The overexpression of Tmprss6 reduces HJV levels by cleaving HJV, thereby inhibiting the phosphorylation of Smad1/5/8 and retention of the latter in the cytoplasm. Due to the decreased P-Smad1/5/8 levels, the transport of P-Smad1/5/8 to the nucleus by Smad4 is reduced. Decreased P-Smad1/5/8 in the nucleus inhibits pro-hepcidin expression. The decreased pro-hepcidin levels block the internalization and degradation of FPN1. The increased FPN1 level leads to the export of cellular Fe2+, limiting the activity of RNR, ultimately arresting the cell cycle before the S phase. Additionally, the decreased amount of P-Smad1/5/8 frees up Smad4 to bind to ATF3 and translocate it to the nucleus, thus activating p38 signaling and the expression of Bnip3, leading to apoptosis.
Neuro-2a cells were transfected with pcDNA3.1 (Vector group) or pcDNA3.1-Smad4-3Flag (Smad4 group) plasmids.
The overexpression of Tmprss6 reduces HJV levels by cleaving HJV, thereby inhibiting the phosphorylation of Smad1/5/8 and retention of the latter in the cytoplasm. Due to the decreased P-Smad1/5/8 levels, the transport of P-Smad1/5/8 to the nucleus by Smad4 is reduced. Decreased P-Smad1/5/8 in the nucleus inhibits pro-hepcidin expression. The decreased pro-hepcidin levels block the internalization and degradation of FPN1. The increased FPN1 level leads to the export of cellular Fe2+, limiting the activity of RNR, ultimately arresting the cell cycle before the S phase. Additionally, the decreased amount of P-Smad1/5/8 frees up Smad4 to bind to ATF3 and translocate it to the nucleus, thus activating p38 signaling and the expression of Bnip3, leading to apoptosis.
Edited by Professor Massimiliano Agostini
Edited by Professor Massimiliano Agostini
SUPPLEMENTAL MATERIAL
Original Data File
The reproducibility checklist
SUPPLEMENTAL MATERIAL
Original Data File
The reproducibility checklist
Wafer bonding technology has evolved significantly, particularly in the field of electronic applications manufactured at the wafer level to achieve high throughput. This advancement is especially critical for the packaging and assembly of MEMS devices, optical systems, and CMOS applications. Among these, wafer bonding of Si and Si-based materials is widely adapted for various applications including the packaging of MEMS sensors##UREF##0##1##, optical system sealing##UREF##1##2##, microchannel devices##REF##34739717##3##, and 3D stacking of memory and logic devices##UREF##2##4##.
","The formation of a layer at the bonding interface is the fundamental and highly effective approach for wafer bonding. One of the representing methods in this regard is hydrophilic bonding, where the Si surface containing physisorbed water and chemisorbed OH groups forms hydrogen and molecular bonds at the bonding interface##UREF##3##5##,##UREF##4##6##. In order to enhance the bonding quality and lower the process temperature, plasma treatments typically using or gas are widely employed to render Si bonding surfaces hydrophilic, facilitating water adsorption. Subsequent post-bonding annealing at 200-600 promotes Si oxidation at the bonding interface, thereby increasing the bond strength##UREF##5##7##,##UREF##6##8##.
","In the field of MEMS packaging, glass frit bonding is also widely adapted through the formation of an interface using low-melting-point glass pastes##UREF##7##9##,##UREF##8##10##. Here, the bonding surfaces are coated with such glass pastes, and thermo compression bonding at 350-600 induces adhesion between the substrates through the reflowed glass layer. Hence, the formation of the layer at the bonding interface is pivotal for ensuring mechanical reliability.
","However, the bonding process temperature is a crucial factor in industrial applications. Elevated temperatures can deteriorate device performance and the bonding interface’s reliability due to mismatches in the coefficient of thermal expansion between substrates. Consequently, low-temperature bonding, particularly at room temperature, is needed to accommodate next-generation electronics.
","Regarding to the formation of , the conversion of polysilazane into is a distinctive characteristic, represented by perhydropolysilazane (PHPS) , which undergoes hydrolysis when it interacts with water. This reaction leads to the formation of , along with the release of byproducts such as and ()##UREF##9##11##,##UREF##10##12##.
","PHPS can be converted into through various methods, including annealing within the temperature range of 200-1000##UREF##10##12##,##UREF##11##13##, infrared (IR) irradiation##UREF##12##14##–##UREF##14##16##, treatment with pH-controlled chemicals##UREF##15##17##, and exposure to moisture##UREF##16##18##,##UREF##17##19##. Since PHPS forms membrane by simple approaches compared to vacuum process such as chemical vapor deposition (CVD), PHPS is widely adapted to the coating of polymer films and metals##UREF##18##20##–##UREF##20##22## and interlayer dielectrics##UREF##21##23##,##UREF##22##24##.
","It is worth noting that the PHPS conversion into proceeds even at room temperature in the presence of water##UREF##9##11##,##UREF##11##13##,##UREF##23##25##,##UREF##24##26##. Hence, the conversion of PHPS to holds the potential for creating a bonding interface consisting of at room temperature, by introducing at the PHPS interface, for instance, through the use of plasma hydrophilic treatment.
","Based on this concept, this study proposes a novel approach of room temperature wafer bonding by forming bonding interface utilizing the PHPS conversion. The plasma hydrophilic treatment is applied to the Si wafer surface or the PHPS layer, thereby introducing adsorbed water to facilitate the PHPS conversion at the bonding interface. This is followed by wafer bonding via PHPS, leading to the formation of a bonding interface at room temperature.
","To investigate the proposed wafer bonding process via PHPS, two approaches were explored. The first approach involved utilizing the PHPS layer as a source of water and oxygen for the conversion of the PHPS bonding interface. In this case, the PHPS layers on the bonding pair wafers were directly subjected to plasma hydrophilic treatment. Wafer bonding was then performed via both side PHPS layers on the both of bonding pair wafers. The effects of plasma treatment were examined by comparing conditions where both sides, one side, or none of the PHPS layers underwent plasma treatment.
","In the second approach, plasma treatment was applied to the Si wafer surfaces. This scenario involved plasma hydrophilic treatment that facilitated water adsorption on the Si wafer surfaces, followed by the PHPS coating on the Si wafers. This implies that the PHPS receives water not from the ambient air but from the Si wafer surface. Subsequently, wafer bonding was performed via one side PHPS layer using the PHPS-coated wafer and the plasma-treated Si wafer. For comparison, wafer bonding was also performed via one side PHPS layer without plasma treatment to the wafer surface.
"],"string":"[\n \"Wafer bonding technology has evolved significantly, particularly in the field of electronic applications manufactured at the wafer level to achieve high throughput. This advancement is especially critical for the packaging and assembly of MEMS devices, optical systems, and CMOS applications. Among these, wafer bonding of Si and Si-based materials is widely adapted for various applications including the packaging of MEMS sensors##UREF##0##1##, optical system sealing##UREF##1##2##, microchannel devices##REF##34739717##3##, and 3D stacking of memory and logic devices##UREF##2##4##.
\",\n \"The formation of a layer at the bonding interface is the fundamental and highly effective approach for wafer bonding. One of the representing methods in this regard is hydrophilic bonding, where the Si surface containing physisorbed water and chemisorbed OH groups forms hydrogen and molecular bonds at the bonding interface##UREF##3##5##,##UREF##4##6##. In order to enhance the bonding quality and lower the process temperature, plasma treatments typically using or gas are widely employed to render Si bonding surfaces hydrophilic, facilitating water adsorption. Subsequent post-bonding annealing at 200-600 promotes Si oxidation at the bonding interface, thereby increasing the bond strength##UREF##5##7##,##UREF##6##8##.
\",\n \"In the field of MEMS packaging, glass frit bonding is also widely adapted through the formation of an interface using low-melting-point glass pastes##UREF##7##9##,##UREF##8##10##. Here, the bonding surfaces are coated with such glass pastes, and thermo compression bonding at 350-600 induces adhesion between the substrates through the reflowed glass layer. Hence, the formation of the layer at the bonding interface is pivotal for ensuring mechanical reliability.
\",\n \"However, the bonding process temperature is a crucial factor in industrial applications. Elevated temperatures can deteriorate device performance and the bonding interface’s reliability due to mismatches in the coefficient of thermal expansion between substrates. Consequently, low-temperature bonding, particularly at room temperature, is needed to accommodate next-generation electronics.
\",\n \"Regarding to the formation of , the conversion of polysilazane into is a distinctive characteristic, represented by perhydropolysilazane (PHPS) , which undergoes hydrolysis when it interacts with water. This reaction leads to the formation of , along with the release of byproducts such as and ()##UREF##9##11##,##UREF##10##12##.
\",\n \"PHPS can be converted into through various methods, including annealing within the temperature range of 200-1000##UREF##10##12##,##UREF##11##13##, infrared (IR) irradiation##UREF##12##14##–##UREF##14##16##, treatment with pH-controlled chemicals##UREF##15##17##, and exposure to moisture##UREF##16##18##,##UREF##17##19##. Since PHPS forms membrane by simple approaches compared to vacuum process such as chemical vapor deposition (CVD), PHPS is widely adapted to the coating of polymer films and metals##UREF##18##20##–##UREF##20##22## and interlayer dielectrics##UREF##21##23##,##UREF##22##24##.
\",\n \"It is worth noting that the PHPS conversion into proceeds even at room temperature in the presence of water##UREF##9##11##,##UREF##11##13##,##UREF##23##25##,##UREF##24##26##. Hence, the conversion of PHPS to holds the potential for creating a bonding interface consisting of at room temperature, by introducing at the PHPS interface, for instance, through the use of plasma hydrophilic treatment.
\",\n \"Based on this concept, this study proposes a novel approach of room temperature wafer bonding by forming bonding interface utilizing the PHPS conversion. The plasma hydrophilic treatment is applied to the Si wafer surface or the PHPS layer, thereby introducing adsorbed water to facilitate the PHPS conversion at the bonding interface. This is followed by wafer bonding via PHPS, leading to the formation of a bonding interface at room temperature.
\",\n \"To investigate the proposed wafer bonding process via PHPS, two approaches were explored. The first approach involved utilizing the PHPS layer as a source of water and oxygen for the conversion of the PHPS bonding interface. In this case, the PHPS layers on the bonding pair wafers were directly subjected to plasma hydrophilic treatment. Wafer bonding was then performed via both side PHPS layers on the both of bonding pair wafers. The effects of plasma treatment were examined by comparing conditions where both sides, one side, or none of the PHPS layers underwent plasma treatment.
\",\n \"In the second approach, plasma treatment was applied to the Si wafer surfaces. This scenario involved plasma hydrophilic treatment that facilitated water adsorption on the Si wafer surfaces, followed by the PHPS coating on the Si wafers. This implies that the PHPS receives water not from the ambient air but from the Si wafer surface. Subsequently, wafer bonding was performed via one side PHPS layer using the PHPS-coated wafer and the plasma-treated Si wafer. For comparison, wafer bonding was also performed via one side PHPS layer without plasma treatment to the wafer surface.
\"\n]"},"methods":{"kind":"list like","value":["4 inch Si wafers, with a thickness of 525 m, were utilized for the wafer bonding experiments. A 20 wt% solution of PHPS in dibutyl ether solvent was purchased.
","In the case of the wafer bonding via both side PHPS layers, first, the wafers were coated with PHPS solution by spin coating at 2000 rpm for 20 s. The coated PHPS layers underwent a baking process on a hot plate at 100 for 5 min to volatilize the solvent. This baking step completely removes the solvent from the PHPS layer##UREF##28##32##,##UREF##39##43##, that is also supported by XPS and EDX analysis (data not shown). Sequentially, the PHPS layers were treated with plasma with 150 W power for 60 s to induce hydrophilicity on the PHPS layer’s surface. After the surface treatment, the PHPS-coated surfaces of the Si wafers were manually brought into contact under ambient air conditions at room temperature. For comparison, wafer bonding was also conducted with plasma treatment applied to both sides of the PHPS layers, one side of the PHPS layers, and without any plasma treatment.
","In the case of the wafer bonding via one side PHPS layer, one wafer of a bonding pair was coated with PHPS. First, the bonding pair of Si wafers were treated with plasma followed by plasma to enhance water adsorption##UREF##40##44##,##UREF##41##45##, with both treatments conducted at a power of 150 W for 60 s each. After the plasma treatment, the wafers underwent a DI water rinse and were subsequently spin-dried. Following these surface preparations, the PHPS solution was spin-coated onto the one of the treated Si wafer pair, and solvent volatilization was conducted under the same conditions as described earlier. Finally, the Si wafer coated with PHPS and the uncoated wafer were bonded together in the ambient air at room temperature. The wafer bonding was also performed without the plasma hydrophilic treatment of Si wafers for comparison.
","The PHPS layer underwent analysis using XPS to investigate surface compositions. The bonding quality was evaluated by IR imaging of the bonded wafers and bond strength measurements using the blade test##UREF##42##46##. The properties of the cross-sectional bonding interface were observed using SEM with EDX.
"],"string":"[\n \"4 inch Si wafers, with a thickness of 525 m, were utilized for the wafer bonding experiments. A 20 wt% solution of PHPS in dibutyl ether solvent was purchased.
\",\n \"In the case of the wafer bonding via both side PHPS layers, first, the wafers were coated with PHPS solution by spin coating at 2000 rpm for 20 s. The coated PHPS layers underwent a baking process on a hot plate at 100 for 5 min to volatilize the solvent. This baking step completely removes the solvent from the PHPS layer##UREF##28##32##,##UREF##39##43##, that is also supported by XPS and EDX analysis (data not shown). Sequentially, the PHPS layers were treated with plasma with 150 W power for 60 s to induce hydrophilicity on the PHPS layer’s surface. After the surface treatment, the PHPS-coated surfaces of the Si wafers were manually brought into contact under ambient air conditions at room temperature. For comparison, wafer bonding was also conducted with plasma treatment applied to both sides of the PHPS layers, one side of the PHPS layers, and without any plasma treatment.
\",\n \"In the case of the wafer bonding via one side PHPS layer, one wafer of a bonding pair was coated with PHPS. First, the bonding pair of Si wafers were treated with plasma followed by plasma to enhance water adsorption##UREF##40##44##,##UREF##41##45##, with both treatments conducted at a power of 150 W for 60 s each. After the plasma treatment, the wafers underwent a DI water rinse and were subsequently spin-dried. Following these surface preparations, the PHPS solution was spin-coated onto the one of the treated Si wafer pair, and solvent volatilization was conducted under the same conditions as described earlier. Finally, the Si wafer coated with PHPS and the uncoated wafer were bonded together in the ambient air at room temperature. The wafer bonding was also performed without the plasma hydrophilic treatment of Si wafers for comparison.
\",\n \"The PHPS layer underwent analysis using XPS to investigate surface compositions. The bonding quality was evaluated by IR imaging of the bonded wafers and bond strength measurements using the blade test##UREF##42##46##. The properties of the cross-sectional bonding interface were observed using SEM with EDX.
\"\n]"},"results":{"kind":"list like","value":["In order to investigate the PHPS conversion into at room temperature by plasma hydrophilic treatment, chemical components of the PHPS coating on wafers were analyzed by x-ray photoelectron spectroscopy (XPS). Figure ##FIG##0##1## presents the XPS core spectra of the PHPS surface before and after plasma treatment. When the PHPS layer is just coated, oxygen peak is not observed, accompanied by a significant peak of nitrogen. The silicon peak at around 101.3 eV indicates silicon nitride##UREF##25##27##,##REF##25585707##28##. This is attributed to the intrinsic composition of PHPS##UREF##14##16##,##UREF##26##29##,##REF##30836334##30##. However, when the PHPS layer is treated with plasma, an increase in the oxygen peak and a decrease in the nitrogen peak are significant. Moreover, the silicon peak shifts towards a higher binding energy around 103.5 eV, indicating the presence of silicon oxide##UREF##25##27##,##REF##25585707##28##. Since the plasma itself does not contain and , it is suggested that the water adsorbs on the PHPS surface after the plasma treatment. Subsequently, the PHPS layer consumes the adsorbed water present on its hydrophilic surface for the conversion reaction.
","The stitched IR images of the typical bonded wafers with PHPS layers are depicted in Fig. ##FIG##1##2##. These images reveal the presence of interfacial voids, which appear as patterns with interference fringes. Basically the bonding interface shows a good adhesion without significant interfacial voids except the bonding via both side PHPS layer with both side plasma treatment (Fig. ##FIG##1##2## (
Conversely, bonding via both sides plasma-treated PHPS layers results in large interfacial voids, as shown in Fig. ##FIG##1##2## (
The results of the bond strength measurements obtained through blade insertion tests are presented in Fig. ##FIG##2##3##. For the bonding via both side PHPS layers, the bond strength is low when both PHPS layers are subjected to plasma treatment or when plasma treatment is not performed, yielding bond strengths of 0.64 J/m and 0.30 J/m, respectively. In the scenario where both sides of the PHPS layers undergo plasma treatment, both surfaces are converted into with surface asperities. Consequently, weak adhesion and a low bond strength result, which aligns with the results of the IR observations. In cases where plasma treatment is not performed, an insufficient supply of water at the bonding interface prevents the PHPS layers from conversion, resulting in a low bond strength.
","Conversely, when only one side of the PHPS layers is treated with plasma, a significantly improved bond strength of 5.54 J/m is achieved. In this case, the bonding interface features the adhesive PHPS layer in contact with the hydrophilic PHPS layer. The hydrophilic layer with adsorbed water facilitates the conversion of the untreated PHPS layer into . As a result, both strong adhesion and high bond strength are attained.
","In the cases of the bondings via the single side PHPS layer, when wafer bonding is conducted without the plasma hydrophilic treatment, the resulting bond strength measures 1.07 J/m. In contrast, bonding with plasma treatment exhibits a significantly improved bond strength of 6.02 J/m. Consistent with the bonding using both side PHPS layers, the plasma treatment introduces adsorbed water to the PHPS layer, thereby promoting its conversion to a mechanically stable interface at room temperature.
","Figure ##FIG##3##4##(a) shows the cross-sectional scanning electron microscopic (SEM) images of the bonding interface for the Si wafers bonded using PHPS layers on both sides with one side subjected to plasma treatment. In the low magnification image, the bonding interface appears uniform and devoid of voids. At higher magnification, the bonding interface is seen to consist of PHPS layers, each with a thickness of 0.4 m, resulting in a total thickness of 0.8 m. For the bonding via single side PHPS layer with plasma treatment shown in Fig. ##FIG##3##4##(b), a uniform and void-free bonding interface is also clearly visible, with the thickness of the PHPS layer measuring 0.4 m, consistent with the thickness observed in the bonding using double side PHPS layers.
","Energy dispersive x-ray spectroscopy (EDX) analysis was performed across the bonding interface, and the results are presented in Fig. ##FIG##4##5##. For the bonding via double side PHPS layers with one side plasma, the presence of O and N is detected at the interface of the PHPS layers, while the Si signal is relatively lower compared to the bulk Si area. This can be attributed to the lower density of Si atoms in the converted PHPS layers. Moreover, the O intensity appears to be uniformly distributed across the PHPS layers, while the N intensity is relatively weak on the left side of the PHPS layers but as strong as O on the right side.
","This distribution of N intensity is attributed to the differing treatment of the two PHPS layers. Specifically, the left side PHPS layer in Fig. ##FIG##4##5##(a) is treated with plasma, while the right side is not. The dominant O signal on the left side is consistent with the PHPS conversion by plasma treatment, while the strong N signal on the right side suggests that the PHPS layer in this region is partially converted to .
","The EDX line profile across the bonding interface of the single PHPS layer is illustrated in Fig. ##FIG##4##5##(b). At the PHPS layer area, the Si intensity is lower, while the presence of O and N is detected at the bonding interface. In comparison to the bonding utilizing both side PHPS layers as shown in Fig. ##FIG##4##5##(a), the composition of the PHPS layer appears to distribute more uniformly across the bonding interface, and the N intensity is closer to the background level. This suggests a more uniform conversion of the PHPS layer to than the bonding via both side PHPS layers. Given that the single PHPS layer at the bonding interface is situated between hydrophilic Si surfaces, the adsorbed water diffuses into the PHPS layer from both sides. As a result, the conversion of PHPS to proceeds more uniformly than the bonding via both sides PHPS layers.
","Furthermore, the N signal in EDX can be partly attributed to the byproducts of resulting from the PHPS conversion. Given that N is generally spread throughout the PHPS layers, it suggests the diffusion of and byproducts into the PHPS layer. As the converted PHPS into has an amorphous structure, both and are expected to diffuse similarly to .
","To investigate the PHPS conversion at the bonding interface, XPS analysis was performed on the debonded surface for both the bondings using both side PHPS layers and a single side PHPS layer, with and without plasma treatment. The atomic ratio of O and N was calculated from the O1s and N1s peaks for each condition, as illustrated in Fig. ##FIG##5##6##.
","For bonding via both side PHPS layers without plasma treatment, the N ratio was 0.88, suggesting that the N in the PHPS molecules was slightly replaced by O from the natural adsorption of water on the Si wafers##UREF##28##32##. However, when one of the both side PHPS layers was treated with plasma, the N ratio decreased to 0.50, indicating that the adsorbed water from the plasma treatment replaced the N at the bonding interface.
","On the other hand, the debonded surface with a single side PHPS layer also exhibited a high N ratio of 0.75 without plasma treatment. Similarly to bonding via both side PHPS layers, plasma treatment reduced the N ratio to 0.30 for bonding via a single side PHPS layer. As indicated by the cross-sectional EDX analysis, the single PHPS layer is sandwiched between the hydrophilic Si surfaces. The conversion of the single side PHPS layer proceeded further due to the relatively larger amount of water present at the bonding interface, as the amount of PHPS was half of that in the bonding via both side PHPS layers. Consequently, the replacement of N by O proceeded more largely compared to the bonding via double side PHPS layers.
"],"string":"[\n \"In order to investigate the PHPS conversion into at room temperature by plasma hydrophilic treatment, chemical components of the PHPS coating on wafers were analyzed by x-ray photoelectron spectroscopy (XPS). Figure ##FIG##0##1## presents the XPS core spectra of the PHPS surface before and after plasma treatment. When the PHPS layer is just coated, oxygen peak is not observed, accompanied by a significant peak of nitrogen. The silicon peak at around 101.3 eV indicates silicon nitride##UREF##25##27##,##REF##25585707##28##. This is attributed to the intrinsic composition of PHPS##UREF##14##16##,##UREF##26##29##,##REF##30836334##30##. However, when the PHPS layer is treated with plasma, an increase in the oxygen peak and a decrease in the nitrogen peak are significant. Moreover, the silicon peak shifts towards a higher binding energy around 103.5 eV, indicating the presence of silicon oxide##UREF##25##27##,##REF##25585707##28##. Since the plasma itself does not contain and , it is suggested that the water adsorbs on the PHPS surface after the plasma treatment. Subsequently, the PHPS layer consumes the adsorbed water present on its hydrophilic surface for the conversion reaction.
\",\n \"The stitched IR images of the typical bonded wafers with PHPS layers are depicted in Fig. ##FIG##1##2##. These images reveal the presence of interfacial voids, which appear as patterns with interference fringes. Basically the bonding interface shows a good adhesion without significant interfacial voids except the bonding via both side PHPS layer with both side plasma treatment (Fig. ##FIG##1##2## (
Conversely, bonding via both sides plasma-treated PHPS layers results in large interfacial voids, as shown in Fig. ##FIG##1##2## (
The results of the bond strength measurements obtained through blade insertion tests are presented in Fig. ##FIG##2##3##. For the bonding via both side PHPS layers, the bond strength is low when both PHPS layers are subjected to plasma treatment or when plasma treatment is not performed, yielding bond strengths of 0.64 J/m and 0.30 J/m, respectively. In the scenario where both sides of the PHPS layers undergo plasma treatment, both surfaces are converted into with surface asperities. Consequently, weak adhesion and a low bond strength result, which aligns with the results of the IR observations. In cases where plasma treatment is not performed, an insufficient supply of water at the bonding interface prevents the PHPS layers from conversion, resulting in a low bond strength.
\",\n \"Conversely, when only one side of the PHPS layers is treated with plasma, a significantly improved bond strength of 5.54 J/m is achieved. In this case, the bonding interface features the adhesive PHPS layer in contact with the hydrophilic PHPS layer. The hydrophilic layer with adsorbed water facilitates the conversion of the untreated PHPS layer into . As a result, both strong adhesion and high bond strength are attained.
\",\n \"In the cases of the bondings via the single side PHPS layer, when wafer bonding is conducted without the plasma hydrophilic treatment, the resulting bond strength measures 1.07 J/m. In contrast, bonding with plasma treatment exhibits a significantly improved bond strength of 6.02 J/m. Consistent with the bonding using both side PHPS layers, the plasma treatment introduces adsorbed water to the PHPS layer, thereby promoting its conversion to a mechanically stable interface at room temperature.
\",\n \"Figure ##FIG##3##4##(a) shows the cross-sectional scanning electron microscopic (SEM) images of the bonding interface for the Si wafers bonded using PHPS layers on both sides with one side subjected to plasma treatment. In the low magnification image, the bonding interface appears uniform and devoid of voids. At higher magnification, the bonding interface is seen to consist of PHPS layers, each with a thickness of 0.4 m, resulting in a total thickness of 0.8 m. For the bonding via single side PHPS layer with plasma treatment shown in Fig. ##FIG##3##4##(b), a uniform and void-free bonding interface is also clearly visible, with the thickness of the PHPS layer measuring 0.4 m, consistent with the thickness observed in the bonding using double side PHPS layers.
\",\n \"Energy dispersive x-ray spectroscopy (EDX) analysis was performed across the bonding interface, and the results are presented in Fig. ##FIG##4##5##. For the bonding via double side PHPS layers with one side plasma, the presence of O and N is detected at the interface of the PHPS layers, while the Si signal is relatively lower compared to the bulk Si area. This can be attributed to the lower density of Si atoms in the converted PHPS layers. Moreover, the O intensity appears to be uniformly distributed across the PHPS layers, while the N intensity is relatively weak on the left side of the PHPS layers but as strong as O on the right side.
\",\n \"This distribution of N intensity is attributed to the differing treatment of the two PHPS layers. Specifically, the left side PHPS layer in Fig. ##FIG##4##5##(a) is treated with plasma, while the right side is not. The dominant O signal on the left side is consistent with the PHPS conversion by plasma treatment, while the strong N signal on the right side suggests that the PHPS layer in this region is partially converted to .
\",\n \"The EDX line profile across the bonding interface of the single PHPS layer is illustrated in Fig. ##FIG##4##5##(b). At the PHPS layer area, the Si intensity is lower, while the presence of O and N is detected at the bonding interface. In comparison to the bonding utilizing both side PHPS layers as shown in Fig. ##FIG##4##5##(a), the composition of the PHPS layer appears to distribute more uniformly across the bonding interface, and the N intensity is closer to the background level. This suggests a more uniform conversion of the PHPS layer to than the bonding via both side PHPS layers. Given that the single PHPS layer at the bonding interface is situated between hydrophilic Si surfaces, the adsorbed water diffuses into the PHPS layer from both sides. As a result, the conversion of PHPS to proceeds more uniformly than the bonding via both sides PHPS layers.
\",\n \"Furthermore, the N signal in EDX can be partly attributed to the byproducts of resulting from the PHPS conversion. Given that N is generally spread throughout the PHPS layers, it suggests the diffusion of and byproducts into the PHPS layer. As the converted PHPS into has an amorphous structure, both and are expected to diffuse similarly to .
\",\n \"To investigate the PHPS conversion at the bonding interface, XPS analysis was performed on the debonded surface for both the bondings using both side PHPS layers and a single side PHPS layer, with and without plasma treatment. The atomic ratio of O and N was calculated from the O1s and N1s peaks for each condition, as illustrated in Fig. ##FIG##5##6##.
\",\n \"For bonding via both side PHPS layers without plasma treatment, the N ratio was 0.88, suggesting that the N in the PHPS molecules was slightly replaced by O from the natural adsorption of water on the Si wafers##UREF##28##32##. However, when one of the both side PHPS layers was treated with plasma, the N ratio decreased to 0.50, indicating that the adsorbed water from the plasma treatment replaced the N at the bonding interface.
\",\n \"On the other hand, the debonded surface with a single side PHPS layer also exhibited a high N ratio of 0.75 without plasma treatment. Similarly to bonding via both side PHPS layers, plasma treatment reduced the N ratio to 0.30 for bonding via a single side PHPS layer. As indicated by the cross-sectional EDX analysis, the single PHPS layer is sandwiched between the hydrophilic Si surfaces. The conversion of the single side PHPS layer proceeded further due to the relatively larger amount of water present at the bonding interface, as the amount of PHPS was half of that in the bonding via both side PHPS layers. Consequently, the replacement of N by O proceeded more largely compared to the bonding via double side PHPS layers.
\"\n]"},"discussion":{"kind":"list like","value":["Based on the experimental results, we propose a room temperature bonding mechanism as illustrated in Fig. ##FIG##6##7##. For bonding with both-side PHPS layers, the plasma treatment initiates the conversion of one PHPS layer to , facilitated by adsorbed water, as confirmed by the XPS analysis. Following the bonding of the plasma-treated PHPS layer with the untreated PHPS layer, the latter adheres to the substrates due to its ability to deform and compensate for surface asperities, as evidenced by IR imaging of the bonding interface. Subsequently, the water from the plasma-treated PHPS layer diffuses into the untreated PHPS layer, proceeding the hydrolysis of the untreated PHPS layer##UREF##26##29##. As the condensation polymerization of Si-OH groups proceeds and enhances the bond strength even at room temperature##UREF##4##6##,##UREF##29##33##,##UREF##30##34##, is formed at the bonding interface proceeds, resulting in a increased bond strength. The plasma-treated PHPS layer serves as a source of water and oxygen, supporting this conversion process, as indicated by cross-sectional EDX analysis.
","In the case of bonding with a single PHPS layer, the PHPS layer is coated on the hydrophilic surface with adsorbed water, thus facilitating PHPS conversion including hydrolysis step starting from the rear side. It has also been reported that the adsorbed water on the substrates react with the PHPS layer coated on the substrate##UREF##28##32##. This conversion is driven by the diffusion of the adsorbed water originating from the Si surface into the PHPS layer. Concurrently, the PHPS layer serves as an adhesive during the bonding of the PHPS-coated wafer to the uncoated wafer. Given that the surface of the uncoated wafer is also hydrophilic due to adsorbed water, water similarly diffuses into the PHPS layer from the front side. Consequently, the PHPS layer, situated between two hydrophilic Si surfaces, undergoes more uniform conversion into compared to bonding via PHPS layers on both sides, as supported by the EDX analysis.
