rus
Issue #7-8/2024
A.V.Moiseenko, N.A.Basalova, D.V.Bagrov, T.S.Trifonova, M.A.Vigovsky, U.D.Dyachkova, O.A.Grigorieva, E.S.Novoseletskaya, A.Yu.Efimenko, O.S.Sokolova
DIRECT VISUALIZATION OF EXTRACELLULAR VESICLES ON THE MEMBRANE OF HUMAN MESENCHYMAL STEM/STROMAL CELLS BY CRYO-ELECTRON MICROSCOPY
DIRECT VISUALIZATION OF EXTRACELLULAR VESICLES ON THE MEMBRANE OF HUMAN MESENCHYMAL STEM/STROMAL CELLS BY CRYO-ELECTRON MICROSCOPY
| DOI: https://doi.org/10.22184/1993-8578.2024.17.7-8.434.443
Extracellular vesicles (EVs) play an important role in intercellular communication and influence a wide range of physiological and pathological processes. Membrane-associated extracellular vesicles (MAVs) represent a distinct and poorly understood class of EVs. This study demonstrates the application of cryo-electron microscopy (cryo-EM) to investigate MAVs secreted by human mesenchymal stem/stromal cells (MSCs). Cryo-EM revealed vesicles ranging in diameter from 50 to 750 nm located near the cell surface. The results obtained will facilitate further studies on the physiological role of MAVs and their association with cell membranes.
Extracellular vesicles (EVs) play an important role in intercellular communication and influence a wide range of physiological and pathological processes. Membrane-associated extracellular vesicles (MAVs) represent a distinct and poorly understood class of EVs. This study demonstrates the application of cryo-electron microscopy (cryo-EM) to investigate MAVs secreted by human mesenchymal stem/stromal cells (MSCs). Cryo-EM revealed vesicles ranging in diameter from 50 to 750 nm located near the cell surface. The results obtained will facilitate further studies on the physiological role of MAVs and their association with cell membranes.
Теги: cryo-electron microscopy extracellular vesicles membrane-associated vesicles stem cells внеклеточные везикулы криоэлектронная микроскопия мембранно-ассоциированные везикулы стволовые клетки
INTRODUCTION
The transfer of biologically active factors in extracellular vesicles (EVs) secreted by mesenchymal stem/stromal cells (MSCs), according to modern data, is the basis of intercellular communication in implementation of many mechanisms of regenerative action of these cells. The use of EVs MSCs is considered as a promising therapeutic approach in various pathological processes treatment. For example, EVs MSCs can stimulate tissue regeneration [1, 2], regulate the immune system [3, 4], help in the treatment of fibrotic, oncological and infectious diseases [5, 6].
The term "extracellular vesicles" refers to particles that are released from cells bounded by a lipid bilayer; they cannot replicate independently (i.e., they do not contain a functional nucleus) [7]. Currently, several classes of EVs are distinguished (e.g., exosomes, microvesicles, and apoptotic cells). However, the classification of EVs subtypes is complicated by the fact that there is no unified consensus of specialists due to the limitations of technical capabilities of methods for separating different subtypes [8].
Membrane vesicle-like particles exist on the surface of many animal cells, ranging in size from tens of nanometres to 1–2 µm. These particles have long been regarded as precursors of EVs (in particular, microvesicles or microparticles), which are attached to the membrane until released into the intercellular space. At present, there is no generally accepted term for these EVs in the Russian-language literature. The group of researchers led by Yong Chen, in whose studies this class of EVs was first described for human cells (umbilical cord endothelial cells (HUVEC), hepatoma cells (HepG-2)), proposed the term "cell-bound membrane vesicles" [9], and hereinafter we will use the term "membrane-associated vesicles" (MAVs). It was shown that MAVs are a separate class of EVs, as MAVs have no co-localisation with putative surface markers of exosomes (proteins CD31, CD63 and others), they are resistant to detergent treatment but soluble in organic compounds, and there is no spontaneous release of MAVs from the cell surface.
