into biochemical signals to complete zero gravity and hence termed microgravity (Huijser, initiate downstream signaling cascades in process known as 2000; Beysens and van Loon, 2015). mechanotransduction. Gravitational force is presumed to play a crucial role in regulating cell and tissue homeostasis by inducing THE IMPACT OF MICROGRAVITY OF mechanical stresses experienced at the cellular level. Thus, the concept of mechanical unloading (a decrease in mechanical CELL CYTOSKELETON stress) is associated with the weightlessness of space and can be replicated by simulating microgravity conditions, allowing Cellular response to mechanical loading has been well for investigation of the mechanobiology aspects of cell function. documented over the decades however, the response that The mechanical unloading of cells under microgravity conditions occurs when cells are placed under conditions of mechanical shifts the balance between physiology and pathophysiology, unloading remains in its infancy. The most apparent cellular accelerating the progression and development of some disease changes that occur following exposure to a microgravity states. For example, kidney stone formation is accelerated environment are alterations to cell shape, size, volume, and under microgravity conditions compared to Earth’s gravity (1 g) adherence properties (Buken et al., 2019; Dietz et al., 2019; Thiel (Pavlakou et al., 2018). Similarly, osteoporosis can take decades to et al., 2019b). These microgravity induced changes to cellular develop under normal gravitational loading, yet this disease can morphology reflect modifications to cytoskeletal structures, be modeled under microgravity over shorter time scales (Pajevic namely microtubules and actin filaments (F-actin), as cells sense et al., 2013). The mechanisms by which human physiology are a reduced gravitational load and therefore mechanical unloading disrupted in microgravity remain unknown, rendering numerous (Crawford-Young, 2006; Corydon et al., 2016a; Thiel et al., open questions regarding the adaptive changes that occur at the 2019a). Microgravity, whether in Space or simulated in the cellular and molecular level in response to microgravity. laboratory, offers a unique mechanical unloading environment to explore cellular mechanotransduction by providing an unparalleled research environment to investigate the relationship between mechanical unloading and cellular response. SIMULATING MICROGRAVITY Numerous studies have been conducted on a myriad of cell One of the key challenges in using microgravity as an types highlighting morphological sensitivity to microgravity investigative tool is creating a microgravity condition that (Ingber, 1999; Vorselen et al., 2014), with the first documented can be applied at a cellular level, on Earth. The process of morphological change reported by Rijken et al. (1991). conducting space research missions is costly and time consuming, Morphological changes as a result of microgravity conditions, thereby limiting the advancement of microgravity research and either real or simulated, have been shown to have altered widespread application of this approach. Currently, there are transcription, translation, and organization of cytoskeletal a number of microgravity devices available for purchase that proteins (Vassy et al., 2001; Infanger et al., 2006b; Tauber et al., are designed to achieve microgravity conditions. Microgravity 2017). Fundamental work carried out by Tabony, Pochon, simulators specific to cellular studies include strong magnetic and Papaseit showed that while tubulin self-assembly into field-induced levitation (i.e., diamagnetic simulation), as well microtubules occurs independent of gravity, the assembly and as two-dimensional and three-dimensional clinostats, rotating organization of the microtubule network is gravity dependent wall vessels and random positioning machines (RPMs) (Huijser, (Papaseit et al., 2000; Tabony et al., 2002). Importantly, this 2000; Russomano et al., 2005; Herranz et al., 2012; Ikeda et al., gravity-dependent organization of the microtubule network 2017). Each of the simulation techniques has shown advantages has since been described in multiple cell lines during both real and disadvantages however, when chosen correctly for a given and simulated microgravity exposures (Vassy et al., 2001; Uva experiment, the results obtained are similar to those observed in et al., 2002; Hughes-Fulford, 2003; Rosner et al., 2006; Janmaleki Space flight studies (Stamenkovic et al., 2010; Herranz et al., 2013; et al., 2016) and possibly be the result of a poorly defined Martinez et al., 2015; Krüger et al., 2019b). For cell culture studies, microtubule organizing center (MTOC) (Lewis et al., 1998). the use of RPM is common as the system achieves microgravity Taken together these data highlight an important regulatory role by continually providing random changes in orientation relative for the microtubule network and the MTOC following exposure to the gravity vector and thus an averaging of the impact of to a microgravity environment. However, the data surrounding the gravity vector to zero occurs over time (Beysens and van the response of the actin cytoskeleton to microgravity exposure Loon, 2015). This averaging is achieved by the independent, yet are less clear. Many studies have reported that microgravity simultaneous, rotation of two axes – with one axis rotating in exposure had decreased expression of actin and actin-associated the X-plane, and the second axis rotating in the Y -plane. It proteins, namely Arp2/3 and RhoA, subsequently resulting in is important that the cell culture flask/sample be placed at the the disorganization of the actin cytoskeleton (Carlsson et al., midpoint of the x-axis, cell culture flasks placed at a distance 2003; Higashibata et al., 2006; Corydon et al., 2016a,b; Louis from the center of the x-axis will be subjected to a greater et al., 2017; Tan et al., 2018). However, other studies have showed rotational force resulting in cells experiencing both centrifugal increased F-actin and stress fiber formation that accompanied the force and an increased gravity load (Beysens and van Loon, development of lamellipodia protrusions following exposure to 2015). Furthermore, the RPM is designed to subject the cells to microgravity (Gruener et al., 1994; Nassef et al., 2019). Contrary Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 