","Compared to other wafer bonding techniques, the wafer bonding via PHPS is comparable to water glass bonding##UREF##31##35##–##UREF##33##37## and sol-gel bonding##UREF##34##38##–##UREF##36##40##. Both of these techniques involve bonding through a liquid precursor of , followed by an annealing step to create the bonding interface composed of . However, a fundamental distinction lies in the mechanism of transformation to . In sol-gel or water glass bonding, the conversion to occurs through condensation polymerization, necessitating annealing to eliminate from the bonding interface. In contrast, the proposed method utilizing a PHPS layer achieves the formation of the bonding interface through hydrolysis, allowing for bonding at room temperature without the need for annealing.
","Regarding to the conventional direct bonding of Si or , wafer bonding via PHPS exhibits a comparable bonding quality. In conventional hydrophilic bonding of Si or with plasma hydrophilic treatment, the bond strength typically increases with post-bonding annealing. For Si/Si and / wafer bonding, the typical bond strength is less than 1 J/m without post-bonding annealing##UREF##4##6##,##UREF##37##41##. The bond strength increases to over 2 J/m after annealing at temperatures no lower than 300 . Despite the conventional hydrophilic wafer bonding requiring a heating step for a robust bonding interface, wafer bonding via PHPS achieves a high bond strength without annealing.
","In addition, conventional hydrophilic wafer bonding is often associated with concerns about bubble formation at the bonding interface. Post-bonding annealing in hydrophilic bonding facilitates the condensation polymerization of Si-OH at the bonding interface. This process generates byproducts such as and residual , resulting in the formation of bubbles and voids at the bonding interface##UREF##37##41##,##UREF##38##42##. In contrast, wafer bonding via PHPS, as depicted in Fig. ##FIG##1##2##, does not exhibit the formation of bubbles, contributing to an improved bonding quality.
"],"string":"[\n \"Based on the experimental results, we propose a room temperature bonding mechanism as illustrated in Fig. ##FIG##6##7##. For bonding with both-side PHPS layers, the plasma treatment initiates the conversion of one PHPS layer to , facilitated by adsorbed water, as confirmed by the XPS analysis. Following the bonding of the plasma-treated PHPS layer with the untreated PHPS layer, the latter adheres to the substrates due to its ability to deform and compensate for surface asperities, as evidenced by IR imaging of the bonding interface. Subsequently, the water from the plasma-treated PHPS layer diffuses into the untreated PHPS layer, proceeding the hydrolysis of the untreated PHPS layer##UREF##26##29##. As the condensation polymerization of Si-OH groups proceeds and enhances the bond strength even at room temperature##UREF##4##6##,##UREF##29##33##,##UREF##30##34##, is formed at the bonding interface proceeds, resulting in a increased bond strength. The plasma-treated PHPS layer serves as a source of water and oxygen, supporting this conversion process, as indicated by cross-sectional EDX analysis.
\",\n \"In the case of bonding with a single PHPS layer, the PHPS layer is coated on the hydrophilic surface with adsorbed water, thus facilitating PHPS conversion including hydrolysis step starting from the rear side. It has also been reported that the adsorbed water on the substrates react with the PHPS layer coated on the substrate##UREF##28##32##. This conversion is driven by the diffusion of the adsorbed water originating from the Si surface into the PHPS layer. Concurrently, the PHPS layer serves as an adhesive during the bonding of the PHPS-coated wafer to the uncoated wafer. Given that the surface of the uncoated wafer is also hydrophilic due to adsorbed water, water similarly diffuses into the PHPS layer from the front side. Consequently, the PHPS layer, situated between two hydrophilic Si surfaces, undergoes more uniform conversion into compared to bonding via PHPS layers on both sides, as supported by the EDX analysis.
\",\n \"Compared to other wafer bonding techniques, the wafer bonding via PHPS is comparable to water glass bonding##UREF##31##35##–##UREF##33##37## and sol-gel bonding##UREF##34##38##–##UREF##36##40##. Both of these techniques involve bonding through a liquid precursor of , followed by an annealing step to create the bonding interface composed of . However, a fundamental distinction lies in the mechanism of transformation to . In sol-gel or water glass bonding, the conversion to occurs through condensation polymerization, necessitating annealing to eliminate from the bonding interface. In contrast, the proposed method utilizing a PHPS layer achieves the formation of the bonding interface through hydrolysis, allowing for bonding at room temperature without the need for annealing.
\",\n \"Regarding to the conventional direct bonding of Si or , wafer bonding via PHPS exhibits a comparable bonding quality. In conventional hydrophilic bonding of Si or with plasma hydrophilic treatment, the bond strength typically increases with post-bonding annealing. For Si/Si and / wafer bonding, the typical bond strength is less than 1 J/m without post-bonding annealing##UREF##4##6##,##UREF##37##41##. The bond strength increases to over 2 J/m after annealing at temperatures no lower than 300 . Despite the conventional hydrophilic wafer bonding requiring a heating step for a robust bonding interface, wafer bonding via PHPS achieves a high bond strength without annealing.
\",\n \"In addition, conventional hydrophilic wafer bonding is often associated with concerns about bubble formation at the bonding interface. Post-bonding annealing in hydrophilic bonding facilitates the condensation polymerization of Si-OH at the bonding interface. This process generates byproducts such as and residual , resulting in the formation of bubbles and voids at the bonding interface##UREF##37##41##,##UREF##38##42##. In contrast, wafer bonding via PHPS, as depicted in Fig. ##FIG##1##2##, does not exhibit the formation of bubbles, contributing to an improved bonding quality.
\"\n]"},"conclusion":{"kind":"list like","value":["In this paper, we investigated and demonstrated wafer bonding via PHPS at room temperature. Through the strategic use of plasma hydrophilic treatment on either the Si surface or the PHPS layer, we have effectively facilitated the conversion of PHPS into a robust bonding interface composed of . This approach achieves the bond strength exceeding 5 J/m and the void-free bonding interfaces, which are attributed to the combined effects of PHPS adhesion and formation. The XPS and EDX analysis also suggest the bonding mechanism that the adsorbed water by hydrophilic plasma treatment diffuses into the PHPS layer which results in the partial conversion of PHPS into , contributing to the robust bonding interface. This technique will provide a new approach for the wafer bonding at room temperature for electronics packaging.
"],"string":"[\n \"In this paper, we investigated and demonstrated wafer bonding via PHPS at room temperature. Through the strategic use of plasma hydrophilic treatment on either the Si surface or the PHPS layer, we have effectively facilitated the conversion of PHPS into a robust bonding interface composed of . This approach achieves the bond strength exceeding 5 J/m and the void-free bonding interfaces, which are attributed to the combined effects of PHPS adhesion and formation. The XPS and EDX analysis also suggest the bonding mechanism that the adsorbed water by hydrophilic plasma treatment diffuses into the PHPS layer which results in the partial conversion of PHPS into , contributing to the robust bonding interface. This technique will provide a new approach for the wafer bonding at room temperature for electronics packaging.
\"\n]"},"front":{"kind":"list like","value":["Room temperature wafer bonding is a desirable approach for the packaging and assembly of diverse electronic devices. The formation of layer at the bonding interface is crucial for a reliable wafer bonding as represented by conventional bonding techniques such as hydrophilic bonding and glass frit bonding. This paper reports a novel concept of room temperature wafer bonding based on the conversion of polysilazane to at the bonding interface. As polysilazane is converted to by hydrolysis, in this work, adsorbed water is introduced to the bonding interface by plasma treatment, thereby facilitating the formation of at the wafer bonding interface. The experimental results indicate that the adsorbed water from the plasma treatment diffuses into the polysilazane layer and facilitates its hydrolysis and conversion. The proposed method demonstrates the successful wafer bonding at room temperature with high bond strength without interfacial voids. This technique will provide a new approach of bonding wafers at room temperature for electronics packaging.
","Room temperature wafer bonding is a desirable approach for the packaging and assembly of diverse electronic devices. The formation of layer at the bonding interface is crucial for a reliable wafer bonding as represented by conventional bonding techniques such as hydrophilic bonding and glass frit bonding. This paper reports a novel concept of room temperature wafer bonding based on the conversion of polysilazane to at the bonding interface. As polysilazane is converted to by hydrolysis, in this work, adsorbed water is introduced to the bonding interface by plasma treatment, thereby facilitating the formation of at the wafer bonding interface. The experimental results indicate that the adsorbed water from the plasma treatment diffuses into the polysilazane layer and facilitates its hydrolysis and conversion. The proposed method demonstrates the successful wafer bonding at room temperature with high bond strength without interfacial voids. This technique will provide a new approach of bonding wafers at room temperature for electronics packaging.
\",\n \"This work is supported by the Amada foundation, Tokyo Ohka Foundation for The Promotion of Science and Technology, and JSPS KAKENHI Grant Number JP23K13554.
","K.T. designed and carried out the experiments, analyzed the experimental results, and wrote the manuscript. K.T., T.S., and E.H. discussed results, commented on, and edited the manuscript.
","The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
","The authors declare no competing interests.
"],"string":"[\n \"This work is supported by the Amada foundation, Tokyo Ohka Foundation for The Promotion of Science and Technology, and JSPS KAKENHI Grant Number JP23K13554.
\",\n \"K.T. designed and carried out the experiments, analyzed the experimental results, and wrote the manuscript. K.T., T.S., and E.H. discussed results, commented on, and edited the manuscript.
\",\n \"The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
\",\n \"The authors declare no competing interests.
\"\n]"},"figure":{"kind":"list like","value":["XPS core spectra of the PHPS surfaces before and after the plasma hydrophilic treatment for (
Stitched IR images of the bonded wafers via PHPS. (
Bond strength of the bonded wafers via PHPS layers at room temperature.
Cross-sectional SEM observation of the bonding interface of (
EDX line profile across the bonding interface via (
Atomic ratio of O and N on the debonded wafer surface of the bonding via both and one side PHPS layers with and without plasma treatment.
The room temperature wafer bonding mechanism via (
XPS core spectra of the PHPS surfaces before and after the plasma hydrophilic treatment for (
Stitched IR images of the bonded wafers via PHPS. (
Bond strength of the bonded wafers via PHPS layers at room temperature.
Cross-sectional SEM observation of the bonding interface of (
EDX line profile across the bonding interface via (
Atomic ratio of O and N on the debonded wafer surface of the bonding via both and one side PHPS layers with and without plasma treatment.
The room temperature wafer bonding mechanism via (
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Reproduction in an environment suitable for genome stability is important for the preservation of species. This requirement is more imperative in poikilotherms animals, in which gametes are directly exposed to undesirable temperatures. As a result, they may have evolved a system that rapidly adapts to such changes. In fish, for example, temperature is generally considered to be an important factor in determining the exact timing of gamete maturation and spawning##UREF##0##1##.
","Inhibition of reproduction by high temperature is observed in a wide range of fish species##REF##20738700##2##. High-temperature environment inhibits female ovulation and male spermatogenesis of Atlantic salmon (
Testes in mammals as well as in fish show clear sensitivities to high-temperature environments. Mammalian testes exposed to temperature >37 °C display abnormal spermatogenesis by way of germ cell apoptosis through various signaling pathways##UREF##2##4##. A proposed model posits that high temperature causes damage to meiotic prophase I and damaged pachytene spermatocytes are eliminated at the checkpoints that monitor meiotic progression##REF##35618762##5##. Checkpoints in multicellular organisms induce apoptosis in DNA-damaged cells and contribute to genome stability across generations. This mechanism contributes to the lower mutation rate of germ cells than somatic cells, which is important for stronger genome integrity##REF##28485371##6##. The mitotic checkpoint functions also in
Leydig cells are found in the interstitial tissue of the testis and regulate spermatogenesis and reproductive function through the secretion of steroid hormones such as testosterone##REF##30980107##8##. In mammalian models, hyperthermic stimulation of the testis by an artificially induced cryptorchidism leads to a decrease in Leydig cell activity as well as in germ cells##REF##19484498##9##. Reduced activity of Leydig cells leads to decreased steroid hormone secretion##REF##8817242##10##,##UREF##3##11##. High temperature also induces apoptosis not only in germ cells but also in Leydig cells##REF##30196893##12##. However, the molecular mechanism and the significance of apoptosis in Leydig cells induced by high-temperature stimulation remain unexplored. In particular, the causal relationship between reduced Leydig cell activity and abnormal spermatogenesis due to the high-temperature environment is not clear.
","In this study, we provide evidence that Leydig cells in
Reproduction in an environment suitable for genome stability is important for the preservation of species. This requirement is more imperative in poikilotherms animals, in which gametes are directly exposed to undesirable temperatures. As a result, they may have evolved a system that rapidly adapts to such changes. In fish, for example, temperature is generally considered to be an important factor in determining the exact timing of gamete maturation and spawning##UREF##0##1##.
\",\n \"Inhibition of reproduction by high temperature is observed in a wide range of fish species##REF##20738700##2##. High-temperature environment inhibits female ovulation and male spermatogenesis of Atlantic salmon (
Testes in mammals as well as in fish show clear sensitivities to high-temperature environments. Mammalian testes exposed to temperature >37 °C display abnormal spermatogenesis by way of germ cell apoptosis through various signaling pathways##UREF##2##4##. A proposed model posits that high temperature causes damage to meiotic prophase I and damaged pachytene spermatocytes are eliminated at the checkpoints that monitor meiotic progression##REF##35618762##5##. Checkpoints in multicellular organisms induce apoptosis in DNA-damaged cells and contribute to genome stability across generations. This mechanism contributes to the lower mutation rate of germ cells than somatic cells, which is important for stronger genome integrity##REF##28485371##6##. The mitotic checkpoint functions also in
Leydig cells are found in the interstitial tissue of the testis and regulate spermatogenesis and reproductive function through the secretion of steroid hormones such as testosterone##REF##30980107##8##. In mammalian models, hyperthermic stimulation of the testis by an artificially induced cryptorchidism leads to a decrease in Leydig cell activity as well as in germ cells##REF##19484498##9##. Reduced activity of Leydig cells leads to decreased steroid hormone secretion##REF##8817242##10##,##UREF##3##11##. High temperature also induces apoptosis not only in germ cells but also in Leydig cells##REF##30196893##12##. However, the molecular mechanism and the significance of apoptosis in Leydig cells induced by high-temperature stimulation remain unexplored. In particular, the causal relationship between reduced Leydig cell activity and abnormal spermatogenesis due to the high-temperature environment is not clear.
\",\n \"In this study, we provide evidence that Leydig cells in
All experiments using animals in this study followed the guidelines of Osaka Medical and Pharmaceutical University. Adult
Up to four individuals were temperature-stimulated in W22 × D12 × H11 cm tanks with 1.8 l of water, and water changes were performed daily. The tanks were placed in a water bath at 35 °C and oxygen was supplied by aquarium air stones. Temperature stimulation was interrupted for feeding from 12:00 to 13:00 daily. Exposure of isolated sperm was for 20 min (Supplementary Fig. ##SUPPL##1##9##).
","Testes were fixed in Bouin solution, and 4 μm plastic sections were prepared using Technobit 8100 (Heraeus Kulzer). Samples were stained with hematoxylin solution for 10 min (Muto pure chemicals, Tokyo, Japan), and stained with 1% eosin (Muto pure chemicals, Tokyo, Japan) for 30 s. The mounted samples were imaged using an Olympus DP74 camera (Evident, Tokyo, Japan).
","RNA samples were extracted from testes. According to the manufacturer’s instructions, 0.5 μg RNA template was used for reverse transcription to synthesize cDNA using a first-strand cDNA synthesis kit (ReverTra Ace, TOYOBO, Tokyo, Japan). The qPCR primers are listed in Supplementary Table ##SUPPL##1##1##. For amplification, the KOD SYBR qPCR/RT (TOYOBO, Tokyo, Japan) and ABI real-time system were used. Statistical significance was evaluated by two-tailed Student’s
RNA in situ hybridization (WISH) was carried out following a standard protocol##REF##16682019##47##. The primers used to amplify the regions used for the probes are shown in Supplementary Table ##SUPPL##1##1##. Briefly, testes were fixed in 4% paraformaldehyde in 1× PBS overnight. On the following day, samples were hybridized at 65 °C overnight. Samples were further incubated with anti-digoxigenin-AP antibody solution (1:2000) overnight at 4 °C and stained with NBT/BCIP. The stained samples were imaged using an Olympus DP74 camera (Evident). Double in situ hybridization was carried out following a standard protocol##REF##16894600##48##. The first probe was labeled with DIG and the second probe was labeled with FITC. After the first staining, incubation was performed with 0.1 M glycine–HCl pH 2.2 for 40 min to remove alkaline phosphatase activity.
","Immunofluorescence staining was performed using an anti-Cleaved caspase 3 antibody (#9661, Cell Signaling Technology, USA). Alexa 488 was used as the secondary antibody. Nuclear DNA was stained with 4’,6-diamidino-2-phenylindol (DAPI). Stained samples were imaged using Leica SP8 (Leica, Germany).
","CRISPR/Cas9 target sites in
Testes were isolated from the adult
Embryos that developed past the epiboly stage were measured as fertilized eggs. Daily survival rates were measured relative to the number of eggs reaching the epiboly stage. Statistical significance was evaluated by two-tailed Student’s
Further information on research design is available in the ##SUPPL##8##Nature Portfolio Reporting Summary## linked to this article.
"],"string":"[\n \"All experiments using animals in this study followed the guidelines of Osaka Medical and Pharmaceutical University. Adult
Up to four individuals were temperature-stimulated in W22 × D12 × H11 cm tanks with 1.8 l of water, and water changes were performed daily. The tanks were placed in a water bath at 35 °C and oxygen was supplied by aquarium air stones. Temperature stimulation was interrupted for feeding from 12:00 to 13:00 daily. Exposure of isolated sperm was for 20 min (Supplementary Fig. ##SUPPL##1##9##).
\",\n \"Testes were fixed in Bouin solution, and 4 μm plastic sections were prepared using Technobit 8100 (Heraeus Kulzer). Samples were stained with hematoxylin solution for 10 min (Muto pure chemicals, Tokyo, Japan), and stained with 1% eosin (Muto pure chemicals, Tokyo, Japan) for 30 s. The mounted samples were imaged using an Olympus DP74 camera (Evident, Tokyo, Japan).
\",\n \"RNA samples were extracted from testes. According to the manufacturer’s instructions, 0.5 μg RNA template was used for reverse transcription to synthesize cDNA using a first-strand cDNA synthesis kit (ReverTra Ace, TOYOBO, Tokyo, Japan). The qPCR primers are listed in Supplementary Table ##SUPPL##1##1##. For amplification, the KOD SYBR qPCR/RT (TOYOBO, Tokyo, Japan) and ABI real-time system were used. Statistical significance was evaluated by two-tailed Student’s
RNA in situ hybridization (WISH) was carried out following a standard protocol##REF##16682019##47##. The primers used to amplify the regions used for the probes are shown in Supplementary Table ##SUPPL##1##1##. Briefly, testes were fixed in 4% paraformaldehyde in 1× PBS overnight. On the following day, samples were hybridized at 65 °C overnight. Samples were further incubated with anti-digoxigenin-AP antibody solution (1:2000) overnight at 4 °C and stained with NBT/BCIP. The stained samples were imaged using an Olympus DP74 camera (Evident). Double in situ hybridization was carried out following a standard protocol##REF##16894600##48##. The first probe was labeled with DIG and the second probe was labeled with FITC. After the first staining, incubation was performed with 0.1 M glycine–HCl pH 2.2 for 40 min to remove alkaline phosphatase activity.
\",\n \"Immunofluorescence staining was performed using an anti-Cleaved caspase 3 antibody (#9661, Cell Signaling Technology, USA). Alexa 488 was used as the secondary antibody. Nuclear DNA was stained with 4’,6-diamidino-2-phenylindol (DAPI). Stained samples were imaged using Leica SP8 (Leica, Germany).
\",\n \"CRISPR/Cas9 target sites in
Testes were isolated from the adult
Embryos that developed past the epiboly stage were measured as fertilized eggs. Daily survival rates were measured relative to the number of eggs reaching the epiboly stage. Statistical significance was evaluated by two-tailed Student’s
Further information on research design is available in the ##SUPPL##8##Nature Portfolio Reporting Summary## linked to this article.
\"\n]"},"results":{"kind":"list like","value":["To determine the appropriate condition for high-temperature stimulation in adult
To analyze the effect of temperature stimulation on testicular structures and spermatogenesis, sections of the testes were observed by HE staining (Fig. ##FIG##0##1a##, Supplementary Fig. ##SUPPL##1##3a##). There are three developmental stages of spermatids: initial (E1), intermediate (E2), and final (E3)##REF##19339708##13##. Temperature stimulation led to a decrease in spermatids, especially at E3 (circled in black lines), and the appearance of abnormal cells (circled in red lines). The abnormal cells had large nuclei relative to the cytoplasm and were different in appearance from any normal types of cells in germ cell development (Supplementary Fig. ##SUPPL##1##4##). Germ cells at pachytene, diplotene, and metaphase stages were present, but the number of spermatids was dramatically reduced in testis exposed to 34 °C. In normal
In addition, there was a significant decrease in the interstitial tissue, which was observed as gaps between cysts. This tendency became more obvious as the duration of exposure to 34 °C became longer. Specifically, distinct Leydig cells were rarely detected after 2 weeks of exposure to 34 °C (Fig. ##FIG##0##1a##, upper panels).
","Transfer of fish from 34 °C back to 29 °C resulted in the disappearance of the abnormal cell population and the appearance of interstitial cells in 7 days (Fig. ##FIG##0##1a##, lower panels). E3 spermatid appeared within 10 days of the transition to 29 °C, and spermatogenesis completely recovered to normal in 1 month. In
We analyzed the expression of several groups of genes in testes exposed to 34 °C. Among them, genes in the
Based on the loss of E3 spermatid at 34 °C (Fig. ##FIG##0##1a##), we predicted a decrease in
Since temperature stimulation strongly affected Leydig cells, they were further analyzed by in situ hybridization using its marker
Suspecting that apoptosis is involved in the different responses between Leydig cells and germ cells, we analyzed the localization of its marker, cleaved caspase 3. The signal was detected in the interstitial cells (circumscribed in white lines) after 1-day exposure to 34 °C and in spermatocytes (in red lines) after 3 days (Fig. ##FIG##0##1e##). These results indicated that apoptosis in response to 34 °C exposure is induced in Leydig cells prior to spermatocytes.
","To identify molecules involved in temperature sensing, we performed qRT-PCR for a group of
To clarify the function of TRPV4, a
To analyze the effects of high-temperature treatment on testis structure and spermatogenesis in
To examine further the effects of
We analyzed the involvement of
Based on these results, we hypothesized that the decrease of 20β-S secreted from Leydig cells after 34 °C treatment impaired sperm motility. The secretion of 20β-S in the testis was analyzed by LC–MS/MS. Unfortunately, 20β-S in the testes was below the detection limit (data not shown). We therefore used qRT-PCR to examine the expression of the
Indeed, when sperm isolated from the testis was directly exposed to high temperature, its motility did not decrease even at 40 °C (Supplementary Fig. ##SUPPL##1##10##). Although the duration of exposure to high temperature was shorter than the incubation of the whole fish (Fig. ##FIG##3##4##) due to technical limitations of maintaining isolated sperm in vitro, these results were consistent with the hypothesis that the endocrine regulation of Leydig cells, not autonomous regulation of sperm, determined the sperm motility in response to high temperature.
","We hypothesized that
Males incubated at 34 °C for 1 day were mated with females normally reared and incubated at 29 °C. Fertilization rates were significantly lower for both WT and
If fertilization occurs at high temperatures, generated embryos are also likely to develop in high temperatures. We therefore examined the effect of 34 °C water temperature on developing embryos of
Finally, to examine whether this mechanism of Leydig cell sensitivity to high temperature is universally conserved among fish, we analyzed
Similar to
We performed qRT-PCR analysis of a group of
To determine the appropriate condition for high-temperature stimulation in adult
To analyze the effect of temperature stimulation on testicular structures and spermatogenesis, sections of the testes were observed by HE staining (Fig. ##FIG##0##1a##, Supplementary Fig. ##SUPPL##1##3a##). There are three developmental stages of spermatids: initial (E1), intermediate (E2), and final (E3)##REF##19339708##13##. Temperature stimulation led to a decrease in spermatids, especially at E3 (circled in black lines), and the appearance of abnormal cells (circled in red lines). The abnormal cells had large nuclei relative to the cytoplasm and were different in appearance from any normal types of cells in germ cell development (Supplementary Fig. ##SUPPL##1##4##). Germ cells at pachytene, diplotene, and metaphase stages were present, but the number of spermatids was dramatically reduced in testis exposed to 34 °C. In normal
In addition, there was a significant decrease in the interstitial tissue, which was observed as gaps between cysts. This tendency became more obvious as the duration of exposure to 34 °C became longer. Specifically, distinct Leydig cells were rarely detected after 2 weeks of exposure to 34 °C (Fig. ##FIG##0##1a##, upper panels).
\",\n \"Transfer of fish from 34 °C back to 29 °C resulted in the disappearance of the abnormal cell population and the appearance of interstitial cells in 7 days (Fig. ##FIG##0##1a##, lower panels). E3 spermatid appeared within 10 days of the transition to 29 °C, and spermatogenesis completely recovered to normal in 1 month. In
We analyzed the expression of several groups of genes in testes exposed to 34 °C. Among them, genes in the
Based on the loss of E3 spermatid at 34 °C (Fig. ##FIG##0##1a##), we predicted a decrease in
Since temperature stimulation strongly affected Leydig cells, they were further analyzed by in situ hybridization using its marker
Suspecting that apoptosis is involved in the different responses between Leydig cells and germ cells, we analyzed the localization of its marker, cleaved caspase 3. The signal was detected in the interstitial cells (circumscribed in white lines) after 1-day exposure to 34 °C and in spermatocytes (in red lines) after 3 days (Fig. ##FIG##0##1e##). These results indicated that apoptosis in response to 34 °C exposure is induced in Leydig cells prior to spermatocytes.
\",\n \"To identify molecules involved in temperature sensing, we performed qRT-PCR for a group of
To clarify the function of TRPV4, a
To analyze the effects of high-temperature treatment on testis structure and spermatogenesis in
To examine further the effects of
We analyzed the involvement of
Based on these results, we hypothesized that the decrease of 20β-S secreted from Leydig cells after 34 °C treatment impaired sperm motility. The secretion of 20β-S in the testis was analyzed by LC–MS/MS. Unfortunately, 20β-S in the testes was below the detection limit (data not shown). We therefore used qRT-PCR to examine the expression of the
Indeed, when sperm isolated from the testis was directly exposed to high temperature, its motility did not decrease even at 40 °C (Supplementary Fig. ##SUPPL##1##10##). Although the duration of exposure to high temperature was shorter than the incubation of the whole fish (Fig. ##FIG##3##4##) due to technical limitations of maintaining isolated sperm in vitro, these results were consistent with the hypothesis that the endocrine regulation of Leydig cells, not autonomous regulation of sperm, determined the sperm motility in response to high temperature.
\",\n \"We hypothesized that
Males incubated at 34 °C for 1 day were mated with females normally reared and incubated at 29 °C. Fertilization rates were significantly lower for both WT and
If fertilization occurs at high temperatures, generated embryos are also likely to develop in high temperatures. We therefore examined the effect of 34 °C water temperature on developing embryos of
Finally, to examine whether this mechanism of Leydig cell sensitivity to high temperature is universally conserved among fish, we analyzed
Similar to
We performed qRT-PCR analysis of a group of
Multiple reports in mammals showed that germ cells are the main target of detrimental high temperatures in testes##REF##12943702##32##. Our study using
TRP channels were identified as sensor molecules for thermal reception, through which cellular responses to temperature changes are regulated##REF##33227348##33##. Each TRP channel has a unique thermoreceptive range. Although TRP proteins are expressed also in the testes##UREF##5##34##,
In
We propose that the Leydig cell-specific temperature-sensing mechanism comprises a system that suppresses fertilization in
Because the meiosis checkpoint controls the differentiation from metaphase to early spermatid, spermatozoa that have matured prior to temperature stimulation cannot be eliminated by the meiosis checkpoint. (Supplementary Figs. ##SUPPL##1##4## and ##SUPPL##1##5##). Indeed, E3 spermatids were abundantly observed in the testis after 1-day incubation at 34 °C (Fig. ##FIG##0##1a## and Supplementary Fig. ##SUPPL##1##3##). Moreover, mature spermatozoa did not lose sperm motility in high temperatures (Supplementary Fig. ##SUPPL##1##10##). Therefore, mature sperm differentiated prior to temperature stimulation are expected to be motile with fertilization capabilities even at 34 °C. However, sperm motility and fertilization rate were significantly reduced by a 1-day exposure to 34 °C (Figs. ##FIG##3##4##a, c and ##FIG##4##5c##). Taken together, we propose that
Neither upregulated expression of
Multiple reports in mammals showed that germ cells are the main target of detrimental high temperatures in testes##REF##12943702##32##. Our study using
TRP channels were identified as sensor molecules for thermal reception, through which cellular responses to temperature changes are regulated##REF##33227348##33##. Each TRP channel has a unique thermoreceptive range. Although TRP proteins are expressed also in the testes##UREF##5##34##,
In
We propose that the Leydig cell-specific temperature-sensing mechanism comprises a system that suppresses fertilization in
Because the meiosis checkpoint controls the differentiation from metaphase to early spermatid, spermatozoa that have matured prior to temperature stimulation cannot be eliminated by the meiosis checkpoint. (Supplementary Figs. ##SUPPL##1##4## and ##SUPPL##1##5##). Indeed, E3 spermatids were abundantly observed in the testis after 1-day incubation at 34 °C (Fig. ##FIG##0##1a## and Supplementary Fig. ##SUPPL##1##3##). Moreover, mature spermatozoa did not lose sperm motility in high temperatures (Supplementary Fig. ##SUPPL##1##10##). Therefore, mature sperm differentiated prior to temperature stimulation are expected to be motile with fertilization capabilities even at 34 °C. However, sperm motility and fertilization rate were significantly reduced by a 1-day exposure to 34 °C (Figs. ##FIG##3##4##a, c and ##FIG##4##5c##). Taken together, we propose that
Neither upregulated expression of
Exposure of testes to high-temperature environment results in defective spermatogenesis.
Under high temperature in zebrafish, the temperature sensitive ion channel
Exposure of testes to high-temperature environment results in defective spermatogenesis.
Under high temperature in zebrafish, the temperature sensitive ion channel
\n\n\n\n\n\n\n\n\n\n
"],"string":"[\n \"\\n\\n\\n\\n\\n\\n\\n\\n\\n\\n
\"\n]"},"back":{"kind":"list like","value":["The online version contains supplementary material available at 10.1038/s42003-023-05740-y.