Nowadays, the structure, composition and functions of MAVs are still poorly understood. In [9, 10], the method of confocal microscopy with differential interference contrast was used to visualise MAVs; in addition, after detachment from the membrane with detergent, they were studied by transmission electron microscopy with negative contrast and using the dynamic light scattering method. According to the latter, their average size was 443±6 nm, which is at the resolution limit of optical microscopy and obviously its use does not always allow to see the EVs. The applicability of atomic force microscopy for visualisation of MAVs on dried cells has been hypothesised [11], but there may be difficulties in identifying burst MAVs in images. Thus, it seems promising to develop approaches involving minimal sample processing and the use of electron microscopy and, in particular, cryo-electron microscopy (cryo-EM) to visualise and study MAVs.
This method involves rapid freezing of samples located on special metal grids covered with a thin carbon substrate. Their peculiarity is that there are numerous holes in the substrate; they can be arranged regularly or chaotically. Cryo-EM has been successfully used to visualise individual EVs [12], whole cells [13], as well as to study interactions between eukaryotic cells and viruses, which are close to EVs in size [14]. However, the use of cryo-EM to visualise MAVs directly on cells is a novel approach. The experiments described in this paper may provide a methodological basis for studying physiological role of MAVs. In addition, considering the growing interest of medicine in the application of EVs from human MSCs as therapeutic agents, description of mechanisms and biological properties of various EVs produced by this cell type is an urgent fundamental and applied task.
MATERIALS AND METHODS
Cell culturing. Immortalised MSCs isolated from human adipose tissue (ATCC, ASC52telo) were used in this work. The cell line was provided by the biobank from the collection of the Centre for Regenerative Medicine, Lomonosov Moscow State University, (https://human.depo.msu.ru). MSCs were grown on the surface of perforated thin carbon substrate C-flat 1.2/1.3 fixed on a gold support grid for electron microscopy (Protochips, USA).
For this purpose, the grids were placed in the wells of a 12-well plate and sterilised by ultraviolet light (3 times for 30 minutes each in a sterile laminar box). MSCs were planted in each well of the plate at a concentration of 40000 cells/well in DMEM-F12 medium (Capricorn, Germany) supplemented with 7% fetal bovine serum (Lacopa, Russia) and 1x penicillin-streptomycin (Gibco, USA). After a day of cultivation, the culture condition was assessed using phase-contrast microscopy (Leica DM IL LED, Germany).
Cryo-EM. Before freezing for cryo-EM, the grids were washed in Hanks’ balanced salt buffer (PanEco, Russia) and then frozen in liquid ethane at -180 °C in an EM GP2 unit (Leica Microsystems, Germany). Excess liquid was removed by blotting with Whatman #1 filter paper from the back side of the carbon substrate for 10 s. This allowed minimising probability of cells detachment from the substrate surface due to contact with filter paper. Cryo-EM studies were performed on a transmission electron microscope JEM-2100 (Jeol, Japan) at an accelerating voltage of 200 kV. Images were acquired using a DE-20 direct electron detection detector (Direct Electron, USA) in the accumulation mode (integration), at a nominal defocus of 10 μm, a total dose of irradiation of each imaging region not more than 20 e/A2 and at a microscope magnification corresponding to a pixel size of 5.7A in the image. Drift correction, Gaussian filtering and contrast correction were performed on the obtained image series.
RESULTS AND DISCUSSION
As a result of these experiments, the protocols for planting MSCs on gold grids and preparing samples for further study by the cryo-EM method were worked out. Photographs were obtained in the process of MSCs cultivation on gold grids (Fig.1a). The cells are spread over the surface of the carbon film covering the gold grid, and their edges are visible in many cells. Next, the meshes with MSCs were subjected to rapid freezing. Figure 1b shows an image of the cell edge obtained by cryo-EM. It can be seen that the carbon substrate is not solid, but contains regularly spaced round holes with a diameter of 1.2 μm filled with ice. In the image, the cell appears darker than the pure substrate, small fragments of crystalline ice are also present, not preventing the study of the peripheral regions of the cells. On a qualitative level, the images we obtained are similar to those reported in the literature for liver cells [15] or neurons [16].