3 Bradbury et al. Modeling the Impact of Microgravity to this, Rosner et al. (2006) reported no changes to actin structure absorption and resorption – a process that is coordinated by or organization and further suggested that the actin cytoskeleton both the actin cytoskeleton and microtubule network (Okumura is only regulated in a hyper-gravity environment. Thus, the et al., 2006). Bone cell morphology is significantly modified data surrounding cellular morphological changes in response following exposure to microgravity when compared to control to microgravity and the role of actin is confounding and at cells (Guignandon et al., 1995; Hughes-Fulford, 2003). To adapt times contradictory. to the new mechanical environment the bone cells have reduced The actin cytoskeleton, its organization and ability to generate transcription and translation of cytoskeletal and cytoskeletal- force are critical for cellular mechanosensing and importantly associated proteins (Xu et al., 2017; Mann et al., 2019), decreased any changes to these processes can initiate pathophysiological focal adhesion formation, together resulting in the increased disruption. Transduction of mechanical forces by integrins formation of osteoclast resorption pits (Nabavi et al., 2011). requires clustering of these transmembrane receptors and the Furthermore, the actin cytoskeleton of osteoblasts subjected to subsequent formation of focal contacts and adhesions that 4 days of microgravity exposure completely collapsed (Hughes- physically link the extracellular matrix (ECM) to the cytoskeleton Fulford, 2003), significantly impacting multiple downstream (Wang et al., 1993; Ingber, 1997; Maniotis et al., 1997). signaling pathways, most notably, the inhibition of bone Binding of integrins to matrix proteins promotes the bundling morphogenic protein (BMP) signaling axis (Patel et al., 2007; of F-actin at the cell-matrix adhesion and the subsequent Xu et al., 2017). The BMP family of proteins regulates the maturation of both the focal adhesion and the actin stress expression of an important mechanosensing protein, sclerostin, fiber (Zaidel-Bar et al., 2003; Wolfenson et al., 2009, 2011) found exclusively in osteocytes (Poole et al., 2005; Kamiya to generate the tension required for cell adherence, migration, et al., 2016), whereby mechanical unloading increases sclerostin and tissue homeostasis. Exposure to microgravity reduces the protein expression to promote bone resorption and cause a formation, number, and total area of focal adhesions per loss of bone density (Robling et al., 2008) – a phenotype that cell (Guignandon et al., 2003; Tan et al., 2018) consequently closely mimics both osteoporosis and osteonecrosis. Thus by affecting cellular adherence, migration capacity, and viability applying the unique mechanical unloading environment offered (Plett et al., 2004; Nabavi et al., 2011; Shi et al., 2015; by both real and simulated-microgravity to bone (specifically, Ahn et al., 2019; Dietz et al., 2019), albeit with contradictory osteoporosis) research has led to the introduction of the FDA results. Mechanical unloading has been shown to significantly approved drug, Evenity, a monoclonal antibody that works as an reduce gene expression of a number of focal adhesion proteins, anabolic agent to increase bone mass via the sclerostin pathway including FAK, DOCK1, and PTEN, while caveolin and (Scheiber et al., 2019). p130Cas expression were shown to be increased (Grenon While a significant number of studies have identified et al., 2013; Ratushnyy and Buravkova, 2017). Thus, the the importance of mechanical unloading in regulating bone activity of the downstream signaling pathways that govern structure and function, the articular cartilage (AC) is also the microgravity-induced cytoskeletal changes are significantly particularly susceptible to the effects of mechanical loading and impaired and are at least in part due to the microgravity- unloading (Sanchez-Adams et al., 2014). The cells found in triggered inhibition of FAK and/or RhoA signaling (Higashibata AC, chondrocytes, sense and respond to changing mechanical et al., 2006; Li et al., 2009; Tan et al., 2018). Furthermore, loads in order to maintain the balanced production of ECM recent data suggest that changes to the cytoskeleton may molecules ensuring that the tissue maintains the ability to also impact signaling via mechanically activated ion channels resist tensile and compressive forces. Both mechanical unloading and contacts in response to both cell-generated (Nourse and and overloading of chondrocytes can disrupt the homeostatic Pathak, 2017; Ellefsen et al., 2019) and externally applied balance in the cartilage leading to cartilage degradation and mechanical inputs (Bavi et al., 2019). Thus, downstream signaling osteoarthritis thereby tipping the balance from homeostatic of the numerous mechanotransduction pathways depend on maintenance to pathophysiology, leading to cartilage degradation the concerted interaction of the cytoskeleton, cell adhesion and osteoarthritis (Vanwanseele et al., 2002; Kurz et al., molecules, and force sensing proteins, including mechanically 2005). To study the effect of extended microgravity on AC activated ion channels. To date, there is little information specifically on the joint tissue, mice were exposed to 30 days regarding the role of mechanically activated ion channels in of spaceflight during the Bion-M1 mission (Fitzgerald et al., microgravity environments. 