","We are grateful to Mrs. Natsuko Okuda at OMPU for her excellent care of
Y.Y. carried out the experiment and analyzed the data. D.H. carried out the LC–MS/MS analysis. Y.Y. designed the study. F.O. supervised the study. Y.Y. and F.O. wrote the paper.
","The video data are available from the authors upon request. The source data behind the figures can be found in Supplementary Data ##SUPPL##7##1##.
","The authors declare no competing interests.
"],"string":"[\n \"The online version contains supplementary material available at 10.1038/s42003-023-05740-y.
\",\n \"We are grateful to Mrs. Natsuko Okuda at OMPU for her excellent care of
Y.Y. carried out the experiment and analyzed the data. D.H. carried out the LC–MS/MS analysis. Y.Y. designed the study. F.O. supervised the study. Y.Y. and F.O. wrote the paper.
\",\n \"The video data are available from the authors upon request. The source data behind the figures can be found in Supplementary Data ##SUPPL##7##1##.
\",\n \"The authors declare no competing interests.
\"\n]"},"figure":{"kind":"list like","value":["Peer Review File
Supplementary Information
Description of Additional Supplementary Files
Supplementary Movie 1
Supplementary Movie 2
Supplementary Movie 3
Supplementary Movie 4
Supplementary Data 1
Reporting Summary
Peer Review File
Supplementary Information
Description of Additional Supplementary Files
Supplementary Movie 1
Supplementary Movie 2
Supplementary Movie 3
Supplementary Movie 4
Supplementary Data 1
Reporting Summary
A considerable body of evidence suggests that disparities in SES—reflecting education, income, occupational prestige, and subjective status—play a critical role in shaping the health trajectories of people, with lower SES associated with elevated morbidity and mortality rates##UREF##0##1##–##REF##7604919##6##. Many of these diseases—including, for example, asthma##REF##36280330##7##, atopic dermatitis##REF##30597208##8##, food allergies##REF##35476967##9##, systemic lupus erythematosus##REF##36987604##10##, and periodontal disease##REF##31860530##11##—vary widely in their pathologies but share a common etiological pathway involving immune dysregulation, and they are more common in lower socioeconomic strata than among people with higher SES##REF##31986347##12##. Three strands of evidence also document associations between SES and biomarkers of the immune system. First, many studies report associations between childhood SES and pro-inflammatory markers in circulating peripheral blood (such as interleukin-6 and C-reactive protein (CRP)##REF##32919037##13##) that, if chronically activated, presage a wide-range of diseases including, for example, diabetes type 2, some cancers, and cardiovascular disease. Second, studies have also examined white blood cell composition, finding that low SES is associated with increased development and circulation of pro-inflammatory immune cells (monocytes and neutrophils##REF##24062448##14##), whereas parental education is positively associated with the proportion of lymphocytes and negatively associated with the proportion of neutrophils and, among older adults, that SES is related to shifts in cell composition indicative of immunosenescence##REF##35696572##15##–##REF##31872259##18##. And finally, a limited number of studies have examined functional assays of immune response, sometimes ex-vivo, and show that, once again, childhood socioeconomic status is a risk factor for immune dysregulation, possibly more so among boys##UREF##2##19##–##REF##19617551##22##. Nevertheless, despite the abundance of evidence connecting socioeconomic inequalities to immune-related diseases and biomarkers, the molecular etiology of SES-mediated immune alterations remains less explored.
","A growing number of studies have examined SES and transcriptional patterns indicative of immune functioning. Research consistently shows people from low SES backgrounds have greater proinflammatory activity##REF##24062448##14##,##REF##19617551##22##–##REF##25658624##26##. Additionally, SES is associated with the expression of genes regulated by the glucocorticoid receptor and interferon response factors, suggesting a suppression of adaptive immunity and innate antiviral immunity##REF##19617551##22##,##REF##28911009##27##. This signature pattern—involving the upregulation of proinflammatory genes and the downregulation of type I interferon innate antiviral response genes among the lower strata of status, called the “Conserved Transcriptional Response to Adversity” (CTRA)—has been observed in numerous populations with a range of research designs. Essentially, SES is associated with CTRA activation, which in turn is associated with the molecular underpinnings of immune-related and inflammatory diseases##REF##19617551##22##,##REF##26166230##28##–##UREF##3##30##.
","The current study seeks to expand on these findings by providing a broader mapping of associations between SES and the molecular signaling pathways that regulate immunity. Understanding the impact of socioeconomic status on immune gene expression requires a systems-oriented approach that extends beyond the functional examination of individually differentially expressed genes and includes the network of upstream regulators. Furthermore, traditional transcription factor binding motif enrichment analysis suffers from the lack of tissue specificity. We aim to address these drawbacks in this paper by detailing upstream tissue-specific transcriptional factors and protein-protein interactors as a networked system, along with differentially regulated genes, that responds to SES. Such an approach offers a comprehensive understanding of how SES is associated with the molecular mechanisms that drive biological processes such as gene expression, cell signaling, and cell fate, which ultimately lead to disease in individuals of lower SES##REF##21414488##31##,##REF##29425488##32##. Significantly, we leverage the comprehensive resource of human tissue-specific gene regulatory network of Marbach et al.##UREF##4##33## to isolate transcription factors that could play a pivotal role in modulating the expression of SES—differentially expressed genes in whole blood. Additionally, by incorporating direct protein-protein interactors, we create a broad, inclusive and holistic set of genes and proteins that act as a networked system in disrupting essential biological processes. Our approach reveals the decisive role of transcription factors in driving SES—associated dysregulation that previously would be attributed to SES—differentially expressed genes.
","Emerging evidence points to the intricate interplay between low socioeconomic position and high body mass index (BMI)##REF##34091232##34##,##REF##33160231##35##. Recent meta-analyses have consistently linked lowered socioeconomic status and elevated inflammatory biomarkers largely via obesity##REF##31628416##36##,##UREF##5##37##. Although BMI is an imperfect indicator of obesity as it does not distinguish fat from fat-free mass, Liu et al.##REF##28490476##38## observed a likely strong mediation of BMI in the negative relationship between childhood SES and adulthood inflammatory marker C reactive protein (CRP). Additionally, previous systematic reviews have shown that improved indicators of fat and obesity follow a similar pattern of health disparity to those seen with BMI##UREF##5##37##. Thus, we examine the potential mediation of BMI, as an indicator of obesity, along with other common social and behavioral mediators of SES, and the entire system of differentially expressed genes, upstream transcription factors and protein-protein interactors that drive immune dysregulation as a result of lowered SES.
","We focus on American adults in their late 1930s, who are ostensibly healthy but nevertheless at-risk for later health challenges. We leverage the mRNA data from 4543 early adults participating in the National Longitudinal Study of Adolescent Health (Add Health)##REF##31257425##39##. First, we identify cell functional pathways and their directionality in SES-related dysregulation of the immune system. To this end, we capitalize on publicly available pathway ontologies to functionally annotate genes that show changes in expression and that cluster together. Second, we identify upstream modulators and regulators of the differentially expressed genes to provide a systems perspective on SES and immunity. Such a view also isolates potential targets for remediation. Finally, we consider the behavioral and health-related factors that may explain associations between SES and the immune cell transcriptome. Results reveal that SES is associated with widespread dysregulation of immunity involving intricately interrelated differentially expressed genes, transcription factors, and protein-protein regulators. Furthermore, BMI is a likely, potent mechanism driving these patterns.
"],"string":"[\n \"A considerable body of evidence suggests that disparities in SES—reflecting education, income, occupational prestige, and subjective status—play a critical role in shaping the health trajectories of people, with lower SES associated with elevated morbidity and mortality rates##UREF##0##1##–##REF##7604919##6##. Many of these diseases—including, for example, asthma##REF##36280330##7##, atopic dermatitis##REF##30597208##8##, food allergies##REF##35476967##9##, systemic lupus erythematosus##REF##36987604##10##, and periodontal disease##REF##31860530##11##—vary widely in their pathologies but share a common etiological pathway involving immune dysregulation, and they are more common in lower socioeconomic strata than among people with higher SES##REF##31986347##12##. Three strands of evidence also document associations between SES and biomarkers of the immune system. First, many studies report associations between childhood SES and pro-inflammatory markers in circulating peripheral blood (such as interleukin-6 and C-reactive protein (CRP)##REF##32919037##13##) that, if chronically activated, presage a wide-range of diseases including, for example, diabetes type 2, some cancers, and cardiovascular disease. Second, studies have also examined white blood cell composition, finding that low SES is associated with increased development and circulation of pro-inflammatory immune cells (monocytes and neutrophils##REF##24062448##14##), whereas parental education is positively associated with the proportion of lymphocytes and negatively associated with the proportion of neutrophils and, among older adults, that SES is related to shifts in cell composition indicative of immunosenescence##REF##35696572##15##–##REF##31872259##18##. And finally, a limited number of studies have examined functional assays of immune response, sometimes ex-vivo, and show that, once again, childhood socioeconomic status is a risk factor for immune dysregulation, possibly more so among boys##UREF##2##19##–##REF##19617551##22##. Nevertheless, despite the abundance of evidence connecting socioeconomic inequalities to immune-related diseases and biomarkers, the molecular etiology of SES-mediated immune alterations remains less explored.
\",\n \"A growing number of studies have examined SES and transcriptional patterns indicative of immune functioning. Research consistently shows people from low SES backgrounds have greater proinflammatory activity##REF##24062448##14##,##REF##19617551##22##–##REF##25658624##26##. Additionally, SES is associated with the expression of genes regulated by the glucocorticoid receptor and interferon response factors, suggesting a suppression of adaptive immunity and innate antiviral immunity##REF##19617551##22##,##REF##28911009##27##. This signature pattern—involving the upregulation of proinflammatory genes and the downregulation of type I interferon innate antiviral response genes among the lower strata of status, called the “Conserved Transcriptional Response to Adversity” (CTRA)—has been observed in numerous populations with a range of research designs. Essentially, SES is associated with CTRA activation, which in turn is associated with the molecular underpinnings of immune-related and inflammatory diseases##REF##19617551##22##,##REF##26166230##28##–##UREF##3##30##.
\",\n \"The current study seeks to expand on these findings by providing a broader mapping of associations between SES and the molecular signaling pathways that regulate immunity. Understanding the impact of socioeconomic status on immune gene expression requires a systems-oriented approach that extends beyond the functional examination of individually differentially expressed genes and includes the network of upstream regulators. Furthermore, traditional transcription factor binding motif enrichment analysis suffers from the lack of tissue specificity. We aim to address these drawbacks in this paper by detailing upstream tissue-specific transcriptional factors and protein-protein interactors as a networked system, along with differentially regulated genes, that responds to SES. Such an approach offers a comprehensive understanding of how SES is associated with the molecular mechanisms that drive biological processes such as gene expression, cell signaling, and cell fate, which ultimately lead to disease in individuals of lower SES##REF##21414488##31##,##REF##29425488##32##. Significantly, we leverage the comprehensive resource of human tissue-specific gene regulatory network of Marbach et al.##UREF##4##33## to isolate transcription factors that could play a pivotal role in modulating the expression of SES—differentially expressed genes in whole blood. Additionally, by incorporating direct protein-protein interactors, we create a broad, inclusive and holistic set of genes and proteins that act as a networked system in disrupting essential biological processes. Our approach reveals the decisive role of transcription factors in driving SES—associated dysregulation that previously would be attributed to SES—differentially expressed genes.
\",\n \"Emerging evidence points to the intricate interplay between low socioeconomic position and high body mass index (BMI)##REF##34091232##34##,##REF##33160231##35##. Recent meta-analyses have consistently linked lowered socioeconomic status and elevated inflammatory biomarkers largely via obesity##REF##31628416##36##,##UREF##5##37##. Although BMI is an imperfect indicator of obesity as it does not distinguish fat from fat-free mass, Liu et al.##REF##28490476##38## observed a likely strong mediation of BMI in the negative relationship between childhood SES and adulthood inflammatory marker C reactive protein (CRP). Additionally, previous systematic reviews have shown that improved indicators of fat and obesity follow a similar pattern of health disparity to those seen with BMI##UREF##5##37##. Thus, we examine the potential mediation of BMI, as an indicator of obesity, along with other common social and behavioral mediators of SES, and the entire system of differentially expressed genes, upstream transcription factors and protein-protein interactors that drive immune dysregulation as a result of lowered SES.
\",\n \"We focus on American adults in their late 1930s, who are ostensibly healthy but nevertheless at-risk for later health challenges. We leverage the mRNA data from 4543 early adults participating in the National Longitudinal Study of Adolescent Health (Add Health)##REF##31257425##39##. First, we identify cell functional pathways and their directionality in SES-related dysregulation of the immune system. To this end, we capitalize on publicly available pathway ontologies to functionally annotate genes that show changes in expression and that cluster together. Second, we identify upstream modulators and regulators of the differentially expressed genes to provide a systems perspective on SES and immunity. Such a view also isolates potential targets for remediation. Finally, we consider the behavioral and health-related factors that may explain associations between SES and the immune cell transcriptome. Results reveal that SES is associated with widespread dysregulation of immunity involving intricately interrelated differentially expressed genes, transcription factors, and protein-protein regulators. Furthermore, BMI is a likely, potent mechanism driving these patterns.
\"\n]"},"methods":{"kind":"list like","value":["The National Longitudinal Study of Adolescent to Adult Health (Add Health) is a representative study of adolescents in the Unites States who were followed into adulthood over five waves of data collection##REF##31257425##39##. Study participants provided informed written consent with respect to all aspects of the Add Health study in accordance with the University of North Carolina School of Public Health Institution Review Board (IRB). Transcriptomic profiles of the consenting participants were collected during Wave V of the Add Health Study (2016–2017) via an intravenous blood draw (age of subjects range from 33 to 43 years). The access to restricted use Add Health transcriptomic data was obtained by completing a contractual and data use agreement. Additional detailed information on the study design, interview procedures, consent procedures, demographic assessments, collection, sequencing and quality control of the blood sample, and derivation of the analytical samples is reported in ##SUPPL##3##Supplementary Methods## and in previous studies##REF##32041883##40##–##REF##36252037##42##. Furthermore, the data analysis and all methods presented in this work were carried out in accordance with the relevant ethical guidelines and regulations. We draw on the mRNA-seq data of 4015 subjects with complete information on the models’ variables. Socioeconomic status composite scores were calculated using the sum of standardized indicators of education, income, occupation, and subjective socioeconomic status of the early adult subjects##REF##36252037##42##–##UREF##7##44##.
","Genes with low counts were excluded from the analysis (see ##SUPPL##3##Supplementary Methods##). After normalizing the raw mRNA-seq counts using a weighted trimmed mean of log expression ratios (TMM normalization)##UREF##8##45## using the
Our overall analytic strategy is to (1) estimate clusters of genes across the whole genome and, within these clusters, identify genes that differentially expressed (DE) by SES (hereafter, SES–DEG); (2) characterize the biological function of these DE genes and gene clusters that are likely to have DE genes; (3) identify transcription factors and their protein neighbors that are associated with these DE genes and act as a networked system; (4) determine the relative functional relevance of SES–DEG and upstream regulators in manifesting immune dysregulation, and, finally, (5) identify behavioral mediators that may account for associations between SES and DE genes and their upstream regulators.
","Processed gene expression data from 14,251 transcripts in 4015 individuals were subject to unsupervised clustering using Weighted Gene Coexpression Network Analysis (WGCNA)##UREF##10##50##. We identified a total of 19 clusters and the number of genes in each cluster and the clusters’ overlaps with the SES–DEG are shown in Supplementary Fig. ##SUPPL##3##S1##. To identify the clusters that have a significant relationship to SES, we modelled the cluster eigengenes (a summarized expression vector of each cluster) as a linear function of SES as in the differential expression analysis. Additionally, we performed a Fisher exact test to identify clusters that show an enrichment for SES–DEG. Together, the two tests resulted in clusters that, (1) have a significant cluster-SES relationship and (2) are enriched for SES—up or downregulated genes (see Supplementary Fig. ##SUPPL##3##S2##). Four clusters (Cluster 7, 11, 13 and 17) had eigengenes that are significantly differentially expressed by SES. Of the 4 clusters, Cluster 11 showed an overrepresentation for SES—downregulated genes, while Clusters 7, 13 and 17 showed overrepresentation of by SES—upregulated genes. In this context, upregulation refers to a positive association between SES and mRNA abundance levels.
","Functional enrichment analysis for the SES–DEG (see Supplementary Fig. ##SUPPL##3##S3## and Supplementary Dataset ##SUPPL##0##S1##) and WGCNA identified cluster genes (see Supplementary Fig. ##SUPPL##3##S4## and Supplementary Dataset ##SUPPL##1##S2##) was performed using R Bioconductor package
Functional enrichment analysis of the SES–DEG and the upstream regulators was performed using the ClueGO##REF##19237447##53## plugin in Cytoscape##REF##14597658##54##. This plugin allows for the combined analysis of multiple gene lists using a preselected ontology. We analyzed the SES–DEG (up- and downregulated gene lists) along with their upstream regulators (transcription factors and protein neighbors) with the Reactome ontology.
","Upstream regulators of the SES–DEG (Set A; see Supplementary Fig. ##SUPPL##3##S5##) were categorized into (1) transcription factors that are themselves differentially expressed (Set B; see Supplementary Fig. ##SUPPL##3##S5##), (2) protein neighbors of the differentially expressed transcription factors (Set C; see Supplementary Fig. ##SUPPL##3##S5##), and (3) transcription factors that putatively modulate the expression of differentially expressed genes (Set D; see Supplementary Fig. ##SUPPL##3##S5##). Marbach et al.##UREF##4##33## constructed tissue-specific regulatory networks that linked transcription factors and genes with a score based on a curated collection of sequence binding motifs. Those transcription factors that had a medium or greater confidence (> 0.4) of modulating the expression of the differentially expressed genes in blood tissue were included in the set of upstream regulators (Sets B and D). A total of 643 transcription factors were identified in the blood tissue-specific gene regulatory network. 304 transcription factors had gene interactions with at least a medium confidence score. Protein neighbors of differentially regulated transcription factors were obtained using the STRING database##REF##23203871##55##. Each protein-protein interaction (PPI) in STRING is annotated with a score that indicates the confidence of the interaction. Only neighbors with scores of at least high confidence (> 0.7) were included in the set of upstream regulators (Set C). Thus, Set A represents the DE genes and Sets B, C and D together constitute their upstream regulators. The 304 tissue-specific transcription factors had interactions with 8543 unique protein-protein neighbors. 1750 neighbors exceeded the confidence threshold for PPI.
","We examined behavioral and psychobiological process that might mediate associations between Wave V early adulthood SES and the expression of the genes and upstream regulators using a counterfactual mediational framework##REF##26917999##56##. The mediators included Body Mass Index (BMI), perceived stress (based on Cohen’s Perceived Stress Scale##REF##25031113##57##), current self-reported smoking status, consumption of alcoholic drinks (days drank over past 30 days; categorized as 0 drinks, 1–2 drinks, 3–5 drinks, and more than 5 drinks per occasion), financial stress (self-reported difficulty in paying bills), and access to health insurance. We also compared the mediation of BMI with the mediation observed for waist circumference, which has been reported to be a more accurate measure of fatness##REF##18166236##58##.
","We quantified the statistical significance of the observed results by performing randomization tests based on 1000 randomly generated sets of differentially expressed genes. Random samples were drawn from the entire genome to obtain a set of genes equal in number to Set A (see Supplementary Fig. ##SUPPL##3##S5##). Sets B, C, and D were derived from every randomly generated Set A using the same procedure used with the SES–DEG. We then computed the significance (empirical
The National Longitudinal Study of Adolescent to Adult Health (Add Health) is a representative study of adolescents in the Unites States who were followed into adulthood over five waves of data collection##REF##31257425##39##. Study participants provided informed written consent with respect to all aspects of the Add Health study in accordance with the University of North Carolina School of Public Health Institution Review Board (IRB). Transcriptomic profiles of the consenting participants were collected during Wave V of the Add Health Study (2016–2017) via an intravenous blood draw (age of subjects range from 33 to 43 years). The access to restricted use Add Health transcriptomic data was obtained by completing a contractual and data use agreement. Additional detailed information on the study design, interview procedures, consent procedures, demographic assessments, collection, sequencing and quality control of the blood sample, and derivation of the analytical samples is reported in ##SUPPL##3##Supplementary Methods## and in previous studies##REF##32041883##40##–##REF##36252037##42##. Furthermore, the data analysis and all methods presented in this work were carried out in accordance with the relevant ethical guidelines and regulations. We draw on the mRNA-seq data of 4015 subjects with complete information on the models’ variables. Socioeconomic status composite scores were calculated using the sum of standardized indicators of education, income, occupation, and subjective socioeconomic status of the early adult subjects##REF##36252037##42##–##UREF##7##44##.
\",\n \"Genes with low counts were excluded from the analysis (see ##SUPPL##3##Supplementary Methods##). After normalizing the raw mRNA-seq counts using a weighted trimmed mean of log expression ratios (TMM normalization)##UREF##8##45## using the
Our overall analytic strategy is to (1) estimate clusters of genes across the whole genome and, within these clusters, identify genes that differentially expressed (DE) by SES (hereafter, SES–DEG); (2) characterize the biological function of these DE genes and gene clusters that are likely to have DE genes; (3) identify transcription factors and their protein neighbors that are associated with these DE genes and act as a networked system; (4) determine the relative functional relevance of SES–DEG and upstream regulators in manifesting immune dysregulation, and, finally, (5) identify behavioral mediators that may account for associations between SES and DE genes and their upstream regulators.
\",\n \"Processed gene expression data from 14,251 transcripts in 4015 individuals were subject to unsupervised clustering using Weighted Gene Coexpression Network Analysis (WGCNA)##UREF##10##50##. We identified a total of 19 clusters and the number of genes in each cluster and the clusters’ overlaps with the SES–DEG are shown in Supplementary Fig. ##SUPPL##3##S1##. To identify the clusters that have a significant relationship to SES, we modelled the cluster eigengenes (a summarized expression vector of each cluster) as a linear function of SES as in the differential expression analysis. Additionally, we performed a Fisher exact test to identify clusters that show an enrichment for SES–DEG. Together, the two tests resulted in clusters that, (1) have a significant cluster-SES relationship and (2) are enriched for SES—up or downregulated genes (see Supplementary Fig. ##SUPPL##3##S2##). Four clusters (Cluster 7, 11, 13 and 17) had eigengenes that are significantly differentially expressed by SES. Of the 4 clusters, Cluster 11 showed an overrepresentation for SES—downregulated genes, while Clusters 7, 13 and 17 showed overrepresentation of by SES—upregulated genes. In this context, upregulation refers to a positive association between SES and mRNA abundance levels.
\",\n \"Functional enrichment analysis for the SES–DEG (see Supplementary Fig. ##SUPPL##3##S3## and Supplementary Dataset ##SUPPL##0##S1##) and WGCNA identified cluster genes (see Supplementary Fig. ##SUPPL##3##S4## and Supplementary Dataset ##SUPPL##1##S2##) was performed using R Bioconductor package
Functional enrichment analysis of the SES–DEG and the upstream regulators was performed using the ClueGO##REF##19237447##53## plugin in Cytoscape##REF##14597658##54##. This plugin allows for the combined analysis of multiple gene lists using a preselected ontology. We analyzed the SES–DEG (up- and downregulated gene lists) along with their upstream regulators (transcription factors and protein neighbors) with the Reactome ontology.
\",\n \"Upstream regulators of the SES–DEG (Set A; see Supplementary Fig. ##SUPPL##3##S5##) were categorized into (1) transcription factors that are themselves differentially expressed (Set B; see Supplementary Fig. ##SUPPL##3##S5##), (2) protein neighbors of the differentially expressed transcription factors (Set C; see Supplementary Fig. ##SUPPL##3##S5##), and (3) transcription factors that putatively modulate the expression of differentially expressed genes (Set D; see Supplementary Fig. ##SUPPL##3##S5##). Marbach et al.##UREF##4##33## constructed tissue-specific regulatory networks that linked transcription factors and genes with a score based on a curated collection of sequence binding motifs. Those transcription factors that had a medium or greater confidence (> 0.4) of modulating the expression of the differentially expressed genes in blood tissue were included in the set of upstream regulators (Sets B and D). A total of 643 transcription factors were identified in the blood tissue-specific gene regulatory network. 304 transcription factors had gene interactions with at least a medium confidence score. Protein neighbors of differentially regulated transcription factors were obtained using the STRING database##REF##23203871##55##. Each protein-protein interaction (PPI) in STRING is annotated with a score that indicates the confidence of the interaction. Only neighbors with scores of at least high confidence (> 0.7) were included in the set of upstream regulators (Set C). Thus, Set A represents the DE genes and Sets B, C and D together constitute their upstream regulators. The 304 tissue-specific transcription factors had interactions with 8543 unique protein-protein neighbors. 1750 neighbors exceeded the confidence threshold for PPI.
\",\n \"We examined behavioral and psychobiological process that might mediate associations between Wave V early adulthood SES and the expression of the genes and upstream regulators using a counterfactual mediational framework##REF##26917999##56##. The mediators included Body Mass Index (BMI), perceived stress (based on Cohen’s Perceived Stress Scale##REF##25031113##57##), current self-reported smoking status, consumption of alcoholic drinks (days drank over past 30 days; categorized as 0 drinks, 1–2 drinks, 3–5 drinks, and more than 5 drinks per occasion), financial stress (self-reported difficulty in paying bills), and access to health insurance. We also compared the mediation of BMI with the mediation observed for waist circumference, which has been reported to be a more accurate measure of fatness##REF##18166236##58##.
\",\n \"We quantified the statistical significance of the observed results by performing randomization tests based on 1000 randomly generated sets of differentially expressed genes. Random samples were drawn from the entire genome to obtain a set of genes equal in number to Set A (see Supplementary Fig. ##SUPPL##3##S5##). Sets B, C, and D were derived from every randomly generated Set A using the same procedure used with the SES–DEG. We then computed the significance (empirical
We performed a differential gene expression analysis followed by an enrichment analysis of the resulting SES–DEG (see Supplementary Dataset ##SUPPL##1##S2## and Supplementary Fig. ##SUPPL##3##S3##). Upregulated genes indicate a significant association between high SES and high expression (i.e., a positive association). Functional enrichment of the SES–DEG (423 upregulated genes and 389 downregulated genes) showed a majority upregulated for pathways involving metabolism, signal transduction and cellular response to stress by a core of ribosomal and translational genes. Interestingly, these cytosolic ribosomal genes (
An inspection of enriched pathways reveals that SES-upregulated pathways include interferon innate immune response and neutrophil degranulation (see Fig. ##FIG##1##2##). Curiously, type II interferon (IFN-γ) signaling is also upregulated. Despite the lack of direct evidence for their involvement, the genes that are tied to the upregulation of type I and type II interferon signaling do share
The biological pathways and molecular mechanisms associated with socioeconomic disparity were also examined via an analysis of upstream regulators (transcriptional factors and protein partners) of the DE genes. A combined functional enrichment analysis of the resulting upstream regulators (239 upstream regulators of SES—upregulated genes and 87 upstream regulators of SES—downregulated genes) and SES–DEG (see Fig. ##FIG##2##3## and Supplementary Dataset ##SUPPL##2##S3##) indicates a significant role of the upstream regulators in structuring the overrepresented pathways in the immune system. The upstream regulators include tissue-specific transcription factors that are (1) differentially expressed (Set B, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##), (2) potentially regulating the expression of a differentially expressed gene (Set D, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##), and (3) PPI neighbors of differentially expressed transcription factors (Set C, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##). Figure ##FIG##2##3## indicates that the immune pathways that are enriched have a larger proportion of upstream targets. Importantly, many of these pathways were also enriched by SES–DEG signifying that the patterns of immune dysregulation observed in Fig. ##FIG##2##3## do not reflect the inclusion of upstream regulators per se, but rather reflect the significant mechanistic role played by these transcription factors and protein partners. Furthermore, the immune dysregulation observed in functional enrichment analysis of the upstream targets without the inclusion of SES–DEG (see Supplementary Fig. ##SUPPL##3##S6##) mirror those enriched by SES–DEG alone and SES–DEG with upstream targets, suggesting that SES–DEG are not wholly responsible for SES-associated immune dysregulation.
","Figure ##FIG##3##4## shows the upstream regulators along with the SES–DEG represented as layers (4 in total, where the innermost circle of genes and upstream regulators is labelled “Layer 1”, sequentially to the outermost group of genes and regulators labelled “Layer 4”) based on their interaction scores derived from the STRING database and the number of times each gene or upstream regulator is involved in functional immune pathways that are enriched in Fig. ##FIG##2##3##. The genes that are responsible for the enrichment of each immune pathway were identified. The innermost layer in Fig. ##FIG##3##4## depicts genes and upstream regulators that are involved in more than ten functional pathways, whereas the outermost layer consists of genes and upstream regulators that are only involved in one enriched biological process. The most essential regulators involved in the dysregulation of the immune system related to SES disparities thus occupy the center of the diagram. Significantly, variations in early adult SES are prominently linked to alterations in cytokine signaling in the immune system involving interleukin and interferon gamma signaling, Toll-like receptor signaling cascade, and TNF pathways (Fig. ##FIG##2##3## and innermost layer in Fig. ##FIG##3##4##).
","Proteins most deeply linked to SES inequalities invariably revolve around the cyclic 3ʹ–5ʹ adenosine monophosphate response element-binding protein (CREB) and NF-κB pathway signaling. Variations in the activity of CREB and NF-κB selectively upregulate the transcription of interferon response factor family while simultaneously inhibiting the activity of proinflammatory interleukins in subjects with high SES. Genes and transcription factors (via the upstream analysis) that are central to the functional response to SES disparities in functional gene regulation (shown in Fig. ##FIG##2##3##) are also shown in Fig. ##FIG##3##4##. Molecules are placed in layers depending on their contribution (instances of enrichment of a pathway) to the functional immune enrichment. The inner most layer consists of transcriptional factors such as
Figure ##FIG##4##5## reports the median percentage mediated ratio for key SES-related social/behavioral processes in every layer of SES-related gene regulation. Intriguingly, the inner most group of genes that are most centrally implicated in SES-associated dysregulation, are also mediated the least (lowest median percentage mediated ratio) by every behavioral risk factor. However, this could reflect the fact that the inner most group of genes are not themselves differentially expressed despite potentially inducing larger mediated changes in the outer layers of genes. BMI presented the strongest explanation of the association between the transcriptional response of the gene groups and SES, followed by smoking tobacco (also see Supplementary Figs. ##SUPPL##3##S7## and ##SUPPL##3##S8##). No significant mediation was observed for financial stress or access to health insurance. Furthermore, we observed no significant differences between BMI and waist circumference in mediating the SES—associated immune transcriptome (see Supplementary Fig. ##SUPPL##3##S9##).