Images obtained by the cryo-EM method have low contrast. This is caused by the fact that the studied objects consist of atoms of elements with low atomic number, weakly scattering electrons at large angles. In addition, the characteristic thickness of preparation to be studied in TEM is nearby the 50–100 nm (for amorphous biological objects consisting of atoms with low atomic number). Therefore, the excessive thickness of even the peripheral region of cells represents a serious limiting factor. To increase the contrast, we used defocusing of the objective lens of the microscope, which allows us to enhance the phase contrast in the images. Also, for clarity and ease of interpretation of the images, the main structures of interest were manually highlighted in colour.
The most contrast structures visible in the images are holes in the carbon substrate and crystalline ice particles, while the biological structures of interest for analysis have low contrast. In this study, we first imaged MAVs produced by MSCs by cryo-EM. At the edge of the cell, where its thickness is minimal, the cell membrane of MSCs and individual vesicles can be seen (Fig.2). In some cases it was possible to observe elongated characteristic structures inside the cell, bundles of fibres, which were interpreted as actin fibrils. Similar structures were observed by cryo-EM in mouse catecholaminergic neuronal cells [16] and human keratinocytes [17].
Cell membranes have a relatively high contrast in cryo-EM, and this allows us to interpret the particles located near the cells, surrounded by the membrane, as EVs. We were able to observe vesicles of different geometries, ranging in size from 50 nm to 750 nm. These values seem realistic, taking into account the general ideas about the size of EVs [8] and the available data on the size of MAVs [9, 10]. Many EVs contained not one lipid bilayer, but two (Fig.3) or more. The presence of several lipid bilayers may be the cause of MAVs resistance to detergents. However, this assumption should be confirmed by additional experiments. Previously, the cryo-EM method allowed detecting the similar multilayered MAVs isolated from samples of different origin [18, 19]; their physiological role is not completely clear.
Both EVs directly in contact with the membrane (Fig.3) and those close to it, at a distance of less than 50 nm (Fig.2), were encountered in the images obtained. The presence of the observed particles near the MSCs can be interpreted in one of three ways.
Firstly, they may be EVs secreted by a cell that is in the field of view. Secondly, they may be EVs that were secreted by another cell and the cell in the field of view has bound them on its surface. To date, most experimental evidence suggests that EVs are usually internalised into the endosomal compartment by endocytosis. However, the exact mechanisms controlling EVs endocytosis remain highly controversial. Various mechanisms of EV uptake by the recipient cell have been proposed, including clathrin-mediated endocytosis, caveolin-dependent endocytosis, micropinocytosis, and phagocytosis. In addition, the role of lipid raft proteins and specific protein-protein interactions in EVs internalisation has been shown. Typically, docking and subsequent endocytosis of EVs is facilitated by protein-protein interactions with membrane receptors, ligands, or contact proteins of recipient cells. Proteins such as tetraspanins, lectins, proteoglycans, and integrins may be involved in these specific interactions affecting EVs internalisation [20]. Fusion is another pathway of EVs internalisation in which the EV membrane directly fuses with the plasma membrane of the recipient cell. In addition to internalisation, EVs can activate intracellular signalling pathways by direct interaction with surface receptors or ligands of target cells. EVs signalling can influence cell phenotype through membrane-bound morphogens such as Wnt and Notch DII4 ligand. Also, EVs signalling can influence on motility, migration and invasiveness of tumour cells [21].
Finally, the EVs we observed may be MAVs at the moment of biogenesis, bound to the cell surface in the field of view. It is this interpretation that seems most likely, since before freezing the cell culture was cleaned from the residues of the culture medium, which could contain freely secreted EVs.
The images obtained in the described experiments do not allow one of these three interpretations to be unambiguously chosen. To prove that the observed particles are indeed MAVs, several experiments seem appropriate. Firstly, it is possible to suppress the secretion of MAVs to exclude them from consideration, but it is extremely difficult to achieve a complete cessation of the cell secretory activity. Secondly, cells can be treated with enzymes or detergents to detach MAV from their surface and the state of the submembrane space after such treatment can be analysed. Third, it may be useful to use colloidal gold immunolabelling, which can help identify particles that have specific protein markers on their surface to clarify the origin of these particles. However, more research is necessary to clarify the protein markers specific to MAVs as a separate subclass of EVs.