2019). Interestingly, tissue degradation was observed only in the AC of load-bearing joints, but not in minimally loaded sternal fibrocartilage highlighting a differential response to mechanical MICROGRAVITY IMPACT BONE CELL unloading and the predisposition of load bearing joints, SIGNALING RESPONSE AND but not structural joints, to mechanical stimuli. Additionally, decreased proteoglycan levels were found in the AC of the CARTILAGE ECM SYNTHESIS mice after the 30 days (Vanwanseele et al., 2002) further The accelerated loss of bone and muscle mass as a result characterizing a mechanical unloading pathology specific to of microgravity has been well documented over the decades AC atrophy. Importantly, reduced proteoglycan levels have also (Burger and Klein-Nulend, 1998; Harris et al., 2000; Fitts et al., been reported in hindlimb unloading and limb immobilization 2001). Osteocytes and osteoblasts are known mechanosensitive studies in various animals (Salter et al., 1980; Haapala et al., 1996; bone cells responsible for maintaining the balance of bone O’Connor, 1997). The reduced proteoglycan levels paired with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 4 Bradbury et al. Modeling the Impact of Microgravity FIGURE 1 | The morphology and physiology alterations of adherently growing cells after microgravity exposure. Cytoskeleton components of actin, microtubules and intermediate filament are displayed in inset circles. In adherent cells, microtubules form radiation arrangement near nuclear. Actin fibers anchor to cell membranes. Intermediate filament forms loose network around nuclear. Among cells under microgravity influence, the microtubules are shortened and curved. Less actin fibers but more condense intermediate filament are observed. This illustration was inspired by long-term thyroid cells culture in simulated microgravity environment (Kopp et al., 2015; Krüger et al., 2019a) . the augmented regulation of ECM-associated genes and proteins markers CD26L and HLA-DR, known regulators of lymphocyte- that help protect against osteoarthritic changes, including endothelial cell adhesion and tissue migration (Crucian et al., collagen type I, II, and X, b integrin, vimentin, and chondrocyte 2011). In vitro studies performed during Space flights have sulfate (Ulbrich et al., 2010; Aleshcheva et al., 2013, 2015), suggest revealed that lymphocytes exhibit important changes in their that while the microgravity-induced osteoarthritic pathology is cytoskeletal properties, suggesting that T cell activation may observed cartilage recovery of the AC is possible. Further to this, be compromised at the level of the T cell receptor (TCR) cell-based studies have shown that primary chondrocytes are to interaction (Sonnenfeld et al., 1992). It has been hypothesized adapt to a microgravity environment within 24 h (Aleshcheva that immunosuppression produced in microgravity is due to et al., 2013). There is a clear need for more research into the impaired TCR activation resulting from cytoskeletal disruption response of AC and specifically chondrocytes as elucidation (Bradley et al., 2019); however, the underlying molecular of the molecular mechanism that underpins chondrocytes mechanisms remain unknown. mechanoadaptation to a microgravity environment, holds great When applied to tumor cells microgravity has been found promise for novel osteoarthritic treatments. to impact tumor cell adhesion, proliferation, migration, and viability (Grimm et al., 2002; Plett et al., 2004; Infanger et al., 2006a; Shi et al., 2015; Tan et al., 2018), and to induce cell autophagy (Jeong et al., 2018). Changes in apoptotic rate MICROGRAVITY-INDUCED were also observed in colorectal cancer cells (DLD-1) and CYTOSKELETAL REGULATION OF a lymphoblast leukemic cell line (MOLT-4), accompanied by reduced transcription of the genes involved in colony formation, IMMUNE AND CANCER CELLS oncogenic progression, and metastatic potential (Vidyasekar The function of the immune system is strongly impacted et al., 2015). The foremost changes to tumor cell following (Frippiat et al., 2016; Smith, 2018) with several studies reporting exposure to microgravity are alterations of cell shape, size, dysregulation or immunosuppression following simulated or and adhesion, indicating changes in cytoskeletal organization real microgravity conditions (Boonyaratanakornkit et al., 2005; (Figure 1). Modulation of the cytoskeletal network have been demonstrated to occur after just minutes (Rijken et al., 1992; Crucian et al., 2015; Martinez et al., 2015; Thiel et al., 2017). Peripheral monocytes collected from astronauts post short- Sciola et al., 1999) or hours (Lewis et al., 1998; Vassy et al., 2001) in microgravity. Microtubule disorganization was observed duration Space missions (13–16 days) showed that there was no change in the numbers of circulating monocytes indicating that in both the breast cancer MCF-7 cells and the thyroid cancer the change to an immunosuppressive phenotype was not due a cell line FTC-133 when exposed to real microgravity (Kopp reduced cell number (Crucian et al., 2011). However, peripheral et al., 2018a,b). In contrast, no changes in Rac-controlled F-actin monocytes showed a significantly decreased expression of surface were detected in the neuroblastoma cell line, SH-Y-5Y (Rosner Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 5 Bradbury et al. Modeling the Impact of Microgravity TABLE 1 | Effects of sub-cellular functions concerning various cell types and exposure duration in microgravity environment. Cell type Effects of cells Microgravity exposure time References Osteosarcoma cells (ROS 17/2.8) Cell morphological change to rounded shape with long 4 days and 6 days Guignandon et al., 1997 cytoplasmic extensions Osteosarcoma cells (ROS 17/2.8) Reduction in cell spread area and vinculin spot area, actin 12 and 24 h Guignandon et al., 2003 and focal adhesion, and stress fibers Breast cancer (MCF-7) Disoriented microtubule 1.5 h Vassy et al., 2003 Thyroid cancer (ML-1) Actin fiber reorganization Parabola flight Ulbrich et al., 2011 Human macrophages No effect on cytoskeletal structure 11 days Tauber et al., 2017 Human chondrocytes Effect on cell cytoplasm, microtubule network disruption, Parabola flight Aleshcheva et al., 2015 loss of stress fibers, actin fiber reorganization. Osteoblasts (MC3T3-E1) Reduction in actin cytoskeletal stress fibers and reduction 4 days Hughes-Fulford and Lewis, 1996 of nuclei size by 30% Primary mouse osteoblasts Thicker microtubule, smaller focal adhesion spots, 5 days Nabavi et al., 2011 reduction in actin stress fibers, and increase in cell spread area Osteocytes Increase in cellular organelles including Golgi complex, 14 days Rodionova et al., 2002 vacuoles, and vesicles et al., 2006), highlighting the differing responses of distinct cell With the privatization and commercialization of the ISS and types to mechanical unloading. The expression of focal adhesion a global push toward the exploration of space, the gateway for proteins, moesin and ezrin, was found to be significantly down- conducting research under simulated and Space microgravity regulated after 24-h of microgravity exposure (Kopp et al., 2018a). is becoming more accessible. In particular the development of There remains a gap