scholarly journals The Effect and Mechanism of KLF7 in the TLR4/NF-κB/IL-6 Inflammatory Signal Pathway of Adipocytes

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Meixiu Zhang ◽  
Cuizhe Wang ◽  
Jinxiu Wu ◽  
Xiaodan Ha ◽  
Yuchun Deng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Inflammatory Cytokine ◽  
Luciferase Reporter ◽  
Signal Pathways ◽  
High Concentration ◽  
Inflammatory Signaling ◽  
Expression Levels ◽  
Mrna And Protein Expression

Objective. To investigate the role and possible molecular mechanism of Krüppel-like factor 7 (KLF7) in the TLR4/NF-κB/IL-6 inflammatory signaling pathway activated by free fatty acids (FFA). Methods. The mRNA and protein expression levels of KLF7 and the factors of TLR4/NF-κB/IL-6 inflammatory signal pathways were detected by qRT-PCR and Western blotting after cell culture with different concentrations of palmitic acid (PA). The expression of KLF7 or TLR4 in adipocytes was upregulated or downregulated; after that, the mRNA and protein expression levels of these key factors were detected. KLF7 expression was downregulated while PA stimulated adipocytes, and then the mRNA and protein expressions of KLF7/p65 and downstream inflammatory cytokine IL-6 were detected. The luciferase reporter assay was used to determine whether KLF7 had a transcriptional activation effect on IL-6. Results. (1) High concentration of PA can promote the expression of TLR4, KLF7, and IL-6 in adipocytes. (2) TLR4 positively regulates KLF7 expression in adipocytes. (3) KLF7 positively regulates IL-6 expression in adipocytes. (4) PA promotes IL-6 expression via KLF7 in adipocytes. (5) KLF7 has a transcriptional activation on IL-6. Conclusion. PA promotes the expression of the inflammatory cytokine IL-6 by activating the TLR4/KLF7/NF-κB inflammatory signaling pathway. In addition, KLF7 may directly bind to the IL-6 promoter region and thus activate IL-6.

The effect of miR-6523a on growth hormone secretion in pituitary cells of Yanbian yellow cattle

2020 ◽  
Vol 100 (4) ◽  
pp. 657-664
Author(s):  
Jiuxiu Ji ◽  
Taihua Jin ◽  
Rui Zhang ◽  
Angang Lou ◽  
Yingying Chen ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Protein Expression ◽  
Luciferase Reporter ◽  
Control Group ◽  
Pituitary Cells ◽  
Expression Levels ◽  
Gh Secretion ◽  
Yellow Cattle ◽  
Mrna And Protein Expression ◽  
In Cells

Yanbian yellow cattle breeding is limited by its slow growth. We previously found that the miRNA miR-6523a is differentially expressed between Yanbian yellow cattle and Han Yan cattle, which differ in growth characteristics. In this study, we evaluated the effects of miR-6523a on growth hormone (GH) secretion in pituitary cells of Yanbian yellow cattle. Bioinformatics analyses using TargetScan and RNAhybrid, as well as dual luciferase reporter assays, showed that miR-6523a targets the 3′ untranslated region of somatostatin receptor 5 (SSTR5). We further found that the mRNA and protein expression levels of GH in pituitary cells were significantly higher in cells treated with miR-6523a mimic than in the control group (P = 0.0082 and P = 0.0069). The GH mRNA and protein expression levels were lower in cells treated with miR-6523a inhibitor than in the control group, but the difference was not significant (P = 0.064 and P = 0.089). SSTR5 mRNA and protein levels were inhibited by miR-6523a mimic compared with the control group (P = 0.0024 and P = 0.0028) and were elevated slightly by miR-6523a inhibitor (P = 0.093 and P = 0.091). These results prove that miR-6523a regulates GH secretion in pituitary cells by SSTR5. More broadly, these findings provide a basis for studies of the roles of miRNAs in animal growth and development.

scholarly journals Effect of CXCR4 on Apoptosis in Osteosarcoma Cells via the PI3K/Akt/NF-κβ Signaling Pathway

2018 ◽  
Vol 46 (6) ◽  
pp. 2250-2260 ◽  
Author(s):  
Chunming Jiang ◽  
Shenglin Ma ◽  
Runlei Hu ◽  
Xuepeng Wang ◽  
Maoqiang Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Osteosarcoma Cells ◽  
Down Regulation ◽  
Expression Levels ◽  
Apoptotic Protein ◽  
Effective Manner ◽  
Human Osteosarcoma Cells

Background/Aims: Osteosarcoma, the most common primary bone malignancy, arises from primitive transformed cells of mesenchymal origin with the worldwide increasing morbidity and mortality. Previous studies found apoptosis of osteosarcoma cells was essential for an effective manner to improve the progress of osteosarcoma, and CXCR4 has been demonstrated to be relevant with various tumor progress and metastasis. Methods: The proliferation of cells transfected with CXCR4 shRNA and control shRNA were measured by BrdU assay. Apoptosis was detected by flow cytometry. Apoptotic protein expression levels were detected by Western blot. Caspase activity was detected by Colorimetric