CONCLUSIONS
EVs are a challenging subject to study because of their heterogeneity and variability [8], and MAVs appear to be a particularly complex specific class of EVs. High-resolution microscopy techniques have been successfully used in EVs studies, and it is hoped that the use of cryo-EM will provide a better understanding of the origin and physiological role of MAVs.
In this study, we first studied feasibility of MAV detection on human MSCs and demonstrated that the cryo-EM method detects EVs located near or directly on the cell surface. For this purpose, cells are cultured on a gold grid for cryo-EM and frozen whole, and studies are carried out on the edges of unfolded cells, where the cytoplasm thickness is relatively small and allows obtaining informative images. Further experiments on the removal of MAVs from the cell surface by enzymatic treatment and analysis of the resulting cell cultures and isolated MAVs by cryo-EM, as well as immunolabelling of these objects, will help to interpret the resulting images more fully.
ACKNOWLEDGEMENTS
This work was supported by the Interdisciplinary Scientific and Educational School of Moscow State University "Molecular Technologies of Living Systems and Synthetic Biology" (#24-Sh04-14). The work was performed using a unique scientific facility "Three-dimensional electron microscopy and spectroscopy" of the Biology Department of Moscow State University.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The transfer of biologically active factors in extracellular vesicles (EVs) secreted by mesenchymal stem/stromal cells (MSCs), according to modern data, is the basis of intercellular communication in implementation of many mechanisms of regenerative action of these cells. The use of EVs MSCs is considered as a promising therapeutic approach in various pathological processes treatment. For example, EVs MSCs can stimulate tissue regeneration [1, 2], regulate the immune system [3, 4], help in the treatment of fibrotic, oncological and infectious diseases [5, 6].
The term "extracellular vesicles" refers to particles that are released from cells bounded by a lipid bilayer; they cannot replicate independently (i.e., they do not contain a functional nucleus) [7]. Currently, several classes of EVs are distinguished (e.g., exosomes, microvesicles, and apoptotic cells). However, the classification of EVs subtypes is complicated by the fact that there is no unified consensus of specialists due to the limitations of technical capabilities of methods for separating different subtypes [8].
Membrane vesicle-like particles exist on the surface of many animal cells, ranging in size from tens of nanometres to 1–2 µm. These particles have long been regarded as precursors of EVs (in particular, microvesicles or microparticles), which are attached to the membrane until released into the intercellular space. At present, there is no generally accepted term for these EVs in the Russian-language literature. The group of researchers led by Yong Chen, in whose studies this class of EVs was first described for human cells (umbilical cord endothelial cells (HUVEC), hepatoma cells (HepG-2)), proposed the term "cell-bound membrane vesicles" [9], and hereinafter we will use the term "membrane-associated vesicles" (MAVs). It was shown that MAVs are a separate class of EVs, as MAVs have no co-localisation with putative surface markers of exosomes (proteins CD31, CD63 and others), they are resistant to detergent treatment but soluble in organic compounds, and there is no spontaneous release of MAVs from the cell surface.
Nowadays, the structure, composition and functions of MAVs are still poorly understood. In [9, 10], the method of confocal microscopy with differential interference contrast was used to visualise MAVs; in addition, after detachment from the membrane with detergent, they were studied by transmission electron microscopy with negative contrast and using the dynamic light scattering method. According to the latter, their average size was 443±6 nm, which is at the resolution limit of optical microscopy and obviously its use does not always allow to see the EVs. The applicability of atomic force microscopy for visualisation of MAVs on dried cells has been hypothesised [11], but there may be difficulties in identifying burst MAVs in images. Thus, it seems promising to develop approaches involving minimal sample processing and the use of electron microscopy and, in particular, cryo-electron microscopy (cryo-EM) to visualise and study MAVs.
This method involves rapid freezing of samples located on special metal grids covered with a thin carbon substrate. Their peculiarity is that there are numerous holes in the substrate; they can be arranged regularly or chaotically. Cryo-EM has been successfully used to visualise individual EVs [12], whole cells [13], as well as to study interactions between eukaryotic cells and viruses, which are close to EVs in size [14]. However, the use of cryo-EM to visualise MAVs directly on cells is a novel approach. The experiments described in this paper may provide a methodological basis for studying physiological role of MAVs. In addition, considering the growing interest of medicine in the application of EVs from human MSCs as therapeutic agents, description of mechanisms and biological properties of various EVs produced by this cell type is an urgent fundamental and applied task.
MATERIALS AND METHODS
Cell culturing. Immortalised MSCs isolated from human adipose tissue (ATCC, ASC52telo) were used in this work. The cell line was provided by the biobank from the collection of the Centre for Regenerative Medicine, Lomonosov Moscow State University, (https://human.depo.msu.ru). MSCs were grown on the surface of perforated thin carbon substrate C-flat 1.2/1.3 fixed on a gold support grid for electron microscopy (Protochips, USA).
For this purpose, the grids were placed in the wells of a 12-well plate and sterilised by ultraviolet light (3 times for 30 minutes each in a sterile laminar box). MSCs were planted in each well of the plate at a concentration of 40000 cells/well in DMEM-F12 medium (Capricorn, Germany) supplemented with 7% fetal bovine serum (Lacopa, Russia) and 1x penicillin-streptomycin (Gibco, USA). After a day of cultivation, the culture condition was assessed using phase-contrast microscopy (Leica DM IL LED, Germany).
Cryo-EM. Before freezing for cryo-EM, the grids were washed in Hanks’ balanced salt buffer (PanEco, Russia) and then frozen in liquid ethane at -180 °C in an EM GP2 unit (Leica Microsystems, Germany). Excess liquid was removed by blotting with Whatman #1 filter paper from the back side of the carbon substrate for 10 s. This allowed minimising probability of cells detachment from the substrate surface due to contact with filter paper. Cryo-EM studies were performed on a transmission electron microscope JEM-2100 (Jeol, Japan) at an accelerating voltage of 200 kV. Images were acquired using a DE-20 direct electron detection detector (Direct Electron, USA) in the accumulation mode (integration), at a nominal defocus of 10 μm, a total dose of irradiation of each imaging region not more than 20 e/A2 and at a microscope magnification corresponding to a pixel size of 5.7A in the image. Drift correction, Gaussian filtering and contrast correction were performed on the obtained image series.
RESULTS AND DISCUSSION
As a result of these experiments, the protocols for planting MSCs on gold grids and preparing samples for further study by the cryo-EM method were worked out. Photographs were obtained in the process of MSCs cultivation on gold grids (Fig.1a). The cells are spread over the surface of the carbon film covering the gold grid, and their edges are visible in many cells. Next, the meshes with MSCs were subjected to rapid freezing. Figure 1b shows an image of the cell edge obtained by cryo-EM. It can be seen that the carbon substrate is not solid, but contains regularly spaced round holes with a diameter of 1.2 μm filled with ice. In the image, the cell appears darker than the pure substrate, small fragments of crystalline ice are also present, not preventing the study of the peripheral regions of the cells. On a qualitative level, the images we obtained are similar to those reported in the literature for liver cells [15] or neurons [16].
Images obtained by the cryo-EM method have low contrast. This is caused by the fact that the studied objects consist of atoms of elements with low atomic number, weakly scattering electrons at large angles. In addition, the characteristic thickness of preparation to be studied in TEM is nearby the 50–100 nm (for amorphous biological objects consisting of atoms with low atomic number). Therefore, the excessive thickness of even the peripheral region of cells represents a serious limiting factor. To increase the contrast, we used defocusing of the objective lens of the microscope, which allows us to enhance the phase contrast in the images. Also, for clarity and ease of interpretation of the images, the main structures of interest were manually highlighted in colour.
The most contrast structures visible in the images are holes in the carbon substrate and crystalline ice particles, while the biological structures of interest for analysis have low contrast. In this study, we first imaged MAVs produced by MSCs by cryo-EM. At the edge of the cell, where its thickness is minimal, the cell membrane of MSCs and individual vesicles can be seen (Fig.2). In some cases it was possible to observe elongated characteristic structures inside the cell, bundles of fibres, which were interpreted as actin fibrils. Similar structures were observed by cryo-EM in mouse catecholaminergic neuronal cells [16] and human keratinocytes [17].
Cell membranes have a relatively high contrast in cryo-EM, and this allows us to interpret the particles located near the cells, surrounded by the membrane, as EVs. We were able to observe vesicles of different geometries, ranging in size from 50 nm to 750 nm. These values seem realistic, taking into account the general ideas about the size of EVs [8] and the available data on the size of MAVs [9, 10]. Many EVs contained not one lipid bilayer, but two (Fig.3) or more. The presence of several lipid bilayers may be the cause of MAVs resistance to detergents. However, this assumption should be confirmed by additional experiments. Previously, the cryo-EM method allowed detecting the similar multilayered MAVs isolated from samples of different origin [18, 19]; their physiological role is not completely clear.
Both EVs directly in contact with the membrane (Fig.3) and those close to it, at a distance of less than 50 nm (Fig.2), were encountered in the images obtained. The presence of the observed particles near the MSCs can be interpreted in one of three ways.
Firstly, they may be EVs secreted by a cell that is in the field of view. Secondly, they may be EVs that were secreted by another cell and the cell in the field of view has bound them on its surface. To date, most experimental evidence suggests that EVs are usually internalised into the endosomal compartment by endocytosis. However, the exact mechanisms controlling EVs endocytosis remain highly controversial. Various mechanisms of EV uptake by the recipient cell have been proposed, including clathrin-mediated endocytosis, caveolin-dependent endocytosis, micropinocytosis, and phagocytosis. In addition, the role of lipid raft proteins and specific protein-protein interactions in EVs internalisation has been shown. Typically, docking and subsequent endocytosis of EVs is facilitated by protein-protein interactions with membrane receptors, ligands, or contact proteins of recipient cells. Proteins such as tetraspanins, lectins, proteoglycans, and integrins may be involved in these specific interactions affecting EVs internalisation [20]. Fusion is another pathway of EVs internalisation in which the EV membrane directly fuses with the plasma membrane of the recipient cell. In addition to internalisation, EVs can activate intracellular signalling pathways by direct interaction with surface receptors or ligands of target cells. EVs signalling can influence cell phenotype through membrane-bound morphogens such as Wnt and Notch DII4 ligand. Also, EVs signalling can influence on motility, migration and invasiveness of tumour cells [21].
Finally, the EVs we observed may be MAVs at the moment of biogenesis, bound to the cell surface in the field of view. It is this interpretation that seems most likely, since before freezing the cell culture was cleaned from the residues of the culture medium, which could contain freely secreted EVs.
The images obtained in the described experiments do not allow one of these three interpretations to be unambiguously chosen. To prove that the observed particles are indeed MAVs, several experiments seem appropriate. Firstly, it is possible to suppress the secretion of MAVs to exclude them from consideration, but it is extremely difficult to achieve a complete cessation of the cell secretory activity. Secondly, cells can be treated with enzymes or detergents to detach MAV from their surface and the state of the submembrane space after such treatment can be analysed. Third, it may be useful to use colloidal gold immunolabelling, which can help identify particles that have specific protein markers on their surface to clarify the origin of these particles. However, more research is necessary to clarify the protein markers specific to MAVs as a separate subclass of EVs.
CONCLUSIONS
EVs are a challenging subject to study because of their heterogeneity and variability [8], and MAVs appear to be a particularly complex specific class of EVs. High-resolution microscopy techniques have been successfully used in EVs studies, and it is hoped that the use of cryo-EM will provide a better understanding of the origin and physiological role of MAVs.
In this study, we first studied feasibility of MAV detection on human MSCs and demonstrated that the cryo-EM method detects EVs located near or directly on the cell surface. For this purpose, cells are cultured on a gold grid for cryo-EM and frozen whole, and studies are carried out on the edges of unfolded cells, where the cytoplasm thickness is relatively small and allows obtaining informative images. Further experiments on the removal of MAVs from the cell surface by enzymatic treatment and analysis of the resulting cell cultures and isolated MAVs by cryo-EM, as well as immunolabelling of these objects, will help to interpret the resulting images more fully.
ACKNOWLEDGEMENTS
This work was supported by the Interdisciplinary Scientific and Educational School of Moscow State University "Molecular Technologies of Living Systems and Synthetic Biology" (#24-Sh04-14). The work was performed using a unique scientific facility "Three-dimensional electron microscopy and spectroscopy" of the Biology Department of Moscow State University.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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