"],"string":"[\n \"We performed a differential gene expression analysis followed by an enrichment analysis of the resulting SES–DEG (see Supplementary Dataset ##SUPPL##1##S2## and Supplementary Fig. ##SUPPL##3##S3##). Upregulated genes indicate a significant association between high SES and high expression (i.e., a positive association). Functional enrichment of the SES–DEG (423 upregulated genes and 389 downregulated genes) showed a majority upregulated for pathways involving metabolism, signal transduction and cellular response to stress by a core of ribosomal and translational genes. Interestingly, these cytosolic ribosomal genes (
An inspection of enriched pathways reveals that SES-upregulated pathways include interferon innate immune response and neutrophil degranulation (see Fig. ##FIG##1##2##). Curiously, type II interferon (IFN-γ) signaling is also upregulated. Despite the lack of direct evidence for their involvement, the genes that are tied to the upregulation of type I and type II interferon signaling do share
The biological pathways and molecular mechanisms associated with socioeconomic disparity were also examined via an analysis of upstream regulators (transcriptional factors and protein partners) of the DE genes. A combined functional enrichment analysis of the resulting upstream regulators (239 upstream regulators of SES—upregulated genes and 87 upstream regulators of SES—downregulated genes) and SES–DEG (see Fig. ##FIG##2##3## and Supplementary Dataset ##SUPPL##2##S3##) indicates a significant role of the upstream regulators in structuring the overrepresented pathways in the immune system. The upstream regulators include tissue-specific transcription factors that are (1) differentially expressed (Set B, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##), (2) potentially regulating the expression of a differentially expressed gene (Set D, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##), and (3) PPI neighbors of differentially expressed transcription factors (Set C, see Supplementary Fig. ##SUPPL##3##S5## and Supplementary Table ##SUPPL##3##S1##). Figure ##FIG##2##3## indicates that the immune pathways that are enriched have a larger proportion of upstream targets. Importantly, many of these pathways were also enriched by SES–DEG signifying that the patterns of immune dysregulation observed in Fig. ##FIG##2##3## do not reflect the inclusion of upstream regulators per se, but rather reflect the significant mechanistic role played by these transcription factors and protein partners. Furthermore, the immune dysregulation observed in functional enrichment analysis of the upstream targets without the inclusion of SES–DEG (see Supplementary Fig. ##SUPPL##3##S6##) mirror those enriched by SES–DEG alone and SES–DEG with upstream targets, suggesting that SES–DEG are not wholly responsible for SES-associated immune dysregulation.
\",\n \"Figure ##FIG##3##4## shows the upstream regulators along with the SES–DEG represented as layers (4 in total, where the innermost circle of genes and upstream regulators is labelled “Layer 1”, sequentially to the outermost group of genes and regulators labelled “Layer 4”) based on their interaction scores derived from the STRING database and the number of times each gene or upstream regulator is involved in functional immune pathways that are enriched in Fig. ##FIG##2##3##. The genes that are responsible for the enrichment of each immune pathway were identified. The innermost layer in Fig. ##FIG##3##4## depicts genes and upstream regulators that are involved in more than ten functional pathways, whereas the outermost layer consists of genes and upstream regulators that are only involved in one enriched biological process. The most essential regulators involved in the dysregulation of the immune system related to SES disparities thus occupy the center of the diagram. Significantly, variations in early adult SES are prominently linked to alterations in cytokine signaling in the immune system involving interleukin and interferon gamma signaling, Toll-like receptor signaling cascade, and TNF pathways (Fig. ##FIG##2##3## and innermost layer in Fig. ##FIG##3##4##).
\",\n \"Proteins most deeply linked to SES inequalities invariably revolve around the cyclic 3ʹ–5ʹ adenosine monophosphate response element-binding protein (CREB) and NF-κB pathway signaling. Variations in the activity of CREB and NF-κB selectively upregulate the transcription of interferon response factor family while simultaneously inhibiting the activity of proinflammatory interleukins in subjects with high SES. Genes and transcription factors (via the upstream analysis) that are central to the functional response to SES disparities in functional gene regulation (shown in Fig. ##FIG##2##3##) are also shown in Fig. ##FIG##3##4##. Molecules are placed in layers depending on their contribution (instances of enrichment of a pathway) to the functional immune enrichment. The inner most layer consists of transcriptional factors such as
Figure ##FIG##4##5## reports the median percentage mediated ratio for key SES-related social/behavioral processes in every layer of SES-related gene regulation. Intriguingly, the inner most group of genes that are most centrally implicated in SES-associated dysregulation, are also mediated the least (lowest median percentage mediated ratio) by every behavioral risk factor. However, this could reflect the fact that the inner most group of genes are not themselves differentially expressed despite potentially inducing larger mediated changes in the outer layers of genes. BMI presented the strongest explanation of the association between the transcriptional response of the gene groups and SES, followed by smoking tobacco (also see Supplementary Figs. ##SUPPL##3##S7## and ##SUPPL##3##S8##). No significant mediation was observed for financial stress or access to health insurance. Furthermore, we observed no significant differences between BMI and waist circumference in mediating the SES—associated immune transcriptome (see Supplementary Fig. ##SUPPL##3##S9##).
\"\n]"},"discussion":{"kind":"list like","value":["The present analyses expand the scope of prior studies of SES-related alteration in transcriptomic profiles of human immune response genes by identifying new genomic functional impacts (e.g., ribosomal biology) and new features of the gene regulatory architecture of SES (e.g.,
The analyses extend previous research by mapping networks of upstream blood-specific transcriptional factors and protein interactions that could play a vital role in structuring the observed transcriptional landscape. The complexity of the impact of socioeconomic inequalities on the immune system and its association with diseases with widely varying pathologies warrants a systems-oriented approach to comprehensively analyze the SES-related perturbations in the immune transcriptome. To our knowledge, most prior research on SES focuses solely on transcriptomic alterations with a particular focus on pro-inflammatory action##UREF##3##30##. Here, we include the upstream regulators to depict an enhanced view of dysregulation with SES, shedding light on a tightly knit group of transcription factors that play a central role in modulating the transcriptomic alterations.
","Our findings map a central network of upstream regulators that vary as a function of early adult SES (central positions in Fig. ##FIG##3##4##). Given the lack of change in the expression of the genes that encode these transcriptional factors, receptor-mediated post-transcriptional modification of these transcriptional factors (e.g., receptor-mediated phosphorylation of
Despite the congruence between our results and the CTRA model in terms of dysregulated pathways, the findings show that SES disparities in early adulthood, interestingly, do not alter the same set of genes identified in previous studies of CTRA. For example, the attenuated interferon innate responses in the CTRA model is possible through the direct suppression of
The observed SES-related transcriptional perturbations are associated with both up- and downregulated functional pathways in the immune system. Social stress-induced gene alterations in humans are associated with diseases that include both upregulation of the immune system as well as suppressed immune responsiveness with lowered social status. This seemingly perplexing pattern has been explained by the selective characteristics of the immune transcriptome, i.e., the increase in expression of certain pro-inflammatory genes and the repression of groups of antiviral immune response genes##REF##32041883##40##,##REF##25166010##61##–##REF##23853742##63##. It is, therefore, unsurprising that a similar pattern of dysregulated immune pathways is emergent in the blood transcriptomic landscape of subjects in Add Health with contributions of enrichment from both up- and downregulated gene clusters. Such a finding calls for additional research that moves beyond the initial general finding that stressors upregulate proinflammatory genes and downregulate antiviral genes.
","Lastly, among the common mediating (or possible explanatory) mechanisms studied here, BMI consistently emerged as a plausible mediator of the SES associations with immune cell gene regulation. Smoking also appears to play a significant role in the SES-related transcriptional alterations. These results underscore the importance of gene regulatory network approach in formulating a comprehensive understanding of psychosocial stressors and their impact on biological mechanisms early in life. Studies have already established that changes caused by socioeconomic disparities in early adulthood could have far-reaching implications for chronic conditions in later adulthood. Identification of novel regulators of such perturbations is an important step in formulating a mitigating strategy.
","We used tissue-specific regulatory networks to link transcription factors to differentially expressed genes, and subsequently the STRING database to find protein interaction partners. There were 643 transcription factors identified in the gene regulatory network, with an even a smaller number (304) having a confidence score that is above the threshold used (0.4). High confidence (threshold > 0.7) protein-protein neighbors derived from the whole network of 643 transcription factors numbered 3051 (18,875 total protein-protein neighbors), of which 1750 were pruned for the 304 transcription factors. Given the central role of many of the transcription factors and protein partners, one could argue that the set of upstream regulators found from the SES–DEG could be equal to a set of upstream regulators derived from a random set of genes. To examine this possibility, we performed randomized trials by starting with randomly selected sets of “DE” genes to then derive these upstream regulators of the random sets. We subsequently compared the upstream regulators of each random set of “DE” genes to our observed results (see Supplementary Figs. ##SUPPL##3##S10## and ##SUPPL##3##S11##). While some of the individual transcription factors may not reach statistical significance (Supplementary Fig. ##SUPPL##3##S10##), the entire set of the upstream regulators is highly significant (Supplementary Fig. ##SUPPL##3##S11##).
"],"string":"[\n \"The present analyses expand the scope of prior studies of SES-related alteration in transcriptomic profiles of human immune response genes by identifying new genomic functional impacts (e.g., ribosomal biology) and new features of the gene regulatory architecture of SES (e.g.,
The analyses extend previous research by mapping networks of upstream blood-specific transcriptional factors and protein interactions that could play a vital role in structuring the observed transcriptional landscape. The complexity of the impact of socioeconomic inequalities on the immune system and its association with diseases with widely varying pathologies warrants a systems-oriented approach to comprehensively analyze the SES-related perturbations in the immune transcriptome. To our knowledge, most prior research on SES focuses solely on transcriptomic alterations with a particular focus on pro-inflammatory action##UREF##3##30##. Here, we include the upstream regulators to depict an enhanced view of dysregulation with SES, shedding light on a tightly knit group of transcription factors that play a central role in modulating the transcriptomic alterations.
\",\n \"Our findings map a central network of upstream regulators that vary as a function of early adult SES (central positions in Fig. ##FIG##3##4##). Given the lack of change in the expression of the genes that encode these transcriptional factors, receptor-mediated post-transcriptional modification of these transcriptional factors (e.g., receptor-mediated phosphorylation of
Despite the congruence between our results and the CTRA model in terms of dysregulated pathways, the findings show that SES disparities in early adulthood, interestingly, do not alter the same set of genes identified in previous studies of CTRA. For example, the attenuated interferon innate responses in the CTRA model is possible through the direct suppression of
The observed SES-related transcriptional perturbations are associated with both up- and downregulated functional pathways in the immune system. Social stress-induced gene alterations in humans are associated with diseases that include both upregulation of the immune system as well as suppressed immune responsiveness with lowered social status. This seemingly perplexing pattern has been explained by the selective characteristics of the immune transcriptome, i.e., the increase in expression of certain pro-inflammatory genes and the repression of groups of antiviral immune response genes##REF##32041883##40##,##REF##25166010##61##–##REF##23853742##63##. It is, therefore, unsurprising that a similar pattern of dysregulated immune pathways is emergent in the blood transcriptomic landscape of subjects in Add Health with contributions of enrichment from both up- and downregulated gene clusters. Such a finding calls for additional research that moves beyond the initial general finding that stressors upregulate proinflammatory genes and downregulate antiviral genes.
\",\n \"Lastly, among the common mediating (or possible explanatory) mechanisms studied here, BMI consistently emerged as a plausible mediator of the SES associations with immune cell gene regulation. Smoking also appears to play a significant role in the SES-related transcriptional alterations. These results underscore the importance of gene regulatory network approach in formulating a comprehensive understanding of psychosocial stressors and their impact on biological mechanisms early in life. Studies have already established that changes caused by socioeconomic disparities in early adulthood could have far-reaching implications for chronic conditions in later adulthood. Identification of novel regulators of such perturbations is an important step in formulating a mitigating strategy.
\",\n \"We used tissue-specific regulatory networks to link transcription factors to differentially expressed genes, and subsequently the STRING database to find protein interaction partners. There were 643 transcription factors identified in the gene regulatory network, with an even a smaller number (304) having a confidence score that is above the threshold used (0.4). High confidence (threshold > 0.7) protein-protein neighbors derived from the whole network of 643 transcription factors numbered 3051 (18,875 total protein-protein neighbors), of which 1750 were pruned for the 304 transcription factors. Given the central role of many of the transcription factors and protein partners, one could argue that the set of upstream regulators found from the SES–DEG could be equal to a set of upstream regulators derived from a random set of genes. To examine this possibility, we performed randomized trials by starting with randomly selected sets of “DE” genes to then derive these upstream regulators of the random sets. We subsequently compared the upstream regulators of each random set of “DE” genes to our observed results (see Supplementary Figs. ##SUPPL##3##S10## and ##SUPPL##3##S11##). While some of the individual transcription factors may not reach statistical significance (Supplementary Fig. ##SUPPL##3##S10##), the entire set of the upstream regulators is highly significant (Supplementary Fig. ##SUPPL##3##S11##).
\"\n]"},"conclusion":{"kind":"list like","value":[],"string":"[]"},"front":{"kind":"list like","value":["Disparities in socio-economic status (SES) predict many immune system-related diseases, and previous research documents relationships between SES and the immune cell transcriptome. Drawing on a bioinformatically-informed network approach, we situate these findings in a broader molecular framework by examining the upstream regulators of SES-associated transcriptional alterations. Data come from the National Longitudinal Study of Adolescent to Adult Health (Add Health), a nationally representative sample of 4543 adults in the United States. Results reveal a network—of differentially expressed genes, transcription factors, and protein neighbors of transcription factors—that shows widespread SES-related dysregulation of the immune system. Mediational models suggest that body mass index (BMI) plays a key role in accounting for many of these associations. Overall, the results reveal the central role of upstream regulators in socioeconomic differences in the molecular basis of immunity, which propagate to increase risk of chronic health conditions in later-life.
","Disparities in socio-economic status (SES) predict many immune system-related diseases, and previous research documents relationships between SES and the immune cell transcriptome. Drawing on a bioinformatically-informed network approach, we situate these findings in a broader molecular framework by examining the upstream regulators of SES-associated transcriptional alterations. Data come from the National Longitudinal Study of Adolescent to Adult Health (Add Health), a nationally representative sample of 4543 adults in the United States. Results reveal a network—of differentially expressed genes, transcription factors, and protein neighbors of transcription factors—that shows widespread SES-related dysregulation of the immune system. Mediational models suggest that body mass index (BMI) plays a key role in accounting for many of these associations. Overall, the results reveal the central role of upstream regulators in socioeconomic differences in the molecular basis of immunity, which propagate to increase risk of chronic health conditions in later-life.
\",\n \"Several limitations are noteworthy. First, the hypotheses and the subsequent results are driven by the mRNA abundance data collected once from every participating subject. The repeated collection of transcriptomic data would be essential to address their highly transient nature, which is likely associated with considerable noise. Second, the identification of upstream transcriptional regulators of gene expression is performed with the aid of tissue-specific gene regulatory networks. These networks link genes to transcription factors based on experimental evidence and assign a confidence score to every identified transcription factor. It is, therefore, possible that transcription factors that play a central role in cell maintenance and cell cycle may be implicated without having a substantive role in the etiology or progression of dysfunction. We tried to account for such effects using a randomization experiment. However, direct measurements of protein abundance are required to concretely determine the role of every transcription factor. In the absence of proteomic assay data, inferring transcription factor abundance from publicly available chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sources that have similar gene expression alterations as those observed with lowered SES, could offer valuable insights and presents an important extension of the current work. Finally, because the design is not experimental, the findings cannot be interpreted as casual relationships. This limitation is especially salient for the casual identification of social and behavioral mediators of SES-related immune dysregulation. Future research could usefully examine them and other mediators in a casual framework to disentangle expected tissue repair response to stress induced by these risk factors (e.g., obesity, smoking) and global immune system dysfunction.
","Nevertheless, results suggest that a network of transcription factors and protein partners play a pivotal role in modulating the SES-related transcriptional response that precipitates dysregulated immune system response in terms of inflammation and interferon innate immunity. These central actors are important targets for future research connecting health disparities and socioeconomic inequalities. The results highlight the need for system-oriented analyses to comprehensively map the biological impact of SES disparities and they represent an essential step forward in identifying targets for possible prevention and intervention.
","\n
"],"string":"[\n \"Several limitations are noteworthy. First, the hypotheses and the subsequent results are driven by the mRNA abundance data collected once from every participating subject. The repeated collection of transcriptomic data would be essential to address their highly transient nature, which is likely associated with considerable noise. Second, the identification of upstream transcriptional regulators of gene expression is performed with the aid of tissue-specific gene regulatory networks. These networks link genes to transcription factors based on experimental evidence and assign a confidence score to every identified transcription factor. It is, therefore, possible that transcription factors that play a central role in cell maintenance and cell cycle may be implicated without having a substantive role in the etiology or progression of dysfunction. We tried to account for such effects using a randomization experiment. However, direct measurements of protein abundance are required to concretely determine the role of every transcription factor. In the absence of proteomic assay data, inferring transcription factor abundance from publicly available chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sources that have similar gene expression alterations as those observed with lowered SES, could offer valuable insights and presents an important extension of the current work. Finally, because the design is not experimental, the findings cannot be interpreted as casual relationships. This limitation is especially salient for the casual identification of social and behavioral mediators of SES-related immune dysregulation. Future research could usefully examine them and other mediators in a casual framework to disentangle expected tissue repair response to stress induced by these risk factors (e.g., obesity, smoking) and global immune system dysfunction.
\",\n \"Nevertheless, results suggest that a network of transcription factors and protein partners play a pivotal role in modulating the SES-related transcriptional response that precipitates dysregulated immune system response in terms of inflammation and interferon innate immunity. These central actors are important targets for future research connecting health disparities and socioeconomic inequalities. The results highlight the need for system-oriented analyses to comprehensively map the biological impact of SES disparities and they represent an essential step forward in identifying targets for possible prevention and intervention.
\",\n \"\\n
\"\n]"},"back":{"kind":"list like","value":["The online version contains supplementary material available at 10.1038/s41598-024-51517-6.
","S.R. conceived of paper, performed data analysis, and wrote paper. S.C., K.H., and M.S. collected the data. S.C., B.L., and M.S. assisted with interpretation and writing.
","This research was supported by NIH Grants R01-HD087061 (MPIs K.H. Harris and M. J. Shanahan) specifically for the present analyses, as well as P30-AG017265, R01-AG043404, and R01-AG033590; by the Swiss National Science Foundation (10531C-197964, Shanahan PI); and by the Jacobs Center for Productive Youth Development (University of Zürich). This research uses data from Add Health, a program directed by Robert Hummer and designed by J. Richard Udry, Peter S. Bearman, and Kathleen Mullan Harris (University of North Carolina at Chapel Hill). The Add Health program is funded by Grant P01-HD31921 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, with cooperative funding from 23 other federal agencies and foundations (
Add Health data are available at
The authors declare no competing interests.
"],"string":"[\n \"The online version contains supplementary material available at 10.1038/s41598-024-51517-6.
\",\n \"S.R. conceived of paper, performed data analysis, and wrote paper. S.C., K.H., and M.S. collected the data. S.C., B.L., and M.S. assisted with interpretation and writing.
\",\n \"This research was supported by NIH Grants R01-HD087061 (MPIs K.H. Harris and M. J. Shanahan) specifically for the present analyses, as well as P30-AG017265, R01-AG043404, and R01-AG033590; by the Swiss National Science Foundation (10531C-197964, Shanahan PI); and by the Jacobs Center for Productive Youth Development (University of Zürich). This research uses data from Add Health, a program directed by Robert Hummer and designed by J. Richard Udry, Peter S. Bearman, and Kathleen Mullan Harris (University of North Carolina at Chapel Hill). The Add Health program is funded by Grant P01-HD31921 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, with cooperative funding from 23 other federal agencies and foundations (
Add Health data are available at
The authors declare no competing interests.
\"\n]"},"figure":{"kind":"list like","value":["Enrichment analysis of the WGCNA clusters exhibiting significant cluster-SES relationships. Eigengenes from individual clusters were identified using WGCNA and then examined for significant associations with SES (adjusted
Immune pathway enrichment of the differentially expressed genes by early adult SES. Significantly enriched Reactome pathways (with immune parent nodes reported to the right, child nodes to the left) for SES–DEG. The size of the circle signifies the number of genes that contribute to the significant enrichment in a pathway and the color of the circle indicates Cramer’s V, a measure of the magnitude of association. The overrepresentation analysis shows a predominantly upregulated antiviral immune response (Interferon signaling and neutrophil degranulation) and downregulation in proinflammatory pathways (NF-κB and toll-like receptor cascade) with high SES.
Combined functional immune enrichment analysis of the SES–DEG and their upstream regulators. Reactome pathways in the immune system are represented here as circular filled nodes. Reactome pathways are hierarchical, and the arrow connects a parent node to its child. The significance of pathways (adjusted
Relative importance of SES–DEG and upstream regulators to the immune dysregulation. The figure shows the upstream regulators along with the SES–DEG represented as layers (4 in total). The innermost layer represents genes and upstream regulators that are involved in the enrichment of more than 10 functional pathways (in Fig. ##FIG##2##3##) and thus they are pivotal in immune dysregulation, whereas the outermost layer consists of genes and upstream regulators that are only involved in 1 enriched biological process.
Mediation analysis for common behavioral risk factors and the layers of upstream regulators of SES-associated immune system dysfunction. The median percent mediated ratio (Average casual mediated effect (ACME)/total effect) is shown for mediational models for the risk factors and the layers of SES–DEG and upstream regulators in Fig. ##FIG##3##4##. All the mediational models were significant (aggregated adjusted
Enrichment analysis of the WGCNA clusters exhibiting significant cluster-SES relationships. Eigengenes from individual clusters were identified using WGCNA and then examined for significant associations with SES (adjusted
Immune pathway enrichment of the differentially expressed genes by early adult SES. Significantly enriched Reactome pathways (with immune parent nodes reported to the right, child nodes to the left) for SES–DEG. The size of the circle signifies the number of genes that contribute to the significant enrichment in a pathway and the color of the circle indicates Cramer’s V, a measure of the magnitude of association. The overrepresentation analysis shows a predominantly upregulated antiviral immune response (Interferon signaling and neutrophil degranulation) and downregulation in proinflammatory pathways (NF-κB and toll-like receptor cascade) with high SES.
Combined functional immune enrichment analysis of the SES–DEG and their upstream regulators. Reactome pathways in the immune system are represented here as circular filled nodes. Reactome pathways are hierarchical, and the arrow connects a parent node to its child. The significance of pathways (adjusted
Relative importance of SES–DEG and upstream regulators to the immune dysregulation. The figure shows the upstream regulators along with the SES–DEG represented as layers (4 in total). The innermost layer represents genes and upstream regulators that are involved in the enrichment of more than 10 functional pathways (in Fig. ##FIG##2##3##) and thus they are pivotal in immune dysregulation, whereas the outermost layer consists of genes and upstream regulators that are only involved in 1 enriched biological process.
Mediation analysis for common behavioral risk factors and the layers of upstream regulators of SES-associated immune system dysfunction. The median percent mediated ratio (Average casual mediated effect (ACME)/total effect) is shown for mediational models for the risk factors and the layers of SES–DEG and upstream regulators in Fig. ##FIG##3##4##. All the mediational models were significant (aggregated adjusted
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information 1.
Supplementary Information 2.
Supplementary Information 3.
Supplementary Information 4.
Supplementary Information 1.
Supplementary Information 2.
Supplementary Information 3.
Supplementary Information 4.
Atherosclerosis is a chronic, progressive disease characterized by the accumulation of lipids, inflammatory cells, and fibrous elements in the arterial wall, leading to the formation of atherosclerotic plaques [##REF##33883728##1##, ##REF##31420554##2##]. These plaques can lead to serious clinical consequences, such as myocardial infarction (MI) and stroke, which are major causes of morbidity and mortality worldwide [##REF##26892956##3##]. The pathogenesis of atherosclerosis involves multiple genetic, environmental, and metabolic factors [##REF##35504280##4##].
","Copper is an essential trace element involved in mitochondrial respiration, antioxidant defense, and neurotransmitter synthesis [##REF##22075424##5##, ##REF##18277979##6##]. Copper homeostasis is tightly regulated. Copper excess or deficiency can lead to pathological alterations, and thus negatively affect human health [##REF##36414625##7##]. Copper dyshomeostasis may contribute to the pathogenesis of atherosclerosis by increasing oxidative stress, inflammation, endothelial dysfunction, and lipid metabolism levels [##REF##36774340##8##–##REF##29874110##10##]. Moreover, a novel type of cell death, which is copper-dependent, has recently been described. This copper-induced cell death, termed cuproptosis, may contribute to atherosclerosis development by causing cell death and impairing mitochondrial function [##REF##35354936##11##, ##REF##35298263##12##].
","In this review, we explore the role of copper homeostasis and cuproptosis in atherosclerosis. We also discuss potential therapeutic strategies against cuproptosis and provide directions for future research on cuproptosis and atherosclerosis.
"],"string":"[\n \"Atherosclerosis is a chronic, progressive disease characterized by the accumulation of lipids, inflammatory cells, and fibrous elements in the arterial wall, leading to the formation of atherosclerotic plaques [##REF##33883728##1##, ##REF##31420554##2##]. These plaques can lead to serious clinical consequences, such as myocardial infarction (MI) and stroke, which are major causes of morbidity and mortality worldwide [##REF##26892956##3##]. The pathogenesis of atherosclerosis involves multiple genetic, environmental, and metabolic factors [##REF##35504280##4##].
\",\n \"Copper is an essential trace element involved in mitochondrial respiration, antioxidant defense, and neurotransmitter synthesis [##REF##22075424##5##, ##REF##18277979##6##]. Copper homeostasis is tightly regulated. Copper excess or deficiency can lead to pathological alterations, and thus negatively affect human health [##REF##36414625##7##]. Copper dyshomeostasis may contribute to the pathogenesis of atherosclerosis by increasing oxidative stress, inflammation, endothelial dysfunction, and lipid metabolism levels [##REF##36774340##8##–##REF##29874110##10##]. Moreover, a novel type of cell death, which is copper-dependent, has recently been described. This copper-induced cell death, termed cuproptosis, may contribute to atherosclerosis development by causing cell death and impairing mitochondrial function [##REF##35354936##11##, ##REF##35298263##12##].
\",\n \"In this review, we explore the role of copper homeostasis and cuproptosis in atherosclerosis. We also discuss potential therapeutic strategies against cuproptosis and provide directions for future research on cuproptosis and atherosclerosis.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":[],"string":"[]"},"discussion":{"kind":"list like","value":[],"string":"[]"},"conclusion":{"kind":"list like","value":["In conclusion, the multifaceted role of copper in atherosclerosis involves complex cellular and systemic metabolic processes. Copper deficiency or excess promotes atherosclerosis by inducing oxidative stress, inflammation, endothelial dysfunction, and adverse effects on lipid metabolism. Further research is required to elucidate the interactions between these factors and their role in the development of atherosclerotic lesions.
","Copper-induced cell death and the subsequent emergence of the concept of cuproptosis have expanded our understanding of the role of copper in atherosclerosis. The involvement of cuproptosis and the associated mitochondrial dysfunction in atherosclerosis highlights the need for further research into the molecular mechanisms underlying these processes. Therapeutic targets and strategies for mitigating the detrimental effects of copper-induced cell death in atherosclerosis have also been identified, including modulating copper levels, inhibiting cuproptosis, or enhancing cellular defenses against cuproptosis. However, potential side effects should also be considered since an excessive reduction in copper levels may impair essential physiological processes or, in itself, lead to atherosclerosis. Additionally, the absence of cuproptosis-specific biomarkers is a significant barrier that hinders further development of clinical applications regarding cuproptosis. Identifying such biomarkers may facilitate risk stratification and the development of personalized therapeutic approaches.
"],"string":"[\n \"In conclusion, the multifaceted role of copper in atherosclerosis involves complex cellular and systemic metabolic processes. Copper deficiency or excess promotes atherosclerosis by inducing oxidative stress, inflammation, endothelial dysfunction, and adverse effects on lipid metabolism. Further research is required to elucidate the interactions between these factors and their role in the development of atherosclerotic lesions.
\",\n \"Copper-induced cell death and the subsequent emergence of the concept of cuproptosis have expanded our understanding of the role of copper in atherosclerosis. The involvement of cuproptosis and the associated mitochondrial dysfunction in atherosclerosis highlights the need for further research into the molecular mechanisms underlying these processes. Therapeutic targets and strategies for mitigating the detrimental effects of copper-induced cell death in atherosclerosis have also been identified, including modulating copper levels, inhibiting cuproptosis, or enhancing cellular defenses against cuproptosis. However, potential side effects should also be considered since an excessive reduction in copper levels may impair essential physiological processes or, in itself, lead to atherosclerosis. Additionally, the absence of cuproptosis-specific biomarkers is a significant barrier that hinders further development of clinical applications regarding cuproptosis. Identifying such biomarkers may facilitate risk stratification and the development of personalized therapeutic approaches.
\"\n]"},"front":{"kind":"list like","value":["Copper is an essential micronutrient that plays a pivotal role in numerous physiological processes in virtually all cell types. Nevertheless, the dysregulation of copper homeostasis, whether towards excess or deficiency, can lead to pathological alterations, such as atherosclerosis. With the advent of the concept of copper-induced cell death, termed cuproptosis, researchers have increasingly focused on the potential role of copper dyshomeostasis in atherosclerosis. In this review, we provide a broad overview of cellular and systemic copper metabolism. We then summarize the evidence linking copper dyshomeostasis to atherosclerosis and elucidate the potential mechanisms underlying atherosclerosis development in terms of both copper excess and copper deficiency. Furthermore, we discuss the evidence for and mechanisms of cuproptosis, discuss its interactions with other modes of cell death, and highlight the role of cuproptosis-related mitochondrial dysfunction in atherosclerosis. Finally, we explore the therapeutic strategy of targeting this novel form of cell death, aiming to provide some insights for the management of atherosclerosis.
","Copper is an essential micronutrient that plays a pivotal role in numerous physiological processes in virtually all cell types. Nevertheless, the dysregulation of copper homeostasis, whether towards excess or deficiency, can lead to pathological alterations, such as atherosclerosis. With the advent of the concept of copper-induced cell death, termed cuproptosis, researchers have increasingly focused on the potential role of copper dyshomeostasis in atherosclerosis. In this review, we provide a broad overview of cellular and systemic copper metabolism. We then summarize the evidence linking copper dyshomeostasis to atherosclerosis and elucidate the potential mechanisms underlying atherosclerosis development in terms of both copper excess and copper deficiency. Furthermore, we discuss the evidence for and mechanisms of cuproptosis, discuss its interactions with other modes of cell death, and highlight the role of cuproptosis-related mitochondrial dysfunction in atherosclerosis. Finally, we explore the therapeutic strategy of targeting this novel form of cell death, aiming to provide some insights for the management of atherosclerosis.
\",\n \"\n Copper is an essential trace element required in various physiological processes in the human body. Dysregulation of copper homeostasis, whether towards excess or deficiency, has been implicated in various health problems, including atherosclerosis. Dysregulation of copper homeostasis and copper-induced cell death (cuproptosis) are acknowledged as potential contributors to the pathogenesis of atherosclerosis.
\n
\n What is the safe window for copper levels to avoid the development of atherosclerosis? How can the suitable copper concentration be determined for the treatment of atherosclerosis? What are the potential candidate biomarkers that can reliably and sensitively indicate the occurrence of cuproptosis in the context of atherosclerosis? What are the potential undiscovered roles of copper in mitochondrial function, and is there interplay between copper and mitochondrial dynamics or mitophagy?
\n
Copper is an essential trace element required in various physiological processes in the human body. Research shows that Cu levels vary across organs and tissues, with values ranging from 3 mg (kidneys) to 46 mg (bone) [##REF##37500296##13##]. Copper serves as a cofactor for numerous enzymes involved in energy metabolism, neurotransmitter synthesis, and antioxidant defense [##REF##22075424##5##, ##REF##18277979##6##, ##REF##34390520##14##]. Excessive or deficient copper levels can, however, lead to cytotoxicity and pathological alterations [##REF##36414625##7##]. Therefore, it is essential to maintain systemic copper levels within a narrow range
Copper is mainly obtained from dietary sources such as organ meat and shellfish [##REF##27049134##15##]. Copper is primarily absorbed by enterocytes in the small intestine, and its uptake is mediated by copper transporter 1 (CTR1), also known as solute carrier family 31 member 1 (SLC31A1) [##REF##36882769##16##]. CTR1 appears to play a primary role in this uptake, as indicated by research showing greatly reduced copper accumulation in the peripheral tissues of neonatal mice lacking CTR1 [##REF##16950140##17##]. The six-transmembrane epithelial antigen of the prostate (STEAP) facilitates this process by reducing divalent Cu2+ to Cu+.
","After uptake by the intestinal epithelial cells, copper is transported and exported into the bloodstream by the ATPase copper transporter 7 A (ATP7A) [##REF##18779306##18##]. Here, copper is transported through the portal system to the liver by binding to soluble chaperones such as human serum albumin (HSA), ceruloplasmin, histidine, and macroglobulin [##UREF##1##19##, ##REF##26934375##20##]. Copper uptake by the hepatocytes in the liver is also mediated by CTR1. The liver is crucial in regulating copper metabolism by storing and excreting copper. Copper storage is mediated by the copper-binding protein metallothionein (MT), a reducing molecule rich in thiol groups with a high affinity for copper ions [##REF##32049413##21##]. MT plays a crucial role in copper homeostasis by storing and releasing excess copper when required. Copper transport is performed by ATPase copper transporter 7B (ATP7B) in hepatocytes, which pumps copper ions from the liver back into the bloodstream. Here, copper ions bind to their soluble partners and are transported to specific tissues and organs [##REF##19922814##22##].
","Copper is primarily eliminated via biliary excretion [##REF##36414625##7##]. Excess copper is secreted by the liver into the bile and then excreted through feces [##REF##15099406##23##]. The ATP7B transporter regulates the elimination of copper from the liver to bile canaliculi [##REF##18395099##24##]. In cases of ATP7B dysfunction, such as Wilson’s disease, copper accumulates in the liver, leading to liver damage and subsequent health issues [##REF##22240481##25##].
","In summary, copper metabolism is a complex process that involves multiple mechanisms to regulate copper absorption, transport, storage, and elimination.
","The maintenance of intracellular copper homeostasis is a complex and tightly regulated process involving the coordinated action of copper transporters, chaperones, and cuproenzymes
In mammalian cells, copper uptake is primarily mediated by CTR1 [##REF##18277979##6##], a transmembrane protein that forms a trimeric channel to facilitate the passage of Cu ions across the plasma membrane [##REF##23658018##26##]. CTR1 expression is regulated by copper levels. Low copper levels upregulate CTR1 expression to increase copper uptake, whereas high levels downregulate CTR1 expression to prevent copper cytotoxicity [##UREF##2##27##, ##REF##16365053##28##]. These findings highlight the importance of CTR1 in copper homeostasis.
","After entering the cell, copper is first delivered to cytoplasmic copper chaperones, which then transport it to intracellular compartments such as the mitochondria, trans-Golgi network (TGN), and nucleus. In mammalian cells, three major copper chaperones have been identified: antioxidant 1 copper chaperone (ATOX1), copper chaperone for superoxide dismutase (CCS), and cytochrome c oxidase copper chaperone 17 (COX17) [##REF##18761103##29##, ##REF##15113935##30##].
","ATOX1 delivers copper to ATP7A and ATP7B in the TGN [##REF##37017692##31##]. Additionally, ATOX1 has been shown to function as a new type of copper-dependent transcription factor that mediates copper-induced cell proliferation [##REF##18245776##32##].
","CCS is a soluble cytoplasmic copper-chaperone protein that transfers copper ions to the copper-binding site of superoxide dismutase 1 (SOD1) [##REF##11263870##33##]. SOD1 is a major antioxidant enzyme that catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide, thereby maintaining reactive oxygen species (ROS) homeostasis and protecting the cells from oxidative stress damage [##REF##30034766##34##]. This function has been shown in SOD1-knockout mice, where the absence of SOD1 increased oxidative stress and led first to liver cell damage and eventually liver cancer [##REF##15531919##35##].
","COX17 is responsible for delivering copper ions to the assembly of cytochrome c oxidase (COX), a key component of the electron transport chain involved in cellular respiration [##REF##35691151##36##]. Subsequently, COX11 and SCO1, which are also important components of the COX assembly, donate copper to Cu(B) and Cu(A) sites in COX2 and COX1 core subunits of COX in the mitochondrial inner membrane, respectively. Additionally, COX17 also acts as a copper donor within the mitochondrial intermembrane space (IMS) [##REF##15199057##37##]. COX17 is thus essential for proper COX assembly, with mutations in COX17 shown to further reduce COX activity, resulting in mitochondrial dysfunction and oxidative stress [##REF##12370308##38##].
","Intracellular copper export is mainly mediated by ATP7A and ATP7B, which are copper transporters located in the TGN that regulate copper delivery to secretory pathways and the plasma membrane [##REF##17615395##39##]. Under normal conditions, ATP7A and ATP7B transport copper ions from the TGN to other cellular compartments for various cellular functions. At excessive intracellular copper levels, ATP7A and ATP7B are activated to export excess copper ions from the TGN and sequester them in copper-binding proteins such as metallothionein [##REF##17531189##40##]. These proteins also regulate copper homeostasis by reducing uptake and increasing efflux. Mutations in ATP7A and ATP7B can lead to copper metabolism disorders such as Menkes disease and Wilson’s disease [##UREF##3##41##, ##UREF##4##42##].
","Atherosclerosis is a chronic inflammatory disease characterized by plaque accumulation on the arterial walls, leading to narrowing and hardening of the arteries [##REF##30653442##43##]. This process can result in serious complications, including coronary artery disease (CAD), stroke, and peripheral artery disease [##REF##22064431##44##–##REF##35420915##46##]. Growing evidence from numerous studies has linked copper dyshomeostasis and the resultant excess or deficiency of copper to atherosclerosis.
","Extensive research has revealed a correlation between elevated copper levels and cardiovascular disease (Table ##TAB##0##1##). For instance, several prospective cohort studies have shown a significant correlation between elevated serum copper levels and higher mortality rates related to cardiovascular diseases, particularly coronary heart disease [##REF##8862478##47##–##REF##3394701##49##]. Elevated copper levels have also been shown to be an independent risk factor for ischemic heart disease [##REF##1877585##50##]. In addition to evidence from prospective studies, Stadler et al. directly detected and quantified transition metal ions in human atherosclerotic plaques and found increased copper levels in the diseased intima [##REF##15001454##51##]. Studies on populations with acute myocardial infarction (AMI) also support these findings, with patients with AMI exhibiting significantly higher serum copper levels than those without AMI [##UREF##5##52##]. Moreover, an increase in serum copper levels among post-MI patients was found to have considerable diagnostic value for the occurrence of MI [##UREF##6##53##]. Furthermore, altered copper bioavailability is negatively correlated with carotid intima-media thickness (IMT), which may serve as a reliable predictor for early atherosclerosis in patients with obesity [##REF##29405466##54##]. Furthermore, serum copper concentrations differ among patients with different carotid atherosclerotic plaque morphologies. Specifically, patients with hemorrhagic plaques have significantly higher serum copper concentrations than those with calcified plaques [##REF##26554112##55##]. These findings suggest a potential involvement of elevated copper levels in the pathogenesis of atherosclerosis.
","Copper deficiency is also recognized as a major contributing factor to the development of atherosclerosis. This is evidenced by the benefit of high dietary copper and copper supplementation. The Institute of Medicine recommends a daily dietary allowance of 900 ug of copper for adults, with a tolerable upper limit of 10,000 μg/day to prevent liver damage [##REF##11269606##56##]. Although recommendations vary between national authorities, most recommend an intake of 800 to 2400 ug/day [##REF##27049134##15##]. Substantial research suggests that adequate copper intake reduces the risk of atherosclerosis. For example, Rock et al. found that copper supplementation in middle-aged individuals enhanced the antioxidative capacity of cells, which may help prevent vascular damage and thus reduce the risk of atherosclerosis [##REF##10699742##57##]. Another cohort study showed an association between adequate dietary intake of copper (equal to or above the estimated average requirement) and a reduced risk of all-cause- and cardiovascular disease-related mortality. However, this association was limited to copper intake from food sources [##REF##30959527##58##]. Studies on animal models corroborate these findings with Lamb et al., showing that dietary copper deficiency or excess increases susceptibility to atherosclerosis of the aorta in cholesterol-fed rabbits [##REF##11703538##59##]. Similarly, copper supplementation was found to reverse pathological changes induced by dietary iron overload in mice, partially normalizing cardiac hypertrophy [##REF##29960117##60##] and improving cardiac function in pressure overload-induced dilated cardiomyopathy [##REF##18679830##61##]. Nevertheless, the effects of copper supplementation on the cardiovascular system remain controversial. A study by Diaf et al. conducted on middle-aged women from Algeria showed that there is little association between dietary copper intake and atherosclerosis prevalence in diabetes [##REF##29740584##62##]. These conflicting results may be due to differences in study design and the dose and duration of copper supplementation.
","The mechanisms of atherosclerosis development due to altered copper levels are not fully understood. Several potential mechanisms include oxidative stress, inflammation, endothelial dysfunction, and lipid metabolism.
","Oxidative stress is a key factor in atherosclerosis development, which typically involves an imbalance in reactive oxygen species (ROS) production and antioxidant defenses [##REF##24300855##63##]. Since copper is a redox-active metal, changes in copper levels can contribute to the generation of ROS and thereby promote oxidative stress [##REF##27049134##15##, ##REF##33316526##64##]
Elevated levels of free copper ions tend to interact more with hydrogen peroxide through Fenton reactions, leading to the production of highly-reactive hydroxyl radicals [##REF##15892631##65##]. These radicals cause lipid peroxidation, protein oxidation, and DNA damage, ultimately contributing to the initiation and progression of atherosclerosis [##REF##10872549##66##]. Lipid peroxidation is a chain reaction initiated by an attack of ROS on polyunsaturated fatty acids in cell membrane lipids, which then leads to the oxidative damage of lipid molecules. Increased copper levels can promote lipid peroxidation and result in the formation of oxidized low-density lipoprotein (ox-LDL), which is an atherosclerosis risk factor [##REF##1398217##67##]. Furthermore, increased copper levels have been shown to reduce the activity of antioxidant enzymes, such as Cu/Zn-SOD, catalase, and glutathione peroxidase, in the red blood cells, whole blood, live tissue, and brain tissue of rats (67, 68, 69). In rat brain tissue, the decrease in SOD activity and glutathione levels arises because copper overload induces lipid peroxidation [##REF##18784908##68##]. Copper is also capable of causing DNA strand breaks and the oxidation of bases via oxygen-derived free radicals [##REF##17914163##69##]. These findings suggest that copper overload causes impairment of the antioxidant defense system in several tissues.
","Copper deficiency has also been suggested to contribute to the development and progression of atherosclerosis via oxidative stress. Copper is an essential cofactor of various antioxidant enzymes, including Cu/Zn-SOD, ceruloplasmin, and lysyl oxidase [##REF##25789247##70##]. Copper deficiency may thus impair the function of these enzymes, leading to an impaired antioxidant defense system and increased susceptibility to oxidative stress [##REF##25789247##70##, ##REF##8615367##71##]. This suggestion is supported by copper deficiency in rats being found to reduce Cu/Zn-SOD activity and increase oxidative damage to various subunits of the erythrocyte spectrin [##REF##9119252##72##]. Decreased activity of
Copper may also promote atherosclerosis development by modulating inflammatory responses associated with the disease.
","Excess copper has been implicated in atherosclerosis pathogenesis due to its ability to induce inflammation. High copper levels were previously reported to stimulate pro-inflammatory cytokine production and thereby promote inflammation within arterial walls [##REF##32727323##75##]. For example, in primary cardiac cells, Cu2+ increases the release of interleukin-6 (IL-6) and activates MAP kinases, which are linked to cardiac inflammation and hypertrophy [##REF##19517273##76##]. Copper-induced oxidative stress also contributes to inflammation because excessive copper generates ROS, which causes oxidative damage to lipids, proteins, and DNA and promotes inflammation [##REF##27049134##15##]. Furthermore, increases in ROS levels due to increased copper levels can, in turn, lead to the activation of nuclear factor-κB (NF-κB), a crucial protein involved in regulating the expression of proinflammatory genes [##REF##35985369##77##]. These findings suggest that high copper levels can exacerbate inflammation of the vascular walls and thus promote atherosclerosis development.
","Copper deficiency has also been linked to atherosclerosis development through its impact on inflammation. Copper deficiency may result in decreased expression of adhesion molecules, such as ICAM-1 and VCAM-1, which facilitate leukocyte adhesion onto activated endothelial cells [##REF##11274733##78##]. Evidence from animal studies also supports the role of copper deficiency in inflammation. For example, neutrophil accumulation increases due to increased ICAM-1 expression in the livers of copper-deficient rats following ischemia/reperfusion [##REF##20544302##79##]. Further, this elevated ICAM-1 expression has been shown to activate neutrophils and endothelial cells, as evidenced by F-actin polymerization and increased accumulation of neutrophils within the lung microcirculation [##REF##16118404##80##–##REF##15186252##82##]. Pulmonary inflammatory responses are also intensified in copper-deficient animals [##REF##11435213##83##]. Finally, copper deficiency impairs the activity of Cu/Zn-SOD as previously mentioned, leading to an increased accumulation of ROS and exacerbation of oxidative stress, which can further promote inflammation and atherosclerosis development [##REF##225457##84##].
","Copper dyshomeostasis has been linked to atherosclerosis, with both an excess and deficiency of copper contributing to endothelial dysfunction, a critical early step in atherogenesis.
","Excessive copper can disrupt the balance between the production and degradation of nitric oxide (NO), a key regulator of vascular tone and endothelial function [##REF##16585403##85##]. Increased copper levels can upregulate inducible NO synthase (iNOS) expression, resulting in excessive NO production and peroxynitrite, a potent oxidant that causes further oxidative damage [##REF##17237348##86##]. Moreover, copper also interacts with atherosclerosis risk factors such as homocysteine and thereby causes increased hydrogen peroxidation and oxidative stress. Specifically, a study shows that incubation with homocysteine and copper for 4 h is able to cause significant damage to endothelial cells [##REF##3514679##87##].
","Copper deficiency is associated with increased endothelial dysfunction as well. Endothelial dysfunction can result in decreased production of NO, a vasodilator and inhibitor of platelet aggregation, and thereby promote a proatherogenic environment [##REF##19875572##88##]. Copper deficiency can also lead to decreased NO levels by reducing the levels of SOD1. Reduced NO levels can then inhibit endothelial function and vasodilation, increase oxidative stress, and thereby promote atherosclerosis [##REF##25789247##70##].
","Excess copper strongly contributes to atherosclerosis development via its effects on the ox-LDL, which plays a central role in atherosclerosis [##REF##29737246##89##]. Specifically, copper participates in the oxidation of LDL particles, and alterations in copper levels may affect the susceptibility of LDL particles to oxidation. Excess copper levels can increase the oxidation of LDL particles and trigger the production of ox-LDL, which then contributes to atherosclerosis development [##REF##1398217##67##].
","In addition to its effects on ox-LDL, copper is involved in several forms of lipid metabolism, including fatty acid and cholesterol synthesis and lipoprotein metabolism. Alterations in copper levels may be associated with changes in lipid and lipoprotein concentrations. For example, serum copper and ceruloplasmin levels were found to be positively associated with lipid peroxides, total cholesterol, triglycerides, and apolipoprotein B in healthy individuals [##REF##7773726##90##]. In another study, the serum copper levels of Iranian patients with angiographically defined CAD were found to be positively correlated with fasting serum triglycerides [##REF##17317522##91##]. In vivo, evidence from animal experiments also supports the role of excess copper in lipid metabolism. A study on yellow catfish demonstrated that copper-induced endoplasmic reticulum stress and disrupted calcium homeostasis alter hepatic lipid metabolism, leading to increased lipid accumulation in the liver [##REF##26615493##92##].
","Insufficient copper levels may also contribute to atherosclerosis development by affecting lipid metabolism. Since copper is involved in the synthesis of fatty acids and cholesterol, insufficient copper levels may lead to impaired lipid metabolism and, thus, the development of fatty liver disease. Copper deficiency increases total cholesterol levels as well [##REF##36414625##7##]. Insufficient copper levels can affect the activity of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2), which are transcription factors involved in fatty acid and cholesterol metabolism [##REF##28271632##93##]. Specifically, both SREBP-1 isoforms (SREBP-1a and SREBP-1c) are involved in the regulation of fatty acid synthesis, whereas SREBP-2 is mainly involved in the regulation of cholesterol biosynthesis [##UREF##7##94##]. Low copper diets were found to induce accumulation of the mature form of SREBP-1 in the nuclei of rat liver cells, yet no change was observed in the DNA-binding site of SREBP-1 [##REF##11110846##95##]. Hence, copper may play a role in regulating the subcellular localization of SREBP-1, potentially affecting its activity and, in turn, lipid metabolism. Further research is needed to fully understand the mechanisms through which copper affects the activity of SREBP-1 and other transcription factors involved in lipid metabolism.
","Copper levels are also associated with other atherosclerosis risk factors, such as high blood pressure. Copper inhibits the activity of angiotensin-converting enzymes, such that copper deficiency leads to increasing angiotensis levels and, thus, water and sodium retention and hypertension [##REF##1711361##96##]. Moreover, copper is a cofactor of SOD, which is a major player in the antioxidant defense system. Hence, copper deficiency may increase the levels of superoxide free radicals, leading to elevated angiotensin levels and consequent hypertension [##REF##22753205##97##].
","The discovery of copper-induced cell death dates back to the early 1980s [##REF##6326753##98##]. Increased copper levels were found to promote ROS generation and thereby lead to oxidative stress, DNA damage, and, ultimately cell death [##REF##12821289##99##]. These findings have led to further investigations into the molecular mechanisms underlying copper-induced cell death and their potential implications for human diseases. Indeed, conflicting findings suggest that, in addition to ROS accumulation, excess copper may induce cell death through apoptosis or caspase-independent cell death [##REF##21452832##100##, ##REF##22542443##101##]. Overall, the mechanisms responsible for copper-induced cell death were not well understood.
","However, in March 2022, Tsvetkov et al. published a groundbreaking study unveiling the mechanism of copper-induced cell death, which was termed cuproptosis [##REF##35298263##12##]. Cuproptosis represents a unique form of cell death triggered by an excess of copper ions. In their study, Tsvetkov et al. showed that treatment with the copper ionophore elesclomol induced cell death. Remarkably, only the copper chelator can rescue cells from elesclomol-induced cell death. In contrast, rescue is not possible with any of the inhibitors for apoptosis, necroptosis, oxidative stress, ROS induced cell death, or ferroptosis. These findings unequivocally establish that cuproptosis is distinct from other known cell death modalities, underscoring its unique mechanisms and signaling pathways. Notably, cuproptosis is regulated by mitochondrial respiration, as supported by research indicating that mitochondria-dependent cells exhibit a sensitivity to copper ionophores nearly 1,000 times higher than cells undergoing glycolysis [##REF##35298263##12##]. The importance of mitochondrial respiration in cuproptosis has been highlighted in further research, revealing a close correlation between cuproptosis and the tricarboxylic acid (TCA) cycle. During cuproptosis, intracellular copper binds to the lipoylated components of the TCA cycle. This leads to the aggregation of copper-bound lipoylated mitochondrial proteins, which can disrupt the TCA cycle and, therefore, interfere with cellular energy production. The upstream regulatory factors FDX1 and LIAS are critical in this process. Aggregation of these proteins and the subsequent reduction of Fe–S clusters, which are essential cofactors for various cellular processes, including electron transport and enzymatic reactions [##REF##31935115##102##], promote proteotoxic stress and ultimately lead to cell death
Accumulating evidence suggests widespread cross-talk and interactions among the primary initiators, effectors, and executioners involved in pyroptosis, necroptosis, ferroptosis, and cuproptosis.
","Pyroptosis is a pro-inflammatory programmed cell death that is primarily driven by inflammasome assembly, accompanied by GSDMD cleavage and IL-1β and IL-18 release [##REF##33776057##103##–##UREF##8##105##]. NLRP1, NLRP3, NLRC4, AIM2, and pyrin are well-established inflammasome sensors that assemble canonical inflammasomes in a process induced by inflammatory stimuli such as those resulting from microbial infections [##REF##27291964##106##, ##REF##23707339##107##]. Copper ions, which are essential micronutrients for many physiological processes, have been shown to trigger ROS production and activate the NF-κB signaling pathway. This leads to the upregulation of pro-inflammatory genes and cytokines, potentially influencing the progression of atherosclerosis [##REF##33485222##108##–##REF##35312958##110##]. Therefore, there is a plausible suggestion of crosstalk between copper ions and pyroptosis.
","Evidence from animal studies demonstrates that copper loading in rat hepatocytes leads to a significant increase in the mRNA expression of pyroptosis-related genes (caspase-1, IL-18, IL-1β, and NLRP3) and the protein expression of caspase-1[##REF##30822667##111##]. Similarly, copper exposure in mouse microglial cells triggers an inflammatory response, resulting in the upregulation of NLRP3/caspase 1/GSDMD axis proteins and subsequent pyroptosis. These effects are likely mediated by the early activation of the ROS/NF-κB pathway and subsequent disruption of mitophagy [##REF##35985369##77##]. Moreover, comparable outcomes were observed in murine macrophages treated with copper oxide nanoparticles. Copper oxide nanoparticle exposure induces oxidative stress and activates NLRP3 inflammasomes, leading to the expression of pro-IL-1β through the MyD88-dependent TLR4 signaling pathway, followed by NF-κB activation in murine macrophages [##REF##33485222##108##].
","In summary, these findings demonstrate the presence of crosstalk between copper-induced cell death and pyroptosis. Copper exposure influences gene and protein expression associated with pyroptosis in various cell types, with the underlying mechanisms being ROS/NF-κB pathway activation and the inflammatory response. Further research is needed to elucidate the precise underlying mechanisms and explore the implications of this interaction.
","Necroptosis is a form of programmed necrosis linked to atherosclerosis via its potential involvement in plaque destabilization and rupture [##REF##35809652##112##, ##REF##35585052##113##]. Necroptosis was recently shown to activate the NLRP3 inflammasome by causing potassium efflux through MLKL pores in macrophages [##REF##28130493##114##]. Bioinformatics analysis further indicated an association between ZBP1 and cuproptosis as well as necroptosis [##REF##36186436##115##]. ZBP1 activation led to the recruitment of RIPK3 and caspase-8 to activate the NLRP3 inflammasome, which in turn triggers necroptosis and pyroptosis [##UREF##9##116##, ##REF##32322058##117##].
","Ferroptosis, a form of iron-dependent cell death triggered by lipid peroxidation and accumulation of lipid-based reactive oxygen species [##REF##22632970##118##–##REF##26653790##120##], appears to be influenced by copper levels due to the redox-active properties of copper ions [##UREF##10##121##, ##REF##34482117##122##]. Cuproptosis can modulate the expression of key genes involved in ferroptosis regulation, such as glutathione peroxidase 4 (GPX4), by eliminating phospholipid hydroperoxides [##REF##31634900##123##, ##REF##24439385##124##] and acylcoenzyme A synthetase long-chain family member 4 (ACSL4) [##REF##27842070##125##], thereby regulating the sensitivity of cells to ferroptosis inducers. Notably, copper chelators can reduce sensitivity to ferroptosis specifically while leaving other forms of cell death unaffected. In a study by Xue et al., copper was found to play a novel role in promoting ferroptotisis through the degradation of GPX4 via macroautophagy/autophagy [##REF##36622894##126##]. Furthermore, research by Gao et al. reveals an interaction between copper-induced cell death and necroptosis. Their findings indicate that elesclomol administration to colorectal cancer cells increased Cu(II) levels in the mitochondria, downregulated ATP7A expression, and increased ROS accumulation. This process triggered SLC7A11 degradation, intensifying oxidative stress and resulting in ferroptosis [##REF##34390123##127##]. Additionally, copper depletion greatly enhanced ferroptosis through mitochondrial perturbation and the disruption of antioxidant mechanisms [##UREF##11##128##]. Specifically, copper depletion limits GPX4 protein expression and reduces cellular sensitivity to ferroptosis inducers, establishing a direct link between copper levels and ferroptosis [##UREF##11##128##].
","Based on the aforementioned studies, it is evident that copper is closely linked to necroptosis, pyroptosis, and ferroptosis, suggesting a significant cross-talk between these different cell death pathways. Understanding the underlying mechanisms that connect these modes of cell death is of paramount importance for the development of novel atherosclerotic therapeutic strategies that can target multiple pathways simultaneously.
","Mitochondria are vulnerable to copper-induced damage, which causes oxidative damage to its membrane [##REF##15629136##129##, ##REF##32979421##130##]. Excessive intracellular copper may also disrupt mitochondrial function by altering the activity of several key enzymes, such as those involved in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation [##REF##15122704##131##]. Severe mitochondrial dysfunction and decreases in the activities of several liver enzymes, including complex I, complex II, complex III, complex IV, and aconitase, were observed in patients with copper overload. These effects are potentially mediated by the accumulation of copper in the mitochondria [##REF##10981891##132##]. Furthermore, copper treatment induces selective changes in metabolic enzymes through lipoylation, which is a highly conserved lysine post-translational modification. Protein lipoylation occurs only on Dihydrolipoamide S-Acetyltransferase (DLAT), Dihydrolipoamide S-Succinyltransferase (DLST), Dihydrolipoamide Branched Chain Transacylase E2 (DBT), and Glycine Cleavage System Protein H (GCSH), all of which are involved in metabolic complexes that regulate carbon entry points to the TCA cycle [##REF##29191830##133##, ##REF##29169048##134##]. Additionally, copper overload may trigger the opening of the mitochondrial permeability transition pore and cause the release of pro-apoptotic factors, ultimately resulting in cell death [##REF##21484409##135##].
","Mitochondrial dysfunction is implicated in atherosclerosis development and progression [##REF##29237304##136##]. Impaired mitochondrial function promotes lipid accumulation, oxidative stress, inflammatory responses, and proliferation of vascular smooth muscle cells, all of which contribute to plaque formation and destabilization [##UREF##12##137##, ##REF##34743301##138##]. Considering its impact on mitochondrial function, cuproptosis may contribute to the progression of atherosclerosis by aggravating mitochondrial dysfunction.
","Copper chelators bind and sequester copper ions. Several copper chelators have shown promise in animal studies and clinical trials for atherosclerosis treatment.
","Tetrathiomolybdate (TTM) has been shown to be an effective copper chelator with potential therapeutic implications in attenuating atherosclerosis progression, as demonstrated in animal models [##REF##22770994##139##, ##REF##21724870##140##]. In a study using ApoE-/- mice, TTM treatment for 10 weeks significantly reduced aortic lesion development, indicating its potential as an anti-atherosclerotic agent [##REF##22770994##139##]. The beneficial effects of TTM were attributed to its ability to reduce bioavailable copper levels and inhibit vascular inflammation [##REF##21724870##140##]. Copper is involved in vascular inflammation as an etiologic factor of atherosclerotic vascular disease, and by chelating copper, TTM may modulate copper-related pathways involved in atherosclerosis and inflammation. Wei et al. demonstrated that TTM copper chelation inhibited lipopolysaccharide (LPS)-induced inflammatory responses in mice aorta and other tissues. This inhibition may occur by suppressing redox-sensitive transcription factors NF-κB and AP-1, which play crucial roles in inflammation [##REF##21724870##140##]. By targeting copper-related pathways and modulating inflammatory processes, TTM exhibits potential as an anti-atherosclerotic agent.
","Ethylenediaminetetraacetic acid disodium salt (EDTA) is a broad-spectrum metal-chelating agent that has also been investigated for its potential to treat atherosclerosis [##REF##13372537##141##]. For instance, a double-blind placebo-controlled trial (
Copper chaperones play a crucial role in maintaining cellular copper homeostasis by facilitating the transfer of copper ions to target proteins and organelles. Modulating the expression of copper chaperone proteins potentially offers an alternative approach to regulate copper levels and limit the contribution of copper-induced cell death to atherosclerosis.
","ATOX1 is a copper chaperone protein that is of major importance in mammalian cells. ATOX1 has been shown to translocate to the nucleus in response to inflammatory cytokines or exogenous copper. Furthermore, ATOX1 is localized in the nucleus of endothelial cells in the inflamed atherosclerotic aorta [##UREF##13##144##]. The migration of VSMCs is crucial for neointimal formation following vascular injury and atherosclerotic lesion formation. ATOX1 was found to promote VSMC migration and inflammatory cell recruitment to injured vessels [##REF##23349186##145##]. Furthermore, copper-dependent binding of ATOX1 to TRAF4 is required to facilitate nuclear translocation of ATOX1 and ROS-dependent inflammatory responses in TNF-α-stimulated endothelial cells (ECs) [##REF##31553645##146##]. This highlights the potential of targeting the ATOX1-TRAF4 axis as a novel therapeutic strategy for the treatment of atherosclerosis. In summary, ATOX1 represents a promising therapeutic target for inflammation-related vascular diseases such as atherosclerosis.
","Copper ionophores transport copper into the cells, leading to an increase in intracellular copper levels and subsequent cell death. However, copper chelators can inhibit this process. Several drugs can act as copper ionophores, including disulfiram, pyrithione, chloroquine, and elesclomol [##UREF##14##147##]. Among these, elesclomol has received the most attention and has been subjected to several clinical trials for use in cancer treatment. Although the majority of these trials have not shown promising results regarding further development of elesclomol as a drug, they have verified its safety [##REF##23401447##148##]. Nanomedicines combining copper ions with copper ionophores are also currently being widely investigated. Recently, the researchers successfully designed multifunctional nanoparticles with pH-responsive and CD44-targeted properties. The utilization of dendritic mesoporous silica nanoparticles capped with copper sulfide and coated with hyaluronic acid enables precise drug delivery and controlled release in the acidic microenvironment of atherosclerotic inflammation [##REF##34982089##149##]. These findings highlight the potential of copper-based nanomedicines in developing innovative approaches for targeted atherosclerosis therapy. Notably, an important aspect to consider in the advancement of copper ionophores for clinical therapeutic applications is the notable impact that slight structural changes can exert on their properties and functions [##REF##36882769##16##]. In addition, copper ionophore-mediated cell death is strongly correlated with mitochondrial metabolism and closely associated with atherosclerosis development. Based on these findings, copper ionophores may represent a novel therapeutic strategy for targeting copper-induced cell death in atherosclerosis.
"],"string":"[\n \"\\n Copper is an essential trace element required in various physiological processes in the human body. Dysregulation of copper homeostasis, whether towards excess or deficiency, has been implicated in various health problems, including atherosclerosis. Dysregulation of copper homeostasis and copper-induced cell death (cuproptosis) are acknowledged as potential contributors to the pathogenesis of atherosclerosis.
\\n
\\n What is the safe window for copper levels to avoid the development of atherosclerosis? How can the suitable copper concentration be determined for the treatment of atherosclerosis? What are the potential candidate biomarkers that can reliably and sensitively indicate the occurrence of cuproptosis in the context of atherosclerosis? What are the potential undiscovered roles of copper in mitochondrial function, and is there interplay between copper and mitochondrial dynamics or mitophagy?
\\n
Copper is an essential trace element required in various physiological processes in the human body. Research shows that Cu levels vary across organs and tissues, with values ranging from 3 mg (kidneys) to 46 mg (bone) [##REF##37500296##13##]. Copper serves as a cofactor for numerous enzymes involved in energy metabolism, neurotransmitter synthesis, and antioxidant defense [##REF##22075424##5##, ##REF##18277979##6##, ##REF##34390520##14##]. Excessive or deficient copper levels can, however, lead to cytotoxicity and pathological alterations [##REF##36414625##7##]. Therefore, it is essential to maintain systemic copper levels within a narrow range
Copper is mainly obtained from dietary sources such as organ meat and shellfish [##REF##27049134##15##]. Copper is primarily absorbed by enterocytes in the small intestine, and its uptake is mediated by copper transporter 1 (CTR1), also known as solute carrier family 31 member 1 (SLC31A1) [##REF##36882769##16##]. CTR1 appears to play a primary role in this uptake, as indicated by research showing greatly reduced copper accumulation in the peripheral tissues of neonatal mice lacking CTR1 [##REF##16950140##17##]. The six-transmembrane epithelial antigen of the prostate (STEAP) facilitates this process by reducing divalent Cu2+ to Cu+.
\",\n \"After uptake by the intestinal epithelial cells, copper is transported and exported into the bloodstream by the ATPase copper transporter 7 A (ATP7A) [##REF##18779306##18##]. Here, copper is transported through the portal system to the liver by binding to soluble chaperones such as human serum albumin (HSA), ceruloplasmin, histidine, and macroglobulin [##UREF##1##19##, ##REF##26934375##20##]. Copper uptake by the hepatocytes in the liver is also mediated by CTR1. The liver is crucial in regulating copper metabolism by storing and excreting copper. Copper storage is mediated by the copper-binding protein metallothionein (MT), a reducing molecule rich in thiol groups with a high affinity for copper ions [##REF##32049413##21##]. MT plays a crucial role in copper homeostasis by storing and releasing excess copper when required. Copper transport is performed by ATPase copper transporter 7B (ATP7B) in hepatocytes, which pumps copper ions from the liver back into the bloodstream. Here, copper ions bind to their soluble partners and are transported to specific tissues and organs [##REF##19922814##22##].
\",\n \"Copper is primarily eliminated via biliary excretion [##REF##36414625##7##]. Excess copper is secreted by the liver into the bile and then excreted through feces [##REF##15099406##23##]. The ATP7B transporter regulates the elimination of copper from the liver to bile canaliculi [##REF##18395099##24##]. In cases of ATP7B dysfunction, such as Wilson’s disease, copper accumulates in the liver, leading to liver damage and subsequent health issues [##REF##22240481##25##].
\",\n \"In summary, copper metabolism is a complex process that involves multiple mechanisms to regulate copper absorption, transport, storage, and elimination.
\",\n \"The maintenance of intracellular copper homeostasis is a complex and tightly regulated process involving the coordinated action of copper transporters, chaperones, and cuproenzymes
In mammalian cells, copper uptake is primarily mediated by CTR1 [##REF##18277979##6##], a transmembrane protein that forms a trimeric channel to facilitate the passage of Cu ions across the plasma membrane [##REF##23658018##26##]. CTR1 expression is regulated by copper levels. Low copper levels upregulate CTR1 expression to increase copper uptake, whereas high levels downregulate CTR1 expression to prevent copper cytotoxicity [##UREF##2##27##, ##REF##16365053##28##]. These findings highlight the importance of CTR1 in copper homeostasis.
\",\n \"After entering the cell, copper is first delivered to cytoplasmic copper chaperones, which then transport it to intracellular compartments such as the mitochondria, trans-Golgi network (TGN), and nucleus. In mammalian cells, three major copper chaperones have been identified: antioxidant 1 copper chaperone (ATOX1), copper chaperone for superoxide dismutase (CCS), and cytochrome c oxidase copper chaperone 17 (COX17) [##REF##18761103##29##, ##REF##15113935##30##].
\",\n \"ATOX1 delivers copper to ATP7A and ATP7B in the TGN [##REF##37017692##31##]. Additionally, ATOX1 has been shown to function as a new type of copper-dependent transcription factor that mediates copper-induced cell proliferation [##REF##18245776##32##].
\",\n \"CCS is a soluble cytoplasmic copper-chaperone protein that transfers copper ions to the copper-binding site of superoxide dismutase 1 (SOD1) [##REF##11263870##33##]. SOD1 is a major antioxidant enzyme that catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide, thereby maintaining reactive oxygen species (ROS) homeostasis and protecting the cells from oxidative stress damage [##REF##30034766##34##]. This function has been shown in SOD1-knockout mice, where the absence of SOD1 increased oxidative stress and led first to liver cell damage and eventually liver cancer [##REF##15531919##35##].
\",\n \"COX17 is responsible for delivering copper ions to the assembly of cytochrome c oxidase (COX), a key component of the electron transport chain involved in cellular respiration [##REF##35691151##36##]. Subsequently, COX11 and SCO1, which are also important components of the COX assembly, donate copper to Cu(B) and Cu(A) sites in COX2 and COX1 core subunits of COX in the mitochondrial inner membrane, respectively. Additionally, COX17 also acts as a copper donor within the mitochondrial intermembrane space (IMS) [##REF##15199057##37##]. COX17 is thus essential for proper COX assembly, with mutations in COX17 shown to further reduce COX activity, resulting in mitochondrial dysfunction and oxidative stress [##REF##12370308##38##].
\",\n \"Intracellular copper export is mainly mediated by ATP7A and ATP7B, which are copper transporters located in the TGN that regulate copper delivery to secretory pathways and the plasma membrane [##REF##17615395##39##]. Under normal conditions, ATP7A and ATP7B transport copper ions from the TGN to other cellular compartments for various cellular functions. At excessive intracellular copper levels, ATP7A and ATP7B are activated to export excess copper ions from the TGN and sequester them in copper-binding proteins such as metallothionein [##REF##17531189##40##]. These proteins also regulate copper homeostasis by reducing uptake and increasing efflux. Mutations in ATP7A and ATP7B can lead to copper metabolism disorders such as Menkes disease and Wilson’s disease [##UREF##3##41##, ##UREF##4##42##].
\",\n \"Atherosclerosis is a chronic inflammatory disease characterized by plaque accumulation on the arterial walls, leading to narrowing and hardening of the arteries [##REF##30653442##43##]. This process can result in serious complications, including coronary artery disease (CAD), stroke, and peripheral artery disease [##REF##22064431##44##–##REF##35420915##46##]. Growing evidence from numerous studies has linked copper dyshomeostasis and the resultant excess or deficiency of copper to atherosclerosis.
\",\n \"Extensive research has revealed a correlation between elevated copper levels and cardiovascular disease (Table ##TAB##0##1##). For instance, several prospective cohort studies have shown a significant correlation between elevated serum copper levels and higher mortality rates related to cardiovascular diseases, particularly coronary heart disease [##REF##8862478##47##–##REF##3394701##49##]. Elevated copper levels have also been shown to be an independent risk factor for ischemic heart disease [##REF##1877585##50##]. In addition to evidence from prospective studies, Stadler et al. directly detected and quantified transition metal ions in human atherosclerotic plaques and found increased copper levels in the diseased intima [##REF##15001454##51##]. Studies on populations with acute myocardial infarction (AMI) also support these findings, with patients with AMI exhibiting significantly higher serum copper levels than those without AMI [##UREF##5##52##]. Moreover, an increase in serum copper levels among post-MI patients was found to have considerable diagnostic value for the occurrence of MI [##UREF##6##53##]. Furthermore, altered copper bioavailability is negatively correlated with carotid intima-media thickness (IMT), which may serve as a reliable predictor for early atherosclerosis in patients with obesity [##REF##29405466##54##]. Furthermore, serum copper concentrations differ among patients with different carotid atherosclerotic plaque morphologies. Specifically, patients with hemorrhagic plaques have significantly higher serum copper concentrations than those with calcified plaques [##REF##26554112##55##]. These findings suggest a potential involvement of elevated copper levels in the pathogenesis of atherosclerosis.
\",\n \"Copper deficiency is also recognized as a major contributing factor to the development of atherosclerosis. This is evidenced by the benefit of high dietary copper and copper supplementation. The Institute of Medicine recommends a daily dietary allowance of 900 ug of copper for adults, with a tolerable upper limit of 10,000 μg/day to prevent liver damage [##REF##11269606##56##]. Although recommendations vary between national authorities, most recommend an intake of 800 to 2400 ug/day [##REF##27049134##15##]. Substantial research suggests that adequate copper intake reduces the risk of atherosclerosis. For example, Rock et al. found that copper supplementation in middle-aged individuals enhanced the antioxidative capacity of cells, which may help prevent vascular damage and thus reduce the risk of atherosclerosis [##REF##10699742##57##]. Another cohort study showed an association between adequate dietary intake of copper (equal to or above the estimated average requirement) and a reduced risk of all-cause- and cardiovascular disease-related mortality. However, this association was limited to copper intake from food sources [##REF##30959527##58##]. Studies on animal models corroborate these findings with Lamb et al., showing that dietary copper deficiency or excess increases susceptibility to atherosclerosis of the aorta in cholesterol-fed rabbits [##REF##11703538##59##]. Similarly, copper supplementation was found to reverse pathological changes induced by dietary iron overload in mice, partially normalizing cardiac hypertrophy [##REF##29960117##60##] and improving cardiac function in pressure overload-induced dilated cardiomyopathy [##REF##18679830##61##]. Nevertheless, the effects of copper supplementation on the cardiovascular system remain controversial. A study by Diaf et al. conducted on middle-aged women from Algeria showed that there is little association between dietary copper intake and atherosclerosis prevalence in diabetes [##REF##29740584##62##]. These conflicting results may be due to differences in study design and the dose and duration of copper supplementation.
\",\n \"The mechanisms of atherosclerosis development due to altered copper levels are not fully understood. Several potential mechanisms include oxidative stress, inflammation, endothelial dysfunction, and lipid metabolism.
\",\n \"Oxidative stress is a key factor in atherosclerosis development, which typically involves an imbalance in reactive oxygen species (ROS) production and antioxidant defenses [##REF##24300855##63##]. Since copper is a redox-active metal, changes in copper levels can contribute to the generation of ROS and thereby promote oxidative stress [##REF##27049134##15##, ##REF##33316526##64##]
Elevated levels of free copper ions tend to interact more with hydrogen peroxide through Fenton reactions, leading to the production of highly-reactive hydroxyl radicals [##REF##15892631##65##]. These radicals cause lipid peroxidation, protein oxidation, and DNA damage, ultimately contributing to the initiation and progression of atherosclerosis [##REF##10872549##66##]. Lipid peroxidation is a chain reaction initiated by an attack of ROS on polyunsaturated fatty acids in cell membrane lipids, which then leads to the oxidative damage of lipid molecules. Increased copper levels can promote lipid peroxidation and result in the formation of oxidized low-density lipoprotein (ox-LDL), which is an atherosclerosis risk factor [##REF##1398217##67##]. Furthermore, increased copper levels have been shown to reduce the activity of antioxidant enzymes, such as Cu/Zn-SOD, catalase, and glutathione peroxidase, in the red blood cells, whole blood, live tissue, and brain tissue of rats (67, 68, 69). In rat brain tissue, the decrease in SOD activity and glutathione levels arises because copper overload induces lipid peroxidation [##REF##18784908##68##]. Copper is also capable of causing DNA strand breaks and the oxidation of bases via oxygen-derived free radicals [##REF##17914163##69##]. These findings suggest that copper overload causes impairment of the antioxidant defense system in several tissues.
\",\n \"Copper deficiency has also been suggested to contribute to the development and progression of atherosclerosis via oxidative stress. Copper is an essential cofactor of various antioxidant enzymes, including Cu/Zn-SOD, ceruloplasmin, and lysyl oxidase [##REF##25789247##70##]. Copper deficiency may thus impair the function of these enzymes, leading to an impaired antioxidant defense system and increased susceptibility to oxidative stress [##REF##25789247##70##, ##REF##8615367##71##]. This suggestion is supported by copper deficiency in rats being found to reduce Cu/Zn-SOD activity and increase oxidative damage to various subunits of the erythrocyte spectrin [##REF##9119252##72##]. Decreased activity of
Copper may also promote atherosclerosis development by modulating inflammatory responses associated with the disease.
\",\n \"Excess copper has been implicated in atherosclerosis pathogenesis due to its ability to induce inflammation. High copper levels were previously reported to stimulate pro-inflammatory cytokine production and thereby promote inflammation within arterial walls [##REF##32727323##75##]. For example, in primary cardiac cells, Cu2+ increases the release of interleukin-6 (IL-6) and activates MAP kinases, which are linked to cardiac inflammation and hypertrophy [##REF##19517273##76##]. Copper-induced oxidative stress also contributes to inflammation because excessive copper generates ROS, which causes oxidative damage to lipids, proteins, and DNA and promotes inflammation [##REF##27049134##15##]. Furthermore, increases in ROS levels due to increased copper levels can, in turn, lead to the activation of nuclear factor-κB (NF-κB), a crucial protein involved in regulating the expression of proinflammatory genes [##REF##35985369##77##]. These findings suggest that high copper levels can exacerbate inflammation of the vascular walls and thus promote atherosclerosis development.
\",\n \"Copper deficiency has also been linked to atherosclerosis development through its impact on inflammation. Copper deficiency may result in decreased expression of adhesion molecules, such as ICAM-1 and VCAM-1, which facilitate leukocyte adhesion onto activated endothelial cells [##REF##11274733##78##]. Evidence from animal studies also supports the role of copper deficiency in inflammation. For example, neutrophil accumulation increases due to increased ICAM-1 expression in the livers of copper-deficient rats following ischemia/reperfusion [##REF##20544302##79##]. Further, this elevated ICAM-1 expression has been shown to activate neutrophils and endothelial cells, as evidenced by F-actin polymerization and increased accumulation of neutrophils within the lung microcirculation [##REF##16118404##80##–##REF##15186252##82##]. Pulmonary inflammatory responses are also intensified in copper-deficient animals [##REF##11435213##83##]. Finally, copper deficiency impairs the activity of Cu/Zn-SOD as previously mentioned, leading to an increased accumulation of ROS and exacerbation of oxidative stress, which can further promote inflammation and atherosclerosis development [##REF##225457##84##].
\",\n \"Copper dyshomeostasis has been linked to atherosclerosis, with both an excess and deficiency of copper contributing to endothelial dysfunction, a critical early step in atherogenesis.
\",\n \"Excessive copper can disrupt the balance between the production and degradation of nitric oxide (NO), a key regulator of vascular tone and endothelial function [##REF##16585403##85##]. Increased copper levels can upregulate inducible NO synthase (iNOS) expression, resulting in excessive NO production and peroxynitrite, a potent oxidant that causes further oxidative damage [##REF##17237348##86##]. Moreover, copper also interacts with atherosclerosis risk factors such as homocysteine and thereby causes increased hydrogen peroxidation and oxidative stress. Specifically, a study shows that incubation with homocysteine and copper for 4 h is able to cause significant damage to endothelial cells [##REF##3514679##87##].
\",\n \"Copper deficiency is associated with increased endothelial dysfunction as well. Endothelial dysfunction can result in decreased production of NO, a vasodilator and inhibitor of platelet aggregation, and thereby promote a proatherogenic environment [##REF##19875572##88##]. Copper deficiency can also lead to decreased NO levels by reducing the levels of SOD1. Reduced NO levels can then inhibit endothelial function and vasodilation, increase oxidative stress, and thereby promote atherosclerosis [##REF##25789247##70##].
\",\n \"Excess copper strongly contributes to atherosclerosis development via its effects on the ox-LDL, which plays a central role in atherosclerosis [##REF##29737246##89##]. Specifically, copper participates in the oxidation of LDL particles, and alterations in copper levels may affect the susceptibility of LDL particles to oxidation. Excess copper levels can increase the oxidation of LDL particles and trigger the production of ox-LDL, which then contributes to atherosclerosis development [##REF##1398217##67##].
\",\n \"In addition to its effects on ox-LDL, copper is involved in several forms of lipid metabolism, including fatty acid and cholesterol synthesis and lipoprotein metabolism. Alterations in copper levels may be associated with changes in lipid and lipoprotein concentrations. For example, serum copper and ceruloplasmin levels were found to be positively associated with lipid peroxides, total cholesterol, triglycerides, and apolipoprotein B in healthy individuals [##REF##7773726##90##]. In another study, the serum copper levels of Iranian patients with angiographically defined CAD were found to be positively correlated with fasting serum triglycerides [##REF##17317522##91##]. In vivo, evidence from animal experiments also supports the role of excess copper in lipid metabolism. A study on yellow catfish demonstrated that copper-induced endoplasmic reticulum stress and disrupted calcium homeostasis alter hepatic lipid metabolism, leading to increased lipid accumulation in the liver [##REF##26615493##92##].
\",\n \"Insufficient copper levels may also contribute to atherosclerosis development by affecting lipid metabolism. Since copper is involved in the synthesis of fatty acids and cholesterol, insufficient copper levels may lead to impaired lipid metabolism and, thus, the development of fatty liver disease. Copper deficiency increases total cholesterol levels as well [##REF##36414625##7##]. Insufficient copper levels can affect the activity of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2), which are transcription factors involved in fatty acid and cholesterol metabolism [##REF##28271632##93##]. Specifically, both SREBP-1 isoforms (SREBP-1a and SREBP-1c) are involved in the regulation of fatty acid synthesis, whereas SREBP-2 is mainly involved in the regulation of cholesterol biosynthesis [##UREF##7##94##]. Low copper diets were found to induce accumulation of the mature form of SREBP-1 in the nuclei of rat liver cells, yet no change was observed in the DNA-binding site of SREBP-1 [##REF##11110846##95##]. Hence, copper may play a role in regulating the subcellular localization of SREBP-1, potentially affecting its activity and, in turn, lipid metabolism. Further research is needed to fully understand the mechanisms through which copper affects the activity of SREBP-1 and other transcription factors involved in lipid metabolism.
\",\n \"Copper levels are also associated with other atherosclerosis risk factors, such as high blood pressure. Copper inhibits the activity of angiotensin-converting enzymes, such that copper deficiency leads to increasing angiotensis levels and, thus, water and sodium retention and hypertension [##REF##1711361##96##]. Moreover, copper is a cofactor of SOD, which is a major player in the antioxidant defense system. Hence, copper deficiency may increase the levels of superoxide free radicals, leading to elevated angiotensin levels and consequent hypertension [##REF##22753205##97##].
\",\n \"The discovery of copper-induced cell death dates back to the early 1980s [##REF##6326753##98##]. Increased copper levels were found to promote ROS generation and thereby lead to oxidative stress, DNA damage, and, ultimately cell death [##REF##12821289##99##]. These findings have led to further investigations into the molecular mechanisms underlying copper-induced cell death and their potential implications for human diseases. Indeed, conflicting findings suggest that, in addition to ROS accumulation, excess copper may induce cell death through apoptosis or caspase-independent cell death [##REF##21452832##100##, ##REF##22542443##101##]. Overall, the mechanisms responsible for copper-induced cell death were not well understood.
\",\n \"However, in March 2022, Tsvetkov et al. published a groundbreaking study unveiling the mechanism of copper-induced cell death, which was termed cuproptosis [##REF##35298263##12##]. Cuproptosis represents a unique form of cell death triggered by an excess of copper ions. In their study, Tsvetkov et al. showed that treatment with the copper ionophore elesclomol induced cell death. Remarkably, only the copper chelator can rescue cells from elesclomol-induced cell death. In contrast, rescue is not possible with any of the inhibitors for apoptosis, necroptosis, oxidative stress, ROS induced cell death, or ferroptosis. These findings unequivocally establish that cuproptosis is distinct from other known cell death modalities, underscoring its unique mechanisms and signaling pathways. Notably, cuproptosis is regulated by mitochondrial respiration, as supported by research indicating that mitochondria-dependent cells exhibit a sensitivity to copper ionophores nearly 1,000 times higher than cells undergoing glycolysis [##REF##35298263##12##]. The importance of mitochondrial respiration in cuproptosis has been highlighted in further research, revealing a close correlation between cuproptosis and the tricarboxylic acid (TCA) cycle. During cuproptosis, intracellular copper binds to the lipoylated components of the TCA cycle. This leads to the aggregation of copper-bound lipoylated mitochondrial proteins, which can disrupt the TCA cycle and, therefore, interfere with cellular energy production. The upstream regulatory factors FDX1 and LIAS are critical in this process. Aggregation of these proteins and the subsequent reduction of Fe–S clusters, which are essential cofactors for various cellular processes, including electron transport and enzymatic reactions [##REF##31935115##102##], promote proteotoxic stress and ultimately lead to cell death
Accumulating evidence suggests widespread cross-talk and interactions among the primary initiators, effectors, and executioners involved in pyroptosis, necroptosis, ferroptosis, and cuproptosis.
\",\n \"Pyroptosis is a pro-inflammatory programmed cell death that is primarily driven by inflammasome assembly, accompanied by GSDMD cleavage and IL-1β and IL-18 release [##REF##33776057##103##–##UREF##8##105##]. NLRP1, NLRP3, NLRC4, AIM2, and pyrin are well-established inflammasome sensors that assemble canonical inflammasomes in a process induced by inflammatory stimuli such as those resulting from microbial infections [##REF##27291964##106##, ##REF##23707339##107##]. Copper ions, which are essential micronutrients for many physiological processes, have been shown to trigger ROS production and activate the NF-κB signaling pathway. This leads to the upregulation of pro-inflammatory genes and cytokines, potentially influencing the progression of atherosclerosis [##REF##33485222##108##–##REF##35312958##110##]. Therefore, there is a plausible suggestion of crosstalk between copper ions and pyroptosis.
\",\n \"Evidence from animal studies demonstrates that copper loading in rat hepatocytes leads to a significant increase in the mRNA expression of pyroptosis-related genes (caspase-1, IL-18, IL-1β, and NLRP3) and the protein expression of caspase-1[##REF##30822667##111##]. Similarly, copper exposure in mouse microglial cells triggers an inflammatory response, resulting in the upregulation of NLRP3/caspase 1/GSDMD axis proteins and subsequent pyroptosis. These effects are likely mediated by the early activation of the ROS/NF-κB pathway and subsequent disruption of mitophagy [##REF##35985369##77##]. Moreover, comparable outcomes were observed in murine macrophages treated with copper oxide nanoparticles. Copper oxide nanoparticle exposure induces oxidative stress and activates NLRP3 inflammasomes, leading to the expression of pro-IL-1β through the MyD88-dependent TLR4 signaling pathway, followed by NF-κB activation in murine macrophages [##REF##33485222##108##].
\",\n \"In summary, these findings demonstrate the presence of crosstalk between copper-induced cell death and pyroptosis. Copper exposure influences gene and protein expression associated with pyroptosis in various cell types, with the underlying mechanisms being ROS/NF-κB pathway activation and the inflammatory response. Further research is needed to elucidate the precise underlying mechanisms and explore the implications of this interaction.
\",\n \"Necroptosis is a form of programmed necrosis linked to atherosclerosis via its potential involvement in plaque destabilization and rupture [##REF##35809652##112##, ##REF##35585052##113##]. Necroptosis was recently shown to activate the NLRP3 inflammasome by causing potassium efflux through MLKL pores in macrophages [##REF##28130493##114##]. Bioinformatics analysis further indicated an association between ZBP1 and cuproptosis as well as necroptosis [##REF##36186436##115##]. ZBP1 activation led to the recruitment of RIPK3 and caspase-8 to activate the NLRP3 inflammasome, which in turn triggers necroptosis and pyroptosis [##UREF##9##116##, ##REF##32322058##117##].
\",\n \"Ferroptosis, a form of iron-dependent cell death triggered by lipid peroxidation and accumulation of lipid-based reactive oxygen species [##REF##22632970##118##–##REF##26653790##120##], appears to be influenced by copper levels due to the redox-active properties of copper ions [##UREF##10##121##, ##REF##34482117##122##]. Cuproptosis can modulate the expression of key genes involved in ferroptosis regulation, such as glutathione peroxidase 4 (GPX4), by eliminating phospholipid hydroperoxides [##REF##31634900##123##, ##REF##24439385##124##] and acylcoenzyme A synthetase long-chain family member 4 (ACSL4) [##REF##27842070##125##], thereby regulating the sensitivity of cells to ferroptosis inducers. Notably, copper chelators can reduce sensitivity to ferroptosis specifically while leaving other forms of cell death unaffected. In a study by Xue et al., copper was found to play a novel role in promoting ferroptotisis through the degradation of GPX4 via macroautophagy/autophagy [##REF##36622894##126##]. Furthermore, research by Gao et al. reveals an interaction between copper-induced cell death and necroptosis. Their findings indicate that elesclomol administration to colorectal cancer cells increased Cu(II) levels in the mitochondria, downregulated ATP7A expression, and increased ROS accumulation. This process triggered SLC7A11 degradation, intensifying oxidative stress and resulting in ferroptosis [##REF##34390123##127##]. Additionally, copper depletion greatly enhanced ferroptosis through mitochondrial perturbation and the disruption of antioxidant mechanisms [##UREF##11##128##]. Specifically, copper depletion limits GPX4 protein expression and reduces cellular sensitivity to ferroptosis inducers, establishing a direct link between copper levels and ferroptosis [##UREF##11##128##].
\",\n \"Based on the aforementioned studies, it is evident that copper is closely linked to necroptosis, pyroptosis, and ferroptosis, suggesting a significant cross-talk between these different cell death pathways. Understanding the underlying mechanisms that connect these modes of cell death is of paramount importance for the development of novel atherosclerotic therapeutic strategies that can target multiple pathways simultaneously.
\",\n \"Mitochondria are vulnerable to copper-induced damage, which causes oxidative damage to its membrane [##REF##15629136##129##, ##REF##32979421##130##]. Excessive intracellular copper may also disrupt mitochondrial function by altering the activity of several key enzymes, such as those involved in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation [##REF##15122704##131##]. Severe mitochondrial dysfunction and decreases in the activities of several liver enzymes, including complex I, complex II, complex III, complex IV, and aconitase, were observed in patients with copper overload. These effects are potentially mediated by the accumulation of copper in the mitochondria [##REF##10981891##132##]. Furthermore, copper treatment induces selective changes in metabolic enzymes through lipoylation, which is a highly conserved lysine post-translational modification. Protein lipoylation occurs only on Dihydrolipoamide S-Acetyltransferase (DLAT), Dihydrolipoamide S-Succinyltransferase (DLST), Dihydrolipoamide Branched Chain Transacylase E2 (DBT), and Glycine Cleavage System Protein H (GCSH), all of which are involved in metabolic complexes that regulate carbon entry points to the TCA cycle [##REF##29191830##133##, ##REF##29169048##134##]. Additionally, copper overload may trigger the opening of the mitochondrial permeability transition pore and cause the release of pro-apoptotic factors, ultimately resulting in cell death [##REF##21484409##135##].
\",\n \"Mitochondrial dysfunction is implicated in atherosclerosis development and progression [##REF##29237304##136##]. Impaired mitochondrial function promotes lipid accumulation, oxidative stress, inflammatory responses, and proliferation of vascular smooth muscle cells, all of which contribute to plaque formation and destabilization [##UREF##12##137##, ##REF##34743301##138##]. Considering its impact on mitochondrial function, cuproptosis may contribute to the progression of atherosclerosis by aggravating mitochondrial dysfunction.
\",\n \"Copper chelators bind and sequester copper ions. Several copper chelators have shown promise in animal studies and clinical trials for atherosclerosis treatment.