in the understanding of the molecular different variations of the RPM device provides a simulated mechanisms that drive changes in the cytoskeleton in response microgravity environment on Earth for investigating the changes to mechanical unloading and the physiological systems that will in cellular function due to mechanical unloading. Over the last be impacted by microgravity. Thus, there is a dual potential several years, experiments involving the use of microgravity of microgravity studies in both elucidating the underlying to study cellular mechanobiology and disease mechanisms importance of mechanical signaling in human physiology and have continued to rise, reinforcing the importance of this in developing understanding and countermeasures for long- platform. This area of research has highlighted the importance of duration space flights. mechanical cues in maintaining cell and tissue homeostasis. The emergence of Space mechanobiology will continue to rise in the foreseeable future as it is evident that the benefits of such research DISCUSSION can catapult survival of astronauts in space for extended duration as well as developing understanding and better treatments for Microgravity research conducted on the International Space Earth-borne diseases. Station (ISS) and in simulated microgravity has highlighted the importance of cellular mechanotransduction in human health and disease (Table 1). Understanding the molecular mechanisms AUTHOR CONTRIBUTIONS by which cells respond to mechanical unloading will not only be important for preparing humans for longer-term space PB contributed to the sections “Simulating Microgravity” exploration but may also contribute to therapeutics for the and “The Impact of Microgravity of Cell Cytoskeleton.” treatment of diseases that depend on mechanical interactions, JuC and JL contributed to the section “Microgravity Impact highlighting opportunities to manipulate and correct certain Bone Cell Signaling Response and Cartilage ECM Synthesis.” disease states. The discovery of sclerostin and the subsequent HW, HZ, and KP contributed to the section “Microgravity- generation and use of the sclerostin monoclonal antibody to Induced Cytoskeletal Regulation of Immune and Cancer Cells.” treat both osteoporotic patients has been a major outcome of AR, KP, and JoC initiated, conceptualized the review, and the field (Martin et al., 2020). Thus, Space and microgravity edited the manuscript. biological research constitute extreme environments in which novel mechanotransduction molecules and mechanosensing mechanisms can be identified and may prove helpful in better FUNDING designing immunotherapies or developing better and more targeted anti-cancer therapies. Studies that leverage these low The author acknowledges the support of the Australian Research gravity environments have the unique potential to unveil Council Discovery Project (ARC DP) (DP190101973), the important physiological changes that occur in response to NHMRC project grant APP1122104, and the University of changing mechanical loads and are of considerable importance Technology Sydney FEIT 2019 BlueSky grants FL160100139 in expanding our understanding of mechanobiology. (Laureate Fellowship AER) and DP190102230 (DP AER). 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Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease

Frontiers in Cell and Developmental Biology , Volume 8 – Feb 21, 2020

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fcell-08-00096 February 20, 2020 Time: 15:20 # 1 MINI REVIEW published: 21 February 2020 doi: 10.3389/fcell.2020.00096 Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease 1 2 3 3 4 Peta Bradbury , Hanjie Wu , Jung Un Choi , Alan E. Rowan , Hongyu Zhang , 5 3 2 Kate Poole , Jan Lauko and Joshua Chou * 1 2 Respiratory Technology, Woolcock Institute of Medical Research, Sydney, NSW, Australia, School of Biomedical Engineering, Faculty of Engineering and Information Technology, University of Technology Sydney, Sydney, NSW, Australia, 3 4 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia, State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing, China, EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia A lack of gravity experienced during space flight has been shown to have profound effects on human physiology including muscle atrophy, reductions in bone density and immune function, and endocrine disorders. At present, these physiological changes present major obstacles to long-term space missions. What is not clear is which pathophysiological disruptions reflect changes at the cellular level versus changes that occur due to the impact of weightlessness on the entire body. This review Edited by: focuses on current research investigating the impact of microgravity at the cellular level Michael Smutny, including cellular morphology, proliferation, and adhesion. As direct research in space University of Warwick, is currently cost prohibitive, we describe here the use of microgravity simulators for United Kingdom studies at the cellular level. Such instruments provide valuable tools for cost-effective Reviewed by: Sarah Boyle, research to better discern the impact of weightlessness on cellular function. Despite Centre for Cancer Biology (CCB), recent advances in understanding the relationship between extracellular forces and Australia Ludmila Buravkova, cell behavior, very little is understood about cellular biology and mechanotransduction Institute of Biomedical Problems under microgravity conditions. This review will examine recent insights into the impact of (RAS), Russia simulated microgravity on cell biology and how this technology may provide new insight *Correspondence: into advancing our understanding of mechanically driven biology and disease. Joshua Chou Joshua.chou@uts.edu.au Keywords: microgravity, mechanobiology, mechanotransduction, cytoskeletal, mechanosensing Specialty section: This article was submitted to INTRODUCTION Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Humans are subjected to persistent gravitational force and the importance of gravity for Biology maintaining physiological function has been revealed by the detrimental impacts of space travel on human health. During space flight, astronauts are exposed to a prolonged state of microgravity Received: 30 September 2019 Accepted: 04 February 2020 and develop a myriad of physiological disruptions including a loss of muscle mass and bone Published: 21 February 2020 density, impaired vision, decreased kidney function, diminished neurological responses, and a Citation: compromised immune system (White and Averner, 2001; Horneck et al., 2003; Crucian et al., 2014; Bradbury P, Wu H, Choi JU, White et al., 2016). This review will discuss recent data that highlight the impact of microgravity Rowan AE, Zhang H, Poole K, at the cellular level. Additionally, this review addresses how such research can be conducted on Lauko J and Chou J (2020) Modeling earth by simulating the microgravity state. These studies are not only important for understanding the Impact of Microgravity how humans are affected by microgravity but have the potential to elucidate the role of mechanical at the Cellular Level: Implications stimuli on cellular function and the development of mechanically driven disease states. for Human Disease. Mechanobiology is the study of how cells are influenced by their physical environment. Front. Cell Dev. Biol. 8:96. doi: 10.3389/fcell.2020.00096 This emerging field of research provides an important perspective on understanding Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 2 Bradbury et al. Modeling the Impact of Microgravity many aspects of cellular function and dysfunction. Cells 10 g (or as close to this value as possible) but cannot achieve can convert mechanical inputs into biochemical signals to complete zero gravity and hence termed microgravity (Huijser, initiate downstream signaling cascades in process known as 2000; Beysens and van Loon, 2015). mechanotransduction. Gravitational force is presumed to play a crucial role in regulating cell and tissue homeostasis by inducing THE IMPACT OF MICROGRAVITY OF mechanical stresses experienced at the cellular level. Thus, the concept of mechanical unloading (a decrease in mechanical CELL CYTOSKELETON stress) is associated with the weightlessness of space and can be replicated by simulating microgravity conditions, allowing Cellular response to mechanical loading has been well for investigation of the mechanobiology aspects of cell function. documented over the decades however, the response that The mechanical unloading of cells under microgravity conditions occurs when cells are placed under conditions of mechanical shifts the balance between physiology and pathophysiology, unloading remains in its infancy. The most apparent cellular accelerating the progression and development of some disease changes that occur following exposure to a microgravity states. For example, kidney stone formation is accelerated environment are alterations to cell shape, size, volume, and under microgravity conditions compared to Earth’s gravity (1 g) adherence properties (Buken et al., 2019; Dietz et al., 2019; Thiel (Pavlakou et al., 2018). Similarly, osteoporosis can take decades to et al., 2019b). These microgravity induced changes to cellular develop under normal gravitational loading, yet this disease can morphology reflect modifications to cytoskeletal structures, be modeled under microgravity over shorter time scales (Pajevic namely microtubules and actin filaments (F-actin), as cells sense et al., 2013). The mechanisms by which human physiology are a reduced gravitational load and therefore mechanical unloading disrupted in microgravity remain unknown, rendering numerous (Crawford-Young, 2006; Corydon et al., 2016a; Thiel et al., open questions regarding the adaptive changes that occur at the 2019a). Microgravity, whether in Space or simulated in the cellular and molecular level in response to microgravity. laboratory, offers a unique mechanical unloading environment to explore cellular mechanotransduction by providing an unparalleled research environment to investigate the relationship between mechanical unloading and cellular response. SIMULATING MICROGRAVITY Numerous studies have been conducted on a myriad of cell One of the key challenges in using microgravity as an types highlighting morphological sensitivity to microgravity investigative tool is creating a microgravity condition that (Ingber, 1999; Vorselen et al., 2014), with the first documented can be applied at a cellular level, on Earth. The process of morphological change reported by Rijken et al. (1991). conducting space research missions is costly and time consuming, Morphological changes as a result of microgravity conditions, thereby limiting the advancement of microgravity research and either real or simulated, have been shown to have altered widespread application of this approach. Currently, there are transcription, translation, and organization of cytoskeletal a number of microgravity devices available for purchase that proteins (Vassy et al., 2001; Infanger et al., 2006b; Tauber et al., are designed to achieve microgravity conditions. Microgravity 2017). Fundamental work carried out by Tabony, Pochon, simulators specific to cellular studies include strong magnetic and Papaseit showed that while tubulin self-assembly into field-induced levitation (i.e., diamagnetic simulation), as well microtubules occurs independent of gravity, the assembly and as two-dimensional and three-dimensional clinostats, rotating organization of the microtubule network is gravity dependent wall vessels and random positioning machines (RPMs) (Huijser, (Papaseit et al., 2000; Tabony et al., 2002). Importantly, this 2000; Russomano et al., 2005; Herranz et al., 2012; Ikeda et al., gravity-dependent organization of the microtubule network 2017). Each of the simulation techniques has shown advantages has since been described in multiple cell lines during both real and disadvantages however, when chosen correctly for a given and simulated microgravity exposures (Vassy et al., 2001; Uva experiment, the results obtained are similar to those observed in et al., 2002; Hughes-Fulford, 2003; Rosner et al., 2006; Janmaleki Space flight studies (Stamenkovic et al., 2010; Herranz et al., 2013; et al., 2016) and possibly be the result of a poorly defined Martinez et al., 2015; Krüger et al., 2019b). For cell culture studies, microtubule organizing center (MTOC) (Lewis et al., 1998). the use of RPM is common as the system achieves microgravity Taken together these data highlight an important regulatory role by continually providing random changes in orientation relative for the microtubule network and the MTOC following exposure to the gravity vector and thus an averaging of the impact of to a microgravity environment. However, the data surrounding the gravity vector to zero occurs over time (Beysens and van the response of the actin cytoskeleton to microgravity exposure Loon, 2015). This averaging is achieved by the independent, yet are less clear. Many studies have reported that microgravity simultaneous, rotation of two axes – with one axis rotating in exposure had decreased expression of actin and actin-associated the X-plane, and the second axis rotating in the Y -plane. It proteins, namely Arp2/3 and RhoA, subsequently resulting in is important that the cell culture flask/sample be placed at the the disorganization of the actin cytoskeleton (Carlsson et al., midpoint of the x-axis, cell culture flasks placed at a distance 2003; Higashibata et al., 2006; Corydon et al., 2016a,b; Louis from the center of the x-axis will be subjected to a greater et al., 2017; Tan et al., 2018). However, other studies have showed rotational force resulting in cells experiencing both centrifugal increased F-actin and stress fiber formation that accompanied the force and an increased gravity load (Beysens and van Loon, development of lamellipodia protrusions following exposure to 2015). Furthermore, the RPM is designed to subject the cells to microgravity (Gruener et al., 1994; Nassef et al., 2019). Contrary Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 3 Bradbury et al. Modeling the Impact of Microgravity to this, Rosner et al. (2006) reported no changes to actin structure absorption and resorption – a process that is coordinated by or organization and further suggested that the actin cytoskeleton both the actin cytoskeleton and microtubule network (Okumura is only regulated in a hyper-gravity environment. Thus, the et al., 2006). Bone cell morphology is significantly modified data surrounding cellular morphological changes in response following exposure to microgravity when compared to control to microgravity and the role of actin is confounding and at cells (Guignandon et al., 1995; Hughes-Fulford, 2003). To adapt times contradictory. to the new mechanical environment the bone cells have reduced The actin cytoskeleton, its organization and ability to generate transcription and translation of cytoskeletal and cytoskeletal- force are critical for cellular mechanosensing and importantly associated proteins (Xu et al., 2017; Mann et al., 2019), decreased any changes to these processes can initiate pathophysiological focal adhesion formation, together resulting in the increased disruption. Transduction of mechanical forces by integrins formation of osteoclast resorption pits (Nabavi et al., 2011). requires clustering of these transmembrane receptors and the Furthermore, the actin cytoskeleton of osteoblasts subjected to subsequent formation of focal contacts and adhesions that 4 days of microgravity exposure completely collapsed (Hughes- physically link the extracellular matrix (ECM) to the cytoskeleton Fulford, 2003), significantly impacting multiple downstream (Wang et al., 1993; Ingber, 1997; Maniotis et al., 1997). signaling pathways, most notably, the inhibition of bone Binding of integrins to matrix proteins promotes the bundling morphogenic protein (BMP) signaling axis (Patel et al., 2007; of F-actin at the cell-matrix adhesion and the subsequent Xu et al., 2017). The BMP family of proteins regulates the maturation of both the focal adhesion and the actin stress expression of an important mechanosensing protein, sclerostin, fiber (Zaidel-Bar et al., 2003; Wolfenson et al., 2009, 2011) found exclusively in osteocytes (Poole et al., 2005; Kamiya to generate the tension required for cell adherence, migration, et al., 2016), whereby mechanical unloading increases sclerostin and tissue homeostasis. Exposure to microgravity reduces the protein expression to promote bone resorption and cause a formation, number, and total area of focal adhesions per loss of bone density (Robling et al., 2008) – a phenotype that cell (Guignandon et al., 2003; Tan et al., 2018) consequently closely mimics both osteoporosis and osteonecrosis. Thus by affecting cellular adherence, migration capacity, and viability applying the unique mechanical unloading environment offered (Plett et al., 2004; Nabavi et al., 2011; Shi et al., 2015; by both real and simulated-microgravity to bone (specifically, Ahn et al., 2019; Dietz et al., 2019), albeit with contradictory osteoporosis) research has led to the introduction of the FDA results. Mechanical unloading has been shown to significantly approved drug, Evenity, a monoclonal antibody that works as an reduce gene expression of a number of focal adhesion proteins, anabolic agent to increase bone mass via the sclerostin pathway including FAK, DOCK1, and PTEN, while caveolin and (Scheiber et al., 2019). p130Cas expression were shown to be increased (Grenon While a significant number of studies have identified et al., 2013; Ratushnyy and Buravkova, 2017). Thus, the the importance of mechanical unloading in regulating bone activity of the downstream signaling pathways that govern structure and function, the articular cartilage (AC) is also the microgravity-induced cytoskeletal changes are significantly particularly susceptible to the effects of mechanical loading and impaired and are at least in part due to the microgravity- unloading (Sanchez-Adams et al., 2014). The cells found in triggered inhibition of FAK and/or RhoA signaling (Higashibata AC, chondrocytes, sense and respond to changing mechanical et al., 2006; Li et al., 2009; Tan et al., 2018). Furthermore, loads in order to maintain the balanced production of ECM recent data suggest that changes to the cytoskeleton may molecules ensuring that the tissue maintains the ability to also impact signaling via mechanically activated ion channels resist tensile and compressive forces. Both mechanical unloading and contacts in response to both cell-generated (Nourse and and overloading of chondrocytes can disrupt the homeostatic Pathak, 2017; Ellefsen et al., 2019) and externally applied balance in the cartilage leading to cartilage degradation and mechanical inputs (Bavi et al., 2019). Thus, downstream signaling osteoarthritis thereby tipping the balance from homeostatic of the numerous mechanotransduction pathways depend on maintenance to pathophysiology, leading to cartilage degradation the concerted interaction of the cytoskeleton, cell adhesion and osteoarthritis (Vanwanseele et al., 2002; Kurz et al., molecules, and force sensing proteins, including mechanically 2005). To study the effect of extended microgravity on AC activated ion channels. To date, there is little information specifically on the joint tissue, mice were exposed to 30 days regarding the role of mechanically activated ion channels in of spaceflight during the Bion-M1 mission (Fitzgerald et al., microgravity environments. 2019). Interestingly, tissue degradation was observed only in the AC of load-bearing joints, but not in minimally loaded sternal fibrocartilage highlighting a differential response to mechanical MICROGRAVITY IMPACT BONE CELL unloading and the predisposition of load bearing joints, SIGNALING RESPONSE AND but not structural joints, to mechanical stimuli. Additionally, decreased proteoglycan levels were found in the AC of the CARTILAGE ECM SYNTHESIS mice after the 30 days (Vanwanseele et al., 2002) further The accelerated loss of bone and muscle mass as a result characterizing a mechanical unloading pathology specific to of microgravity has been well documented over the decades AC atrophy. Importantly, reduced proteoglycan levels have also (Burger and Klein-Nulend, 1998; Harris et al., 2000; Fitts et al., been reported in hindlimb unloading and limb immobilization 2001). Osteocytes and osteoblasts are known mechanosensitive studies in various animals (Salter et al., 1980; Haapala et al., 1996; bone cells responsible for maintaining the balance of bone O’Connor, 1997). The reduced proteoglycan levels paired with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 4 Bradbury et al. Modeling the Impact of Microgravity FIGURE 1 | The morphology and physiology alterations of adherently growing cells after microgravity exposure. Cytoskeleton components of actin, microtubules and intermediate filament are displayed in inset circles. In adherent cells, microtubules form radiation arrangement near nuclear. Actin fibers anchor to cell membranes. Intermediate filament forms loose network around nuclear. Among cells under microgravity influence, the microtubules are shortened and curved. Less actin fibers but more condense intermediate filament are observed. This illustration was inspired by long-term thyroid cells culture in simulated microgravity environment (Kopp et al., 2015; Krüger et al., 2019a) . the augmented regulation of ECM-associated genes and proteins markers CD26L and HLA-DR, known regulators of lymphocyte- that help protect against osteoarthritic changes, including endothelial cell adhesion and tissue migration (Crucian et al., collagen type I, II, and X, b integrin, vimentin, and chondrocyte 2011). In vitro studies performed during Space flights have sulfate (Ulbrich et al., 2010; Aleshcheva et al., 2013, 2015), suggest revealed that lymphocytes exhibit important changes in their that while the microgravity-induced osteoarthritic pathology is cytoskeletal properties, suggesting that T cell activation may observed cartilage recovery of the AC is possible. Further to this, be compromised at the level of the T cell receptor (TCR) cell-based studies have shown that primary chondrocytes are to interaction (Sonnenfeld et al., 1992). It has been hypothesized adapt to a microgravity environment within 24 h (Aleshcheva that immunosuppression produced in microgravity is due to et al., 2013). There is a clear need for more research into the impaired TCR activation resulting from cytoskeletal disruption response of AC and specifically chondrocytes as elucidation (Bradley et al., 2019); however, the underlying molecular of the molecular mechanism that underpins chondrocytes mechanisms remain unknown. mechanoadaptation to a microgravity environment, holds great When applied to tumor cells microgravity has been found promise for novel osteoarthritic treatments. to impact tumor cell adhesion, proliferation, migration, and viability (Grimm et al., 2002; Plett et al., 2004; Infanger et al., 2006a; Shi et al., 2015; Tan et al., 2018), and to induce cell autophagy (Jeong et al., 2018). Changes in apoptotic rate MICROGRAVITY-INDUCED were also observed in colorectal cancer cells (DLD-1) and CYTOSKELETAL REGULATION OF a lymphoblast leukemic cell line (MOLT-4), accompanied by reduced transcription of the genes involved in colony formation, IMMUNE AND CANCER CELLS oncogenic progression, and metastatic potential (Vidyasekar The function of the immune system is strongly impacted et al., 2015). The foremost changes to tumor cell following (Frippiat et al., 2016; Smith, 2018) with several studies reporting exposure to microgravity are alterations of cell shape, size, dysregulation or immunosuppression following simulated or and adhesion, indicating changes in cytoskeletal organization real microgravity conditions (Boonyaratanakornkit et al., 2005; (Figure 1). Modulation of the cytoskeletal network have been demonstrated to occur after just minutes (Rijken et al., 1992; Crucian et al., 2015; Martinez et al., 2015; Thiel et al., 2017). Peripheral monocytes collected from astronauts post short- Sciola et al., 1999) or hours (Lewis et al., 1998; Vassy et al., 2001) in microgravity. Microtubule disorganization was observed duration Space missions (13–16 days) showed that there was no change in the numbers of circulating monocytes indicating that in both the breast cancer MCF-7 cells and the thyroid cancer the change to an immunosuppressive phenotype was not due a cell line FTC-133 when exposed to real microgravity (Kopp reduced cell number (Crucian et al., 2011). However, peripheral et al., 2018a,b). In contrast, no changes in Rac-controlled F-actin monocytes showed a significantly decreased expression of surface were detected in the neuroblastoma cell line, SH-Y-5Y (Rosner Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 February 2020 | Volume 8 | Article 96 fcell-08-00096 February 20, 2020 Time: 15:20 # 5 Bradbury et al. Modeling the Impact of Microgravity TABLE 1 | Effects of sub-cellular functions concerning various cell types and exposure duration in microgravity environment. Cell type Effects of cells Microgravity exposure time References Osteosarcoma cells (ROS 17/2.8) Cell morphological change to rounded shape with long 4 days and 6 days Guignandon et al., 1997 cytoplasmic extensions Osteosarcoma cells (ROS 17/2.8) Reduction in cell spread area and vinculin spot area, actin 12 and 24 h Guignandon et al., 2003 and focal adhesion, and stress fibers Breast cancer (MCF-7) Disoriented microtubule 1.5 h Vassy et al., 2003 Thyroid cancer (ML-1) Actin fiber reorganization Parabola flight Ulbrich et al., 2011 Human macrophages No effect on cytoskeletal structure 11 days Tauber et al., 2017 Human chondrocytes Effect on cell cytoplasm, microtubule network disruption, Parabola flight Aleshcheva et al., 2015 loss of stress fibers, actin fiber reorganization. Osteoblasts (MC3T3-E1) Reduction in actin cytoskeletal stress fibers and reduction 4 days Hughes-Fulford and Lewis, 1996 of nuclei size by 30% Primary mouse osteoblasts Thicker microtubule, smaller focal adhesion spots, 5 days Nabavi et al., 2011 reduction in actin stress fibers, and increase in cell spread area Osteocytes Increase in cellular organelles including Golgi complex, 14 days Rodionova et al., 2002 vacuoles, and vesicles et al., 2006), highlighting the differing responses of distinct cell With the privatization and commercialization of the ISS and types to mechanical unloading. The expression of focal adhesion a global push toward the exploration of space, the gateway for proteins, moesin and ezrin, was found to be significantly down- conducting research under simulated and Space microgravity regulated after 24-h of microgravity exposure (Kopp et al., 2018a). is becoming more accessible. In particular the development of There remains a gap in the understanding of the molecular different variations of the RPM device provides a simulated mechanisms that drive changes in the cytoskeleton in response microgravity environment on Earth for investigating the changes to mechanical unloading and the physiological systems that will in cellular function due to mechanical unloading. Over the last be impacted by microgravity. Thus, there is a dual potential several years, experiments involving the use of microgravity of microgravity studies in both elucidating the underlying to study cellular mechanobiology and disease mechanisms importance of mechanical signaling in human physiology and have continued to rise, reinforcing the importance of this in developing understanding and countermeasures for long- platform. This area of research has highlighted the importance of duration space flights. mechanical cues in maintaining cell and tissue homeostasis. The emergence of Space mechanobiology will continue to rise in the foreseeable future as it is evident that the benefits of such research DISCUSSION can catapult survival of astronauts in space for extended duration as well as developing understanding and better treatments for Microgravity research conducted on the International Space Earth-borne diseases. Station (ISS) and in simulated microgravity has highlighted the importance of cellular mechanotransduction in human health and disease (Table 1). Understanding the molecular mechanisms AUTHOR CONTRIBUTIONS by which cells respond to mechanical unloading will not only be important for preparing humans for longer-term space PB contributed to the sections “Simulating Microgravity” exploration but may also contribute to therapeutics for the and “The Impact of Microgravity of Cell Cytoskeleton.” treatment of diseases that depend on mechanical interactions, JuC and JL contributed to the section “Microgravity Impact highlighting opportunities to manipulate and correct certain Bone Cell Signaling Response and Cartilage ECM Synthesis.” disease states. The discovery of sclerostin and the subsequent HW, HZ, and KP contributed to the section “Microgravity- generation and use of the sclerostin monoclonal antibody to Induced Cytoskeletal Regulation of Immune and Cancer Cells.” treat both osteoporotic patients has been a major outcome of AR, KP, and JoC initiated, conceptualized the review, and the field (Martin et al., 2020). Thus, Space and microgravity edited the manuscript. biological research constitute extreme environments in which novel mechanotransduction molecules and mechanosensing mechanisms can be identified and may prove helpful in better FUNDING designing immunotherapies or developing better and more targeted anti-cancer therapies. Studies that leverage these low The author acknowledges the support of the Australian Research gravity environments have the unique potential to unveil Council Discovery Project (ARC DP) (DP190101973), the important physiological changes that occur in response to NHMRC project grant APP1122104, and the University of changing mechanical loads and are of considerable importance Technology Sydney FEIT 2019 BlueSky grants FL160100139 in expanding our understanding of mechanobiology. (Laureate Fellowship AER) and DP190102230 (DP AER). 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Conflict of Interest: The authors declare that the research was conducted in the Rapid morphological and cytoskeletal response to microgravity in human absence of any commercial or financial relationships that could be construed as a primary macrophages. Int. J. Mol. Sci. 20:2402. doi: 10.3390/ijms20102402 potential conflict of interest. Thiel, C. S., Tauber, S., Seebacher, C., Schropp, M., Uhl, R., Lauber, B., et al. (2019b). Real-time 3D high-resolution microscopy of human cells on the international Copyright © 2020 Bradbury, Wu, Choi, Rowan, Zhang, Poole, Lauko and Chou. space station. Int. J. Mol. Sci. 20:2033. doi: 10.3390/ijms20082033 This is an open-access article distributed under the terms of the Creative Commons Ulbrich, C., Westphal, K., Pietsch, J., Winkler, H. D., Leder, A., Bauer, J., Attribution License (CC BY). The use, distribution or reproduction in other forums et al. (2010). Characterization of human chondrocytes exposed to simulated is permitted, provided the original author(s) and the copyright owner(s) are credited microgravity. Cell Physiol. Biochem. 25, 551–560. doi: 10.1159/000303059 and that the original publication in this journal is cited, in accordance with accepted Ulbrich, C., Pietsch, J., Grosse, J., Wehland, M., Schulz, H., Saar, K., et al. academic practice. No use, distribution or reproduction is permitted which does not (2011). Differential gene regulation under altered gravity conditions in follicular comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 February 2020 | Volume 8 | Article 96

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