\",\n \"Tetrathiomolybdate (TTM) has been shown to be an effective copper chelator with potential therapeutic implications in attenuating atherosclerosis progression, as demonstrated in animal models [##REF##22770994##139##, ##REF##21724870##140##]. In a study using ApoE-/- mice, TTM treatment for 10 weeks significantly reduced aortic lesion development, indicating its potential as an anti-atherosclerotic agent [##REF##22770994##139##]. The beneficial effects of TTM were attributed to its ability to reduce bioavailable copper levels and inhibit vascular inflammation [##REF##21724870##140##]. Copper is involved in vascular inflammation as an etiologic factor of atherosclerotic vascular disease, and by chelating copper, TTM may modulate copper-related pathways involved in atherosclerosis and inflammation. Wei et al. demonstrated that TTM copper chelation inhibited lipopolysaccharide (LPS)-induced inflammatory responses in mice aorta and other tissues. This inhibition may occur by suppressing redox-sensitive transcription factors NF-κB and AP-1, which play crucial roles in inflammation [##REF##21724870##140##]. By targeting copper-related pathways and modulating inflammatory processes, TTM exhibits potential as an anti-atherosclerotic agent.
\",\n \"Ethylenediaminetetraacetic acid disodium salt (EDTA) is a broad-spectrum metal-chelating agent that has also been investigated for its potential to treat atherosclerosis [##REF##13372537##141##]. For instance, a double-blind placebo-controlled trial (
Copper chaperones play a crucial role in maintaining cellular copper homeostasis by facilitating the transfer of copper ions to target proteins and organelles. Modulating the expression of copper chaperone proteins potentially offers an alternative approach to regulate copper levels and limit the contribution of copper-induced cell death to atherosclerosis.
\",\n \"ATOX1 is a copper chaperone protein that is of major importance in mammalian cells. ATOX1 has been shown to translocate to the nucleus in response to inflammatory cytokines or exogenous copper. Furthermore, ATOX1 is localized in the nucleus of endothelial cells in the inflamed atherosclerotic aorta [##UREF##13##144##]. The migration of VSMCs is crucial for neointimal formation following vascular injury and atherosclerotic lesion formation. ATOX1 was found to promote VSMC migration and inflammatory cell recruitment to injured vessels [##REF##23349186##145##]. Furthermore, copper-dependent binding of ATOX1 to TRAF4 is required to facilitate nuclear translocation of ATOX1 and ROS-dependent inflammatory responses in TNF-α-stimulated endothelial cells (ECs) [##REF##31553645##146##]. This highlights the potential of targeting the ATOX1-TRAF4 axis as a novel therapeutic strategy for the treatment of atherosclerosis. In summary, ATOX1 represents a promising therapeutic target for inflammation-related vascular diseases such as atherosclerosis.
\",\n \"Copper ionophores transport copper into the cells, leading to an increase in intracellular copper levels and subsequent cell death. However, copper chelators can inhibit this process. Several drugs can act as copper ionophores, including disulfiram, pyrithione, chloroquine, and elesclomol [##UREF##14##147##]. Among these, elesclomol has received the most attention and has been subjected to several clinical trials for use in cancer treatment. Although the majority of these trials have not shown promising results regarding further development of elesclomol as a drug, they have verified its safety [##REF##23401447##148##]. Nanomedicines combining copper ions with copper ionophores are also currently being widely investigated. Recently, the researchers successfully designed multifunctional nanoparticles with pH-responsive and CD44-targeted properties. The utilization of dendritic mesoporous silica nanoparticles capped with copper sulfide and coated with hyaluronic acid enables precise drug delivery and controlled release in the acidic microenvironment of atherosclerotic inflammation [##REF##34982089##149##]. These findings highlight the potential of copper-based nanomedicines in developing innovative approaches for targeted atherosclerosis therapy. Notably, an important aspect to consider in the advancement of copper ionophores for clinical therapeutic applications is the notable impact that slight structural changes can exert on their properties and functions [##REF##36882769##16##]. In addition, copper ionophore-mediated cell death is strongly correlated with mitochondrial metabolism and closely associated with atherosclerosis development. Based on these findings, copper ionophores may represent a novel therapeutic strategy for targeting copper-induced cell death in atherosclerosis.
\"\n]"},"back":{"kind":"list like","value":["MW and LL provided the guidance for this study. SY prepared the original manuscript and the illustrations. YL, LZ, and XW assisted with manuscript review and revision. All the authors have read and approved the final version of the manuscript.
","This study was supported by the National Natural Science Foundation of China (Nos. 81202805, 82074254, 82374281), the Beijing Natural Science Foundation (No. 7172185), and the Science and Technology Innovation Project of China Academy of Chinese Medical Science (C12021A01413).
","The authors declare no competing interests.
"],"string":"[\n \"MW and LL provided the guidance for this study. SY prepared the original manuscript and the illustrations. YL, LZ, and XW assisted with manuscript review and revision. All the authors have read and approved the final version of the manuscript.
\",\n \"This study was supported by the National Natural Science Foundation of China (Nos. 81202805, 82074254, 82374281), the Beijing Natural Science Foundation (No. 7172185), and the Science and Technology Innovation Project of China Academy of Chinese Medical Science (C12021A01413).
\",\n \"The authors declare no competing interests.
\"\n]"},"figure":{"kind":"list like","value":["Copper absorption occurs primarily in the small intestine, a process mediated by SLC31A1. Copper is then transported and exported to the bloodstream through the action of ATP7A, combined with soluble chaperones, and transported through the portal system to the liver, where it is stored and further transported. Excess copper is excreted by the liver into the bile. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, ATP7A and ATP7B ATPase copper transporters 7 A and 7B, STEAP six-transmembrane epithelial antigen of the prostate, HSA human serum albumin, CP ceruloplasmin, MT metallothionein, HIS histidine, MG macroglobulin.
The maintenance of intracellular copper homeostasis is a complex and tightly regulated process. Copper ions are primarily taken up by cells via SLC31A1, whereas their export is facilitated by ATP7A/B. Once inside the cell, copper is transported to interact with cytoplasmic copper chaperones, including COX17, CCS, and ATOX1. These chaperones transport copper to specific cellular compartments, such as the mitochondria, TGN, and nucleus. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, ATP7A and ATP7B ATPase copper transporter 7A and 7B, ATOX1 antioxidant 1 copper chaperone, CCS copper chaperone for superoxide dismutase, COX17 cytochrome c oxidase copper chaperone 17, COX11 cytochrome c oxidase copper chaperone 11, COX cytochrome c oxidase, ROS reactive oxygen species, SCO1 synthesis of cytochrome c oxidase 1, SOD1 superoxide dismutase 1, TGN trans-Golgi network, GSH glutathione, MT1/2 metallothionein 1/2, and STEAP six-transmembrane epithelial antigen of the prostate.
Excessive copper interacts with H2O2 via the Fenton reaction, generating highly reactive hydroxyl radicals. These radicals induce lipid peroxidation, DNA strand breaks, and base oxidation and impair the function of antioxidant enzymes, ultimately leading to increased oxidative stress and potential damage to cells. On the other hand, copper deficiency also impairs the function of certain antioxidant enzymes, causing a decrease in SOD1 and COX activity. This reduced activity results in lowered NO levels, inactivation of complex I, and increased production of ROS, thereby exacerbating oxidative stress within cells. Together, these processes promote the development of atherosclerosis. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, STEAP six-transmembrane epithelial antigen of the prostate, H2O2 hydrogen peroxide, •OH reactive hydroxyl radicals, ROS reactive oxygen species, ATP7A and ATP7B ATPase copper transporter 7A and 7B, CAT catalase, GSH-Px glutathione peroxidase, CP ceruloplasmin, LOX lysyl oxidase, SOD1 superoxide dismutase 1, COX cytochrome c oxidase, and AS atherosclerosis.
The copper ionophore ES transports extracellular copper into the cell. Subsequently, intracellular copper binds to lipoylated mitochondrial enzymes (such as DLAT) involved in the TCA cycle. This binding leads to the aggregation of these proteins, which can disrupt the TCA cycle and, therefore, interfere with cellular energy production. The upstream regulatory factors FDX1 and LIAS play a critical role in this process. The aggregation of lipoylated mitochondrial proteins and loss of Fe-S clusters promote proteotoxic stress and ultimately lead to cell death. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, STEAP the six-transmembrane epithelial antigen of the prostate, ATP7A and ATP7B ATPase copper transporter 7A and 7B, ES elesclomol, α-KG α-ketoglutarate, DLAT dihydrolipoamide S-acetyltransferase, FDX1 ferredoxin-1, Fe–S iron–sulfur, LIAS lipoic acid synthetase, TCA tricarboxylic acid, and GSH glutathione.
Copper absorption occurs primarily in the small intestine, a process mediated by SLC31A1. Copper is then transported and exported to the bloodstream through the action of ATP7A, combined with soluble chaperones, and transported through the portal system to the liver, where it is stored and further transported. Excess copper is excreted by the liver into the bile. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, ATP7A and ATP7B ATPase copper transporters 7 A and 7B, STEAP six-transmembrane epithelial antigen of the prostate, HSA human serum albumin, CP ceruloplasmin, MT metallothionein, HIS histidine, MG macroglobulin.
The maintenance of intracellular copper homeostasis is a complex and tightly regulated process. Copper ions are primarily taken up by cells via SLC31A1, whereas their export is facilitated by ATP7A/B. Once inside the cell, copper is transported to interact with cytoplasmic copper chaperones, including COX17, CCS, and ATOX1. These chaperones transport copper to specific cellular compartments, such as the mitochondria, TGN, and nucleus. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, ATP7A and ATP7B ATPase copper transporter 7A and 7B, ATOX1 antioxidant 1 copper chaperone, CCS copper chaperone for superoxide dismutase, COX17 cytochrome c oxidase copper chaperone 17, COX11 cytochrome c oxidase copper chaperone 11, COX cytochrome c oxidase, ROS reactive oxygen species, SCO1 synthesis of cytochrome c oxidase 1, SOD1 superoxide dismutase 1, TGN trans-Golgi network, GSH glutathione, MT1/2 metallothionein 1/2, and STEAP six-transmembrane epithelial antigen of the prostate.
Excessive copper interacts with H2O2 via the Fenton reaction, generating highly reactive hydroxyl radicals. These radicals induce lipid peroxidation, DNA strand breaks, and base oxidation and impair the function of antioxidant enzymes, ultimately leading to increased oxidative stress and potential damage to cells. On the other hand, copper deficiency also impairs the function of certain antioxidant enzymes, causing a decrease in SOD1 and COX activity. This reduced activity results in lowered NO levels, inactivation of complex I, and increased production of ROS, thereby exacerbating oxidative stress within cells. Together, these processes promote the development of atherosclerosis. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, STEAP six-transmembrane epithelial antigen of the prostate, H2O2 hydrogen peroxide, •OH reactive hydroxyl radicals, ROS reactive oxygen species, ATP7A and ATP7B ATPase copper transporter 7A and 7B, CAT catalase, GSH-Px glutathione peroxidase, CP ceruloplasmin, LOX lysyl oxidase, SOD1 superoxide dismutase 1, COX cytochrome c oxidase, and AS atherosclerosis.
The copper ionophore ES transports extracellular copper into the cell. Subsequently, intracellular copper binds to lipoylated mitochondrial enzymes (such as DLAT) involved in the TCA cycle. This binding leads to the aggregation of these proteins, which can disrupt the TCA cycle and, therefore, interfere with cellular energy production. The upstream regulatory factors FDX1 and LIAS play a critical role in this process. The aggregation of lipoylated mitochondrial proteins and loss of Fe-S clusters promote proteotoxic stress and ultimately lead to cell death. Figure created with BioRender. SLC31A1 solute carrier family 31 member 1, STEAP the six-transmembrane epithelial antigen of the prostate, ATP7A and ATP7B ATPase copper transporter 7A and 7B, ES elesclomol, α-KG α-ketoglutarate, DLAT dihydrolipoamide S-acetyltransferase, FDX1 ferredoxin-1, Fe–S iron–sulfur, LIAS lipoic acid synthetase, TCA tricarboxylic acid, and GSH glutathione.
Evidence linking copper excess and atherosclerosis.
| Country, author, year | Methods and study population | Result |
|---|---|---|
| Finland, Reunanen et al. [##REF##8862478##47##] | 230 men who died from CVDs and 298 matched controls; 10 years follow up. | The adjusted relative risk of CHD mortality between the highest and lowest tertiles of serum copper were 2.86 ( |
| United States, Ford, et al. [##REF##10905530##48##] | 4,574 participants aged ≥ 30 years; 151 died from CHD during 16-year follow-up. | There was an ~5% increase in serum copper levels among individuals who died from CHD compared to those who did not ( |
| Dutch, Kok et al. [##REF##3394701##49##] | Cancer ( | Individuals with elevated serum copper levels (above 1.43 mg/liter) exhibited a roughly four times higher adjusted risk of mortality from cancer and CVD compared to those with serum copper levels within the normal range. |
| Eastern Finland, Salonen et al. [##REF##1877585##50##] | 1666 randomly selected men aged 42, 48, 54, or 60 years with no symptomatic IHD. | Elevated serum copper level is a risk factor for IHD that acts independently, as shown by a 3.5-fold to 4.0-fold increased risk of AMI associated with high serum copper levels (1.02-1.16 mg/liter and 1.17 mg/liter or more). |
| Nadina, Robyn et al. [##REF##15001454##51##] | Atherosclerotic patients aged 67.9 ± 8.2 years and healthy controls. | Copper levels in the intima of lesions were notably higher in atherosclerotic patients compared to those in healthy controls (7.51 pmol/mg vs 2.01 pmol/mg tissue, |
| Bangladesh, Begum et al. [##UREF##5##52##] | 60 patients diagnosed with AMI and 60 healthy controls. | The mean serum copper level was significantly higher in AMI patients compared to that in controls (146.49 ± 23.52 μg/dl vs 105.44 ± 24.15 μg/dl, |
| Grzegorz, Barbara et al. [##UREF##6##53##] | 74 participants of MI and 72 healthy controls. | Higher serum copper level was significantly associated with MI ( |
| Italy, Tarantino et al. [##REF##29405466##54##] | 100 obese patients who had a low prevalence of comorbidities. | Altered copper bioavailability was negatively associated with carotid IMT ( |
| Serbia, Tasić et al. [##REF##26554112##55##] | 91 patients (mean age 64 ± 7) with carotid atherosclerosis and 27 patients (mean age 58 ± 9) without carotid atherosclerosis. | Patients with hemorrhagic plaque had significantly higher serum copper levels compared to those with calcified plaque; high copper concentrations may contribute to atherosclerosis. |
Evidence linking copper excess and atherosclerosis.
| Country, author, year | Methods and study population | Result |
|---|---|---|
| Finland, Reunanen et al. [##REF##8862478##47##] | 230 men who died from CVDs and 298 matched controls; 10 years follow up. | The adjusted relative risk of CHD mortality between the highest and lowest tertiles of serum copper were 2.86 ( |
| United States, Ford, et al. [##REF##10905530##48##] | 4,574 participants aged ≥ 30 years; 151 died from CHD during 16-year follow-up. | There was an ~5% increase in serum copper levels among individuals who died from CHD compared to those who did not ( |
| Dutch, Kok et al. [##REF##3394701##49##] | Cancer ( | Individuals with elevated serum copper levels (above 1.43 mg/liter) exhibited a roughly four times higher adjusted risk of mortality from cancer and CVD compared to those with serum copper levels within the normal range. |
| Eastern Finland, Salonen et al. [##REF##1877585##50##] | 1666 randomly selected men aged 42, 48, 54, or 60 years with no symptomatic IHD. | Elevated serum copper level is a risk factor for IHD that acts independently, as shown by a 3.5-fold to 4.0-fold increased risk of AMI associated with high serum copper levels (1.02-1.16 mg/liter and 1.17 mg/liter or more). |
| Nadina, Robyn et al. [##REF##15001454##51##] | Atherosclerotic patients aged 67.9 ± 8.2 years and healthy controls. | Copper levels in the intima of lesions were notably higher in atherosclerotic patients compared to those in healthy controls (7.51 pmol/mg vs 2.01 pmol/mg tissue, |
| Bangladesh, Begum et al. [##UREF##5##52##] | 60 patients diagnosed with AMI and 60 healthy controls. | The mean serum copper level was significantly higher in AMI patients compared to that in controls (146.49 ± 23.52 μg/dl vs 105.44 ± 24.15 μg/dl, |
| Grzegorz, Barbara et al. [##UREF##6##53##] | 74 participants of MI and 72 healthy controls. | Higher serum copper level was significantly associated with MI ( |
| Italy, Tarantino et al. [##REF##29405466##54##] | 100 obese patients who had a low prevalence of comorbidities. | Altered copper bioavailability was negatively associated with carotid IMT ( |
| Serbia, Tasić et al. [##REF##26554112##55##] | 91 patients (mean age 64 ± 7) with carotid atherosclerosis and 27 patients (mean age 58 ± 9) without carotid atherosclerosis. | Patients with hemorrhagic plaque had significantly higher serum copper levels compared to those with calcified plaque; high copper concentrations may contribute to atherosclerosis. |
CVDs, cardiovascular disease; CHD, coronary heart disease; AMI, acute myocardial infarction; IHD, ischemic heart disease; IMT, intima-media thickness.
CVDs, cardiovascular disease; CHD, coronary heart disease; AMI, acute myocardial infarction; IHD, ischemic heart disease; IMT, intima-media thickness.
Fish skin has been used as a biological dressing in the management of burns##UREF##0##1##–##REF##34332229##9## and wounds##REF##33426019##10##. It has also been used as a biological graft in neovaginoplasty in cases of vaginal agenesis##REF##31546063##11##,##REF##31103284##12##. The histological characteristics of fish skin and human skin are similar##REF##29380095##13##. The fish skin contains a high amount of collagen, making it a suitable biomaterial for tissue engineering##REF##34332229##9##,##REF##29380095##13##,##REF##26798644##14##.
","Fish skin perishability still poses the most enormous preservation difficulties##REF##32466909##15##. It must be kept chilled or frozen, and even then, it has a very short shelf life##UREF##3##16##. Several diverse damage mechanisms, including microbiological spoilage, autolytic degradation, and lipid oxidation, are responsible for the depreciating of fresh fish skin during storage##UREF##4##17##. Therefore, most clinical practices have used fish skin grafts in fresh form for wound and burn management##UREF##0##1##,##REF##32603408##3##,##REF##31900475##5##–##REF##33426019##10##.
","Lyophilization, known also as freeze drying or cryodesiccation, is a low-temperature dehydration process involving freezing the product, lowering the pressure, then removing the ice by sublimation. This is in contrast to dehydration by most conventional methods use heat to evaporate water##UREF##5##18##.
","Lyophilization can be a potential method for the long-term storage of fish skin grafts. In a previous study, lyophilization was addressed as a method for preserving fish skin grafts##REF##33052795##2##. However, applying several detergents and sterilizing agents (e.g., chlorhexidine) to fish skin before lyophilization raises concerns about the integrity of collagen and the targeted material in the fish skin##REF##33052795##2##,##REF##34332229##9##,##REF##33426019##10##,##REF##29380095##13##. Moreover, the histopathology and microbiology of these lyophilized fish skin grafts were not evaluated before being used clinically.
","The microbial colonization of the wound is an essential factor that affects the healing process of wounds##REF##29649179##19##. Different sterilization methods have been described for biological dressings. These include physical methods such as irradiation and chemical techniques including treatment with chlorhexidine, povidone iodine, ethylene oxide gas, silver nanoparticles, and ozone##REF##33426019##10##,##REF##29380095##13##,##REF##16023938##20##. Gamma irradiation represents an effective sterilization method as it has direct and indirect effects on microbial DNA##REF##15916651##21##. Different lengths of Gamma irradiation have been used to sterilize fish skin (25, 30, and 50 kGy) without reference to its impact on collagen content##UREF##0##1##,##REF##33052795##2##,##REF##29380095##13## or antimicrobial efficiency##REF##33052795##2##,##REF##34332229##9##.
","The current study’s objectives are (1): to describe the optimized method of Tilapia skin lyophilization and (2): to define the standard length of gamma irradiation for sterilization of lyophilized Tilapia skin with special consideration to collagen integrity and microbial count of fish skin.
"],"string":"[\n \"Fish skin has been used as a biological dressing in the management of burns##UREF##0##1##–##REF##34332229##9## and wounds##REF##33426019##10##. It has also been used as a biological graft in neovaginoplasty in cases of vaginal agenesis##REF##31546063##11##,##REF##31103284##12##. The histological characteristics of fish skin and human skin are similar##REF##29380095##13##. The fish skin contains a high amount of collagen, making it a suitable biomaterial for tissue engineering##REF##34332229##9##,##REF##29380095##13##,##REF##26798644##14##.
\",\n \"Fish skin perishability still poses the most enormous preservation difficulties##REF##32466909##15##. It must be kept chilled or frozen, and even then, it has a very short shelf life##UREF##3##16##. Several diverse damage mechanisms, including microbiological spoilage, autolytic degradation, and lipid oxidation, are responsible for the depreciating of fresh fish skin during storage##UREF##4##17##. Therefore, most clinical practices have used fish skin grafts in fresh form for wound and burn management##UREF##0##1##,##REF##32603408##3##,##REF##31900475##5##–##REF##33426019##10##.
\",\n \"Lyophilization, known also as freeze drying or cryodesiccation, is a low-temperature dehydration process involving freezing the product, lowering the pressure, then removing the ice by sublimation. This is in contrast to dehydration by most conventional methods use heat to evaporate water##UREF##5##18##.
\",\n \"Lyophilization can be a potential method for the long-term storage of fish skin grafts. In a previous study, lyophilization was addressed as a method for preserving fish skin grafts##REF##33052795##2##. However, applying several detergents and sterilizing agents (e.g., chlorhexidine) to fish skin before lyophilization raises concerns about the integrity of collagen and the targeted material in the fish skin##REF##33052795##2##,##REF##34332229##9##,##REF##33426019##10##,##REF##29380095##13##. Moreover, the histopathology and microbiology of these lyophilized fish skin grafts were not evaluated before being used clinically.
\",\n \"The microbial colonization of the wound is an essential factor that affects the healing process of wounds##REF##29649179##19##. Different sterilization methods have been described for biological dressings. These include physical methods such as irradiation and chemical techniques including treatment with chlorhexidine, povidone iodine, ethylene oxide gas, silver nanoparticles, and ozone##REF##33426019##10##,##REF##29380095##13##,##REF##16023938##20##. Gamma irradiation represents an effective sterilization method as it has direct and indirect effects on microbial DNA##REF##15916651##21##. Different lengths of Gamma irradiation have been used to sterilize fish skin (25, 30, and 50 kGy) without reference to its impact on collagen content##UREF##0##1##,##REF##33052795##2##,##REF##29380095##13## or antimicrobial efficiency##REF##33052795##2##,##REF##34332229##9##.
\",\n \"The current study’s objectives are (1): to describe the optimized method of Tilapia skin lyophilization and (2): to define the standard length of gamma irradiation for sterilization of lyophilized Tilapia skin with special consideration to collagen integrity and microbial count of fish skin.
\"\n]"},"methods":{"kind":"list like","value":["The Research Ethics Committee (REC) of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt, has approved all the procedures in this study in accordance with the Egyptian bylaws and OIE animal welfare standards for animal care and use in research and education. All methods were performed in accordance with relevant guidelines and regulations.
","The fish skin was collected from fresh Nile tilapia
Lyophilization was carried out in a freeze dryer (LYOQUEST, SPAIN, Serial No.: 1812, manufacture company: Telstar). Fresh fish skin strips were placed in the flasks of the freeze dryer apparatus at − 80 °C for 24 h. Skin strips were then subjected to a cold vacuum for 5–6 h. Lyophilized skin strips were then vacuum-packed (Fig. ##FIG##0##1##A–C).
","Gamma irradiation was performed at a gamma station (60Co gamma cell (2000 Ci), 30 ± 5 °C, 1.5 Gy/s, 150 rad/s), the National Center for Radiation Research and Technology (NCRRT) of the Egyptian Atomic Energy Authority, Cairo, Egypt. The vacuum-packed lyophilized Tilapia skin was subjected to Cobalt 60 γ sterilization at 5, 10, and 25 kGy##REF##33052795##2##. Sterilized skin strips subjected to microbiological and histopathological evaluations after each treatment 15- and 30-days post-sterilization (3 skin strips each).
","Following Morton et al.##UREF##6##22##, the dilution plate method was employed to count the lyophilized fish skin microbiology under different lengths of gamma irradiation (0, 5, 10, and 25 KGy) after 15 and 30 days of irradiation. Fish skin strips were swabbed with a sterilized swab needle and suspended in 10 mL of sterile saline solution. For microbiological counting and identification, various media types were employed; nutrient agar and MacConkey agar media to count all aerobic bacteria, yeast malt extract medium (YME) to count the aerobic yeasts, and potato dextrose agar medium (PDA) to count the fungi##UREF##7##23##–##UREF##9##25##. One milliliter (1 mL) of the saline-swabbed solution was added to sterilized Petri dishes before the dishes were filled with sterilized isolation medium, three replicates were performed for each medium, and left to solidify. For bacteria, plates were incubated for 24 h at 35 °C, for yeasts, for 72 h at 28 °C, and for fungi, 7 days at 28 °C. The developed colony count was estimated as CFU/cm2. Using the same media, developed colonies with variations in their morphological characteristics, such as size, color, colony edge, and pigmentation, were sub-cultured and purified for identification using Bergey’s Manual of Systematic Bacteriology##UREF##10##26##, Yeasts: characteristics and identification##UREF##8##24## and fungal identification##UREF##11##27##.
","Fish skin strips (0.5 × 0.5 cm) were fixed in 10% neutral buffered formalin, routinely processed, and subsequently embedded in paraffin. Afterwards, they were sectioned into 5 μm thick sections and stained with Mayer’s hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma, Missouri, USA). After microscopically examining the slides, histological evaluations were performed blindly on coded samples, with a comparison to control group. Collagen fibers integrity and organization were assessed histologically based on 0–3 scale##REF##33426019##10##,##REF##21495863##28##,##REF##28255657##29##. Collagen fibers integrity scores: 0 = continue, long fiber, 1 = slightly fragmented, 2 = moderately fragmented, 3 = severely fragmented. Collagen fibers organization scores: 0 = compact and parallel, 1 = slightly loose and wave, 2 = moderately loose, wavy and cross to each other, 3 = no identifiable pattern.
","The collagen content evaluation was carried out by the Gomori’s trichrome stain##REF##15432364##30##. After deparaffinization in xylene, paraffin-embedded sections were and rehydrated in a graded series of ethanol solutions (into 0.1 M phosphate-buffered saline (PBS), pH 7.2) to distilled water. Sections were stained with Gomori’s trichrome according to the manufacturer’s protocol, dehydrated in graded alcohol, made transparent with xylene, and mounted. Slides were then microscopically examined to verify collagen staining with green. The collagen content was evaluated based on the depth of the green staining.
","The percentage of collagen-positive area was calculated by ImageJ (1.48v) using threshold area fraction determination. The amount of collagen was expressed as a percentage from the total number of pixels in the optical view as a percentage and expressed as mean ± SEM.
","One-way ANOVA followed by Tukey's post hoc test was performed using GraphPad Prism software version 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA).
The Research Ethics Committee (REC) of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt, has approved all the procedures in this study in accordance with the Egyptian bylaws and OIE animal welfare standards for animal care and use in research and education. All methods were performed in accordance with relevant guidelines and regulations.
\",\n \"The fish skin was collected from fresh Nile tilapia
Lyophilization was carried out in a freeze dryer (LYOQUEST, SPAIN, Serial No.: 1812, manufacture company: Telstar). Fresh fish skin strips were placed in the flasks of the freeze dryer apparatus at − 80 °C for 24 h. Skin strips were then subjected to a cold vacuum for 5–6 h. Lyophilized skin strips were then vacuum-packed (Fig. ##FIG##0##1##A–C).
\",\n \"Gamma irradiation was performed at a gamma station (60Co gamma cell (2000 Ci), 30 ± 5 °C, 1.5 Gy/s, 150 rad/s), the National Center for Radiation Research and Technology (NCRRT) of the Egyptian Atomic Energy Authority, Cairo, Egypt. The vacuum-packed lyophilized Tilapia skin was subjected to Cobalt 60 γ sterilization at 5, 10, and 25 kGy##REF##33052795##2##. Sterilized skin strips subjected to microbiological and histopathological evaluations after each treatment 15- and 30-days post-sterilization (3 skin strips each).
\",\n \"Following Morton et al.##UREF##6##22##, the dilution plate method was employed to count the lyophilized fish skin microbiology under different lengths of gamma irradiation (0, 5, 10, and 25 KGy) after 15 and 30 days of irradiation. Fish skin strips were swabbed with a sterilized swab needle and suspended in 10 mL of sterile saline solution. For microbiological counting and identification, various media types were employed; nutrient agar and MacConkey agar media to count all aerobic bacteria, yeast malt extract medium (YME) to count the aerobic yeasts, and potato dextrose agar medium (PDA) to count the fungi##UREF##7##23##–##UREF##9##25##. One milliliter (1 mL) of the saline-swabbed solution was added to sterilized Petri dishes before the dishes were filled with sterilized isolation medium, three replicates were performed for each medium, and left to solidify. For bacteria, plates were incubated for 24 h at 35 °C, for yeasts, for 72 h at 28 °C, and for fungi, 7 days at 28 °C. The developed colony count was estimated as CFU/cm2. Using the same media, developed colonies with variations in their morphological characteristics, such as size, color, colony edge, and pigmentation, were sub-cultured and purified for identification using Bergey’s Manual of Systematic Bacteriology##UREF##10##26##, Yeasts: characteristics and identification##UREF##8##24## and fungal identification##UREF##11##27##.
\",\n \"Fish skin strips (0.5 × 0.5 cm) were fixed in 10% neutral buffered formalin, routinely processed, and subsequently embedded in paraffin. Afterwards, they were sectioned into 5 μm thick sections and stained with Mayer’s hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma, Missouri, USA). After microscopically examining the slides, histological evaluations were performed blindly on coded samples, with a comparison to control group. Collagen fibers integrity and organization were assessed histologically based on 0–3 scale##REF##33426019##10##,##REF##21495863##28##,##REF##28255657##29##. Collagen fibers integrity scores: 0 = continue, long fiber, 1 = slightly fragmented, 2 = moderately fragmented, 3 = severely fragmented. Collagen fibers organization scores: 0 = compact and parallel, 1 = slightly loose and wave, 2 = moderately loose, wavy and cross to each other, 3 = no identifiable pattern.
\",\n \"The collagen content evaluation was carried out by the Gomori’s trichrome stain##REF##15432364##30##. After deparaffinization in xylene, paraffin-embedded sections were and rehydrated in a graded series of ethanol solutions (into 0.1 M phosphate-buffered saline (PBS), pH 7.2) to distilled water. Sections were stained with Gomori’s trichrome according to the manufacturer’s protocol, dehydrated in graded alcohol, made transparent with xylene, and mounted. Slides were then microscopically examined to verify collagen staining with green. The collagen content was evaluated based on the depth of the green staining.
\",\n \"The percentage of collagen-positive area was calculated by ImageJ (1.48v) using threshold area fraction determination. The amount of collagen was expressed as a percentage from the total number of pixels in the optical view as a percentage and expressed as mean ± SEM.
\",\n \"One-way ANOVA followed by Tukey's post hoc test was performed using GraphPad Prism software version 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA).
Gamma irradiation exhibited an efficient sterilizing effect on fish skin surface microbiota. Different lengths of gamma irradiation (5, 10, and 25 KGy) were applied to the lyophilized fish skin. The microbial counts of aerobic bacteria, aerobic yeasts, and fungi were detected 15- and 30- days after the irradiation as cleared in Fig. ##FIG##1##2##A–C. It was clear that gamma irradiation is a great microbial sterilizer, especially with the high length of 25 KGy that inhibited the aerobic bacterial counts significantly by 98.53% and 98.96%, aerobic yeast counts by 99.2% and 99.8%, and fungal counts by 98.48% and 99.25% 15- and 30- days after irradiation, respectively, (
By increasing the gamma irradiation, the total counts of aerobic bacteria decrease dramatically giving 92.67 ± 2.62 (88.36% inhibition), 39.33 ± 4.92 (95.06% inhibition), and 11.67 ± 1.7 (98.53% inhibition) at 5, 10, and 25 KGy 15 days post-irradiation, respectively, (
For yeasts, it was clear that by increasing the length of gamma irradiation, the total counts of yeasts decreased significantly after 15- and 30-days irradiation giving 208.67 ± 6.13 (55.25% inhibition), 19 ± 1.63 (95.93% inhibition), and 3.67 ± 0.47 (99.2% inhibition) at 5, 10, and 25 KGy, respectively, at 15 days (
Filamentous fungi had the same criteria as bacteria and yeasts, the total counts of fungi decreased significantly (
By investigating the microbial species present on the fish skin,
In the control group, fresh fish skin was examined histologically, and the collagen fibers were tightly packed, well-organized, parallel-distributed, and no evidence of disaggregation (Fig. ##FIG##2##3##A,B). In addition, the lyophilized fish skin rehydrated in normal saline for 15 min showed the preservation of the collagen fibers in a well-organized pattern with no signs of disaggregation (Fig. ##FIG##2##3##C,D).
","In the lyophilized fish skin subjected to gamma irradiation at 5, 10, and 25 kGy, histological examination of the fish skin 15-day post-sterilization revealed slightly to moderately disorganized and disaggregated in irradiated skin at 5 and 25 kGy (Figs. ##FIG##3##4##A,E, ##FIG##5##6##A,B) (
The collagen fibers were also stained with Gomori’s trichrome stain and the collagen intensity in the skin was measured using ImageJ (1.48v). At 15- and 30-days post-sterilization, the collagen disposition and intensity in skin samples irradiated at 5 and 10 kGy did not show significant differences compared with the control (Figs. ##FIG##3##4##B,D, ##FIG##4##5##B,D, ##FIG##5##6##C). However, collagen deposition and intensity were significantly decreased in skin samples exposed to 25 kGy after 15 and 30 days of irradiation (Figs. ##FIG##3##4##F, ##FIG##4##5##F, ##FIG##5##6##C) (
Gamma irradiation exhibited an efficient sterilizing effect on fish skin surface microbiota. Different lengths of gamma irradiation (5, 10, and 25 KGy) were applied to the lyophilized fish skin. The microbial counts of aerobic bacteria, aerobic yeasts, and fungi were detected 15- and 30- days after the irradiation as cleared in Fig. ##FIG##1##2##A–C. It was clear that gamma irradiation is a great microbial sterilizer, especially with the high length of 25 KGy that inhibited the aerobic bacterial counts significantly by 98.53% and 98.96%, aerobic yeast counts by 99.2% and 99.8%, and fungal counts by 98.48% and 99.25% 15- and 30- days after irradiation, respectively, (
By increasing the gamma irradiation, the total counts of aerobic bacteria decrease dramatically giving 92.67 ± 2.62 (88.36% inhibition), 39.33 ± 4.92 (95.06% inhibition), and 11.67 ± 1.7 (98.53% inhibition) at 5, 10, and 25 KGy 15 days post-irradiation, respectively, (
For yeasts, it was clear that by increasing the length of gamma irradiation, the total counts of yeasts decreased significantly after 15- and 30-days irradiation giving 208.67 ± 6.13 (55.25% inhibition), 19 ± 1.63 (95.93% inhibition), and 3.67 ± 0.47 (99.2% inhibition) at 5, 10, and 25 KGy, respectively, at 15 days (
Filamentous fungi had the same criteria as bacteria and yeasts, the total counts of fungi decreased significantly (
By investigating the microbial species present on the fish skin,
In the control group, fresh fish skin was examined histologically, and the collagen fibers were tightly packed, well-organized, parallel-distributed, and no evidence of disaggregation (Fig. ##FIG##2##3##A,B). In addition, the lyophilized fish skin rehydrated in normal saline for 15 min showed the preservation of the collagen fibers in a well-organized pattern with no signs of disaggregation (Fig. ##FIG##2##3##C,D).
\",\n \"In the lyophilized fish skin subjected to gamma irradiation at 5, 10, and 25 kGy, histological examination of the fish skin 15-day post-sterilization revealed slightly to moderately disorganized and disaggregated in irradiated skin at 5 and 25 kGy (Figs. ##FIG##3##4##A,E, ##FIG##5##6##A,B) (
The collagen fibers were also stained with Gomori’s trichrome stain and the collagen intensity in the skin was measured using ImageJ (1.48v). At 15- and 30-days post-sterilization, the collagen disposition and intensity in skin samples irradiated at 5 and 10 kGy did not show significant differences compared with the control (Figs. ##FIG##3##4##B,D, ##FIG##4##5##B,D, ##FIG##5##6##C). However, collagen deposition and intensity were significantly decreased in skin samples exposed to 25 kGy after 15 and 30 days of irradiation (Figs. ##FIG##3##4##F, ##FIG##4##5##F, ##FIG##5##6##C) (
Recently, biological dressings have become indispensable in modern strategies of burn and wound management. The current study investigated the use of lyophilization as a method for the long-term preservation of fish skin grafts in a manner that maintains the integrity of fish skin collagen. Moreover, the study revealed that gamma irradiation at a length of 10 KGy is optimal for sterilizing the lyophilized fish skin grafts without disrupting the collagen content.
","Autologous skin grafts have a limited availability and add an additional scaring##REF##15241002##31##. Allografts have many limitations such as the finding of suitable donors and the risk of infection transmission, especially, viral infections##REF##16023938##20##. Therefore, various xenografts have been described as biological wound dressings in humans. This includes the bovine embryo skin##UREF##12##32##, bovine amnion##UREF##13##33##, canine skin##UREF##14##34##, frog skin##UREF##15##35##, and porcine skin##REF##11996864##36##–##REF##33946298##38##. However, there are concerns about their clinical application due to immunological causes, zoonotic risk, and religious beliefs##REF##16023938##20##,##REF##34834962##39##,##REF##10484730##40##.
","The fish skin was found to be consistent with human skin##UREF##2##8##,##REF##29380095##13## and has a higher collagen content than other skins##REF##34332229##9##,##REF##29380095##13##,##REF##26798644##14##, which is a potential wound healing promotor##UREF##16##41##. Moreover, fish skin has advantages such as low antigenicity, a high potential adherent to the skin, a lower risk of transmitting diseases, and a degree of moisture symbolizing the human skin##UREF##2##8##,##UREF##17##42##. Fish skin graft is highly porous. Since the pore size is within the range of typical cell size, fish skin is well suited to support cell ingrowth##REF##28291503##43##. Therefore, the fish skin has recently been suggested as a potential xenograft##UREF##0##1##–##UREF##1##7##,##REF##34332229##9##–##REF##31103284##12##.
","Although the tilapia skin contains a normal non-infectious microbiota, it may be exposed to microbial contaminants from a contaminated water environment##UREF##1##7##,##REF##28757200##44##. Bacteria, yeasts, fungi, and viruses represent serious fish contaminants##REF##32781103##45##. Enterobacteriaceae, yeast, and aerobic spoilers are the main spoilage microorganisms of fish during the storage##REF##8913813##46##. Due to the high-water activity, and low acidity in the fish skin, bacteria proliferate quickly causing its spoilage##REF##33916441##47##. Therefore, in most studies, Tilapia skin was used as a biological dressing graft for burn or wound treatment shortly after its processing##UREF##0##1##,##REF##32603408##3##–##UREF##1##7##,##REF##34332229##9##,##REF##33426019##10##. This creates an urgent need to find a way for long-term preservation and storage of the fish skin grafts.
","Various methods have been used for the preservation of different biological dressings, such as cryopreservation##REF##24018216##48##,##REF##9568334##49## and glycerolization##REF##33890902##4##,##REF##31900475##5##,##UREF##1##7##,##REF##34332229##9##,##REF##28196742##50##–##REF##24070848##52##. Although these methods could prolong the shelf life of biological dressings, they may also affect their effectiveness##REF##9063797##53##.
","Lyophilization or freeze-drying is a specific dehydration process, which provides benefits to the final product through improved composition stability and reduced water content to low levels, which allow the cell to survive during long storage##UREF##5##18##,##UREF##18##54##. Microbial cells need high stabilization processes to survive on the lyophilized parts. Otherwise, they will be damaged and/or die##REF##28446905##55##.
","Lyophilization has been addressed in previous studies for fish skin preservation##REF##33052795##2##,##REF##32603408##3##. However, using a rigorous process of chemical sterilization of the fish skin before the lyophilization raises concerns about collagen integrity and other biological components potential for healing##REF##28291503##43##, especially, since these studies were not followed by histological evaluation before clinical application##UREF##0##1##,##REF##33052795##2##,##UREF##1##7##. Moreover, using several sterilizing steps with several chemicals make such procedures cost-effective and impractical for industrial application. Chlorhexidine is commonly used to sterilize and disinfect grafting procedures. However, many bacterial spores or mycobacteria are chlorhexidine-resistant##REF##11556507##56##,##REF##17908945##57##. It also has a low activity against viruses. A potential limitation of chlorhexidine is its cytotoxicity and alteration in the biochemical properties of collagen##REF##33426019##10##,##REF##22523372##58##.
","Here, this study describes the lyophilization of fish skin without using any disinfectants during the processing of fish skin grafts to ensure the collagen safety and subsequently its effectiveness. This was confirmed by histological and histochemical evaluations of the lyophilized fish skin after rehydration in normal saline for 15 min that there was no change in the arrangement and content of the collagen fibers of the fish skin.
","Sterilization of fish skin with gamma irradiation represents an efficient sterilizing method for the fish skin microbiota. High-energy gamma irradiations are released by several radioisotopes, including the comparatively cheap byproducts of nuclear fission Caesium-137 (137Cs) and Cobalt-60 (60Co). By subjecting the plentiful, non-radioactive Co-59 isotope to neutron irradiation inside a nuclear reactor, radioactive Co is created. Then, after emitting one electron and two gamma rays with energies of 1.17 MeV and 1.33 MeV, Co-60 atoms decompose into nonradioactive Ni-60 atoms. Gamma rays don't have enough energy to cause radioactivity in other materials because they are released isotropically. Similar to x-rays, gamma rays have a shorter wavelength and higher energy. Thus, Gamma rays are appealing for industrial sterilization of materials with a significant thickness or volume, such as packaged food, medical equipment, or medical supplies##UREF##19##59##.
","The combination of lyophilization and gamma irradiation inhibited the microbial growth by 99.8%. Gram-negative and Gram-positive bacteria, as proteobacteria and the Lactobacillales, are more vulnerable to Gamma irradiation than spore-forming bacteria, such as Bacilli and Clostridia##REF##31121676##60##. This is in accordance with our findings that
An infection is deemed to exist when there are more than 105 CFU of bacteria per gram of skin graft##REF##11292638##63##. On wound beds with more than 105 bacteria/g of tissue, the absorption of a skin graft is decreased##UREF##21##64##. In our results, the total bacterial counts were less than 35 CFU/cm2 at 10 and 25 kGy after 30 days of sterilization, indicating the high sterilizing efficiency of these irradiations; however, 10 kGy is preferred as it maintains the collagen integrity and content. Also, the total counts of yeasts and fungi were less than 10 and 16 CFU/cm2, respectively, at 10 and 25 kGy after 30 days. In accordance with our results, clinical investigations found that skin transplant failure occurred in wounds that were significantly infected with 107 pathogens##REF##1098503##65##,##UREF##22##66##. Also, Nsaful et al.##UREF##23##67## found that wound graft failure mainly occurs by the microbial contamination, especially bacterial contamination with the bacteria counts ranged from 3.7 × 105 to 9 × 105 CFU/cm2.
","In accordance, histological evaluation of the fish skin grafts revealed that the exposure to gamma irradiation at 25 kGy caused the collagen fibers to dissociate and disintegrate with a reduction in the collagen intensity, whereas exposure at 5 kGy altered collagen fiber arrangement and integrity. However, the exposure to gamma irradiation at 10 kGy showed the preferred intensity since it preserved the collagen fiber content and intensity in the skin graft.
","The limitation of this study is the lack of histological and microbiological assessment of the sterilized vacuum-packed lyophilized fish skin grafts on further long period (6 and 12 months). Therefore, future studies should be conducted to address this concern.
"],"string":"[\n \"Recently, biological dressings have become indispensable in modern strategies of burn and wound management. The current study investigated the use of lyophilization as a method for the long-term preservation of fish skin grafts in a manner that maintains the integrity of fish skin collagen. Moreover, the study revealed that gamma irradiation at a length of 10 KGy is optimal for sterilizing the lyophilized fish skin grafts without disrupting the collagen content.
\",\n \"Autologous skin grafts have a limited availability and add an additional scaring##REF##15241002##31##. Allografts have many limitations such as the finding of suitable donors and the risk of infection transmission, especially, viral infections##REF##16023938##20##. Therefore, various xenografts have been described as biological wound dressings in humans. This includes the bovine embryo skin##UREF##12##32##, bovine amnion##UREF##13##33##, canine skin##UREF##14##34##, frog skin##UREF##15##35##, and porcine skin##REF##11996864##36##–##REF##33946298##38##. However, there are concerns about their clinical application due to immunological causes, zoonotic risk, and religious beliefs##REF##16023938##20##,##REF##34834962##39##,##REF##10484730##40##.
\",\n \"The fish skin was found to be consistent with human skin##UREF##2##8##,##REF##29380095##13## and has a higher collagen content than other skins##REF##34332229##9##,##REF##29380095##13##,##REF##26798644##14##, which is a potential wound healing promotor##UREF##16##41##. Moreover, fish skin has advantages such as low antigenicity, a high potential adherent to the skin, a lower risk of transmitting diseases, and a degree of moisture symbolizing the human skin##UREF##2##8##,##UREF##17##42##. Fish skin graft is highly porous. Since the pore size is within the range of typical cell size, fish skin is well suited to support cell ingrowth##REF##28291503##43##. Therefore, the fish skin has recently been suggested as a potential xenograft##UREF##0##1##–##UREF##1##7##,##REF##34332229##9##–##REF##31103284##12##.
\",\n \"Although the tilapia skin contains a normal non-infectious microbiota, it may be exposed to microbial contaminants from a contaminated water environment##UREF##1##7##,##REF##28757200##44##. Bacteria, yeasts, fungi, and viruses represent serious fish contaminants##REF##32781103##45##. Enterobacteriaceae, yeast, and aerobic spoilers are the main spoilage microorganisms of fish during the storage##REF##8913813##46##. Due to the high-water activity, and low acidity in the fish skin, bacteria proliferate quickly causing its spoilage##REF##33916441##47##. Therefore, in most studies, Tilapia skin was used as a biological dressing graft for burn or wound treatment shortly after its processing##UREF##0##1##,##REF##32603408##3##–##UREF##1##7##,##REF##34332229##9##,##REF##33426019##10##. This creates an urgent need to find a way for long-term preservation and storage of the fish skin grafts.
\",\n \"Various methods have been used for the preservation of different biological dressings, such as cryopreservation##REF##24018216##48##,##REF##9568334##49## and glycerolization##REF##33890902##4##,##REF##31900475##5##,##UREF##1##7##,##REF##34332229##9##,##REF##28196742##50##–##REF##24070848##52##. Although these methods could prolong the shelf life of biological dressings, they may also affect their effectiveness##REF##9063797##53##.
\",\n \"Lyophilization or freeze-drying is a specific dehydration process, which provides benefits to the final product through improved composition stability and reduced water content to low levels, which allow the cell to survive during long storage##UREF##5##18##,##UREF##18##54##. Microbial cells need high stabilization processes to survive on the lyophilized parts. Otherwise, they will be damaged and/or die##REF##28446905##55##.
\",\n \"Lyophilization has been addressed in previous studies for fish skin preservation##REF##33052795##2##,##REF##32603408##3##. However, using a rigorous process of chemical sterilization of the fish skin before the lyophilization raises concerns about collagen integrity and other biological components potential for healing##REF##28291503##43##, especially, since these studies were not followed by histological evaluation before clinical application##UREF##0##1##,##REF##33052795##2##,##UREF##1##7##. Moreover, using several sterilizing steps with several chemicals make such procedures cost-effective and impractical for industrial application. Chlorhexidine is commonly used to sterilize and disinfect grafting procedures. However, many bacterial spores or mycobacteria are chlorhexidine-resistant##REF##11556507##56##,##REF##17908945##57##. It also has a low activity against viruses. A potential limitation of chlorhexidine is its cytotoxicity and alteration in the biochemical properties of collagen##REF##33426019##10##,##REF##22523372##58##.
\",\n \"Here, this study describes the lyophilization of fish skin without using any disinfectants during the processing of fish skin grafts to ensure the collagen safety and subsequently its effectiveness. This was confirmed by histological and histochemical evaluations of the lyophilized fish skin after rehydration in normal saline for 15 min that there was no change in the arrangement and content of the collagen fibers of the fish skin.
\",\n \"Sterilization of fish skin with gamma irradiation represents an efficient sterilizing method for the fish skin microbiota. High-energy gamma irradiations are released by several radioisotopes, including the comparatively cheap byproducts of nuclear fission Caesium-137 (137Cs) and Cobalt-60 (60Co). By subjecting the plentiful, non-radioactive Co-59 isotope to neutron irradiation inside a nuclear reactor, radioactive Co is created. Then, after emitting one electron and two gamma rays with energies of 1.17 MeV and 1.33 MeV, Co-60 atoms decompose into nonradioactive Ni-60 atoms. Gamma rays don't have enough energy to cause radioactivity in other materials because they are released isotropically. Similar to x-rays, gamma rays have a shorter wavelength and higher energy. Thus, Gamma rays are appealing for industrial sterilization of materials with a significant thickness or volume, such as packaged food, medical equipment, or medical supplies##UREF##19##59##.
\",\n \"The combination of lyophilization and gamma irradiation inhibited the microbial growth by 99.8%. Gram-negative and Gram-positive bacteria, as proteobacteria and the Lactobacillales, are more vulnerable to Gamma irradiation than spore-forming bacteria, such as Bacilli and Clostridia##REF##31121676##60##. This is in accordance with our findings that
An infection is deemed to exist when there are more than 105 CFU of bacteria per gram of skin graft##REF##11292638##63##. On wound beds with more than 105 bacteria/g of tissue, the absorption of a skin graft is decreased##UREF##21##64##. In our results, the total bacterial counts were less than 35 CFU/cm2 at 10 and 25 kGy after 30 days of sterilization, indicating the high sterilizing efficiency of these irradiations; however, 10 kGy is preferred as it maintains the collagen integrity and content. Also, the total counts of yeasts and fungi were less than 10 and 16 CFU/cm2, respectively, at 10 and 25 kGy after 30 days. In accordance with our results, clinical investigations found that skin transplant failure occurred in wounds that were significantly infected with 107 pathogens##REF##1098503##65##,##UREF##22##66##. Also, Nsaful et al.##UREF##23##67## found that wound graft failure mainly occurs by the microbial contamination, especially bacterial contamination with the bacteria counts ranged from 3.7 × 105 to 9 × 105 CFU/cm2.
\",\n \"In accordance, histological evaluation of the fish skin grafts revealed that the exposure to gamma irradiation at 25 kGy caused the collagen fibers to dissociate and disintegrate with a reduction in the collagen intensity, whereas exposure at 5 kGy altered collagen fiber arrangement and integrity. However, the exposure to gamma irradiation at 10 kGy showed the preferred intensity since it preserved the collagen fiber content and intensity in the skin graft.
\",\n \"The limitation of this study is the lack of histological and microbiological assessment of the sterilized vacuum-packed lyophilized fish skin grafts on further long period (6 and 12 months). Therefore, future studies should be conducted to address this concern.
\"\n]"},"conclusion":{"kind":"list like","value":["This study established an optimized method for tilapia skin lyophilization. It defined the standard length of gamma irradiation for sterilizing lyophilized tilapia skin, considering the collagen integrity and microbial count of fish skin. These processed, sterilized, vacuum-packed, lyophilized fish skin patches could be suitable for long-term storage for burn and wound management in hospitals and medical centers. Further clinical studies are still being conducted on this product.
"],"string":"[\n \"This study established an optimized method for tilapia skin lyophilization. It defined the standard length of gamma irradiation for sterilizing lyophilized tilapia skin, considering the collagen integrity and microbial count of fish skin. These processed, sterilized, vacuum-packed, lyophilized fish skin patches could be suitable for long-term storage for burn and wound management in hospitals and medical centers. Further clinical studies are still being conducted on this product.
\"\n]"},"front":{"kind":"list like","value":["The introduction of fish skin as a biological dressing for treating burns and wounds holds great promise, offering an alternative to existing management strategies. However, the risk of disease transmission is a significant concern. Therefore, this study aimed to examine how established sterilization and preservation procedures affected fish skin grafts' microbiological and histological properties for long-term usage. Lyophilization of the fish skin graft followed by rehydration in normal saline for 15 min did not change the collagen content. Furthermore, gamma irradiation of the lyophilized fish skin graft at different lengths 5, 10, and 25 KGy showed a significant reduction in microbial growth (aerobic bacteria, aerobic yeasts, and fungi) at 15- and 30 days after the irradiation. However, exposure to 10 KGy was found to be the most effective intensity among the different gamma irradiation lengths since it preserved the collagen fiber content and intensity in the lyophilized fish skin grafts at 15- and 30 days after the irradiation. These findings provide efficient preservation and sterilization methods for long-term usage of the fresh Tilapia skin grafts used for biological dressings.
","Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
"],"string":"[\n \"The introduction of fish skin as a biological dressing for treating burns and wounds holds great promise, offering an alternative to existing management strategies. However, the risk of disease transmission is a significant concern. Therefore, this study aimed to examine how established sterilization and preservation procedures affected fish skin grafts' microbiological and histological properties for long-term usage. Lyophilization of the fish skin graft followed by rehydration in normal saline for 15 min did not change the collagen content. Furthermore, gamma irradiation of the lyophilized fish skin graft at different lengths 5, 10, and 25 KGy showed a significant reduction in microbial growth (aerobic bacteria, aerobic yeasts, and fungi) at 15- and 30 days after the irradiation. However, exposure to 10 KGy was found to be the most effective intensity among the different gamma irradiation lengths since it preserved the collagen fiber content and intensity in the lyophilized fish skin grafts at 15- and 30 days after the irradiation. These findings provide efficient preservation and sterilization methods for long-term usage of the fresh Tilapia skin grafts used for biological dressings.
\",\n \"Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
\"\n]"},"body":{"kind":"list like","value":[],"string":"[]"},"back":{"kind":"list like","value":["A.I.: Conceptualization, Methodology, Validation, Data curation, Visualization, Investigation, Supervision, Writing- Original draft preparation, Writing- Reviewing and Editing. H.F.: Conceptualization, Methodology, Data curation, Investigation, Writing- Original draft preparation. G.A.-E.M.: Methodology, Visualization, Investigation, Writing- Original draft preparation. A.E.: Methodology, Validation, Data curation, Investigation, Writing- Original draft preparation. M.S.: Methodology, Validation, Data curation, Software, Visualization, Investigation, Supervision, Writing- Original draft preparation, Writing- Reviewing and Editing.
","Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
","All data generated or analyzed during this study are included in this published article.
","The authors declare no competing interests.
"],"string":"[\n \"A.I.: Conceptualization, Methodology, Validation, Data curation, Visualization, Investigation, Supervision, Writing- Original draft preparation, Writing- Reviewing and Editing. H.F.: Conceptualization, Methodology, Data curation, Investigation, Writing- Original draft preparation. G.A.-E.M.: Methodology, Visualization, Investigation, Writing- Original draft preparation. A.E.: Methodology, Validation, Data curation, Investigation, Writing- Original draft preparation. M.S.: Methodology, Validation, Data curation, Software, Visualization, Investigation, Supervision, Writing- Original draft preparation, Writing- Reviewing and Editing.
\",\n \"Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
\",\n \"All data generated or analyzed during this study are included in this published article.
\",\n \"The authors declare no competing interests.
\"\n]"},"figure":{"kind":"list like","value":["(
Total accounts (CFU/cm2 ± S.D) of aerobic bacteria (
(
Histological and histochemical evaluations of the lyophilized Tilapia fish skin submitted to different irradiation dosages 15-days post-sterilization. Hematoxylin and eosin stained sections were irradiated at 5 (
Histological and histochemical evaluations of the lyophilized Tilapia fish skin submitted to different irradiation dosages 30-days post-sterilization. Hematoxylin and eosin-stained sections were irradiated at 5 (
Evaluation of the collagen integrity, organization, and intensity in the lyophilized Tilapia fish skin submitted to different irradiation dosages. Collagen integrity and organization were evaluated based on a 0–3 scale as described in the materials and methods. The collagen intensity was quantified using ImageJ. The results are expressed as a percentage of the total number of pixels and are normalized to the control-treated group. Differences were evaluated using one
(
Total accounts (CFU/cm2 ± S.D) of aerobic bacteria (
(
Histological and histochemical evaluations of the lyophilized Tilapia fish skin submitted to different irradiation dosages 15-days post-sterilization. Hematoxylin and eosin stained sections were irradiated at 5 (
Histological and histochemical evaluations of the lyophilized Tilapia fish skin submitted to different irradiation dosages 30-days post-sterilization. Hematoxylin and eosin-stained sections were irradiated at 5 (
Evaluation of the collagen integrity, organization, and intensity in the lyophilized Tilapia fish skin submitted to different irradiation dosages. Collagen integrity and organization were evaluated based on a 0–3 scale as described in the materials and methods. The collagen intensity was quantified using ImageJ. The results are expressed as a percentage of the total number of pixels and are normalized to the control-treated group. Differences were evaluated using one
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
When matter vibrates, it transmits energy waves into our ears and produces sound. The sound changes depending on two factors: the material of the object and how slow or fast the object vibrates, making different sound waves. In other words, the object’s material affects the timbre, while the speed of object vibration changes the pitch. Sound event classification is a technique used to distinguish the actions involved in the audio. The action generated from our environment, for example: music genre, human speech, water running, animal sound, etc. Nowadays, sound event classification is a common issue, and is usually addressed in the following pipeline: (1) audio pre-processing: the audio data needs to be transfer to the proper signal that can be recognized by learning algorithm. (2) feature extraction: to make the classification algorithm more efficient and accurate, the high level represented features are extracted from the raw signal. For example, the Short-time Fourier Transform (STFT), and Constant-Q transform are famous feature extraction techniques. (3) Sound event algorithm: once the extracted feature is prepared, the algorithm is developed to predict the label of the sound event with the input features. In the early 20 s, experts focused on generating powerful audio descriptors##UREF##0##1##. These are usually divided into three sets: low-level features, score features, and mid-level features. Some hand-created music features are involved in these sets, for example: spectral centroid##UREF##1##2##, and pitch salience##REF##30949547##3##. Score features, such as mode##UREF##2##4## and pitch density##UREF##3##5##, are directly calculated from the musical score. In general, mid-level features##UREF##4##6## are intuitively understandable by the typical learner. Nowadays, the deep learning algorithm has made tremendous progress by integrating feature extraction and decision-making algorithms for classification. With many trainable parameters, the network can directly learn to map the extracted features from input to target labels. Also, as the number of labeled samples increases, the network becomes more robust and reliable. Reference##UREF##5##7## built a sound event classification system by adapting the traditional convolutional neural network from the image domain to the audio domain. The sound event classification system performs well, even in a noisy environment. In common, it is challenging to recognize non-stationary sound under the interference of ambient noise. Reference##UREF##6##8## proposed a two-stream convolutional neural network. One stream directly processes the clip of the raw audio sequence, while the other tries to learn rich representation features from the log-Mel spectrogram. Recently, the transformer network has been receiving more attention. Reference##UREF##7##9## explores the transformer structure to detect the sound event based on audio tagging and temporal location information.
","However, in addition to such known sound events, there exist many unknown sound events in our daily lives, which include rhythm, scene, instrument sound, etc. So, the question, “what is that sound?” has frequently come up. Usually, the machine learning algorithm tries to learn categories on a closed set, where the training and test data share the same feature embedding spaces and labels. The training data includes samples with target labels (A, B, C), while the test data restricts the labels to (A, B, C). The classes like D, F, etc. cannot appear on the test. However, in realistic scenarios, it is inevitable to introduce unknown observations D, F, or others into the dataset. The task to learn only with classes (A, B, C) and predict to categorize (A, B, C) or other unexpected D, F, or other classes is called open set recognition. In the open set recognition task, there are several categories (Fig. ##FIG##0##1##) assumed to characterize the learning models##UREF##8##10##: (1) Known Known classes (KKCs): the samples are annotated by humans and given with the corresponding labels. (2) Known Unknown classes (KUCs)##UREF##9##11##: The KUC is an umbrella term for different events, objects or concepts. It is hard to define the attribute of the KUC, which is usually a particular product incidental to the description of the subject of the thing. (3) Unknown Known classes##UREF##10##12##: Classes for which no samples are available during training, but side information, such as semantic/attribute information, is available. The algorithm to detect unknown known classes is called few-shot open set recognition, a subset scope##UREF##11##13##–##UREF##14##16## of open set recognition. (4) Unknown Unknown classes (UUCs)##UREF##15##17##: The sample of category does not exist during the training, as either the corresponding attribution or semantic information is not known. Our task focuses on unknown classes. The algorithm only learns in labeled known classes, and is able to detect unknown classes D, F, or others, even though we do not know, and cannot describe, what they are.
","In this paper, we deal with classifier unknown sound events or their related patterns. In summary, our work makes the flowing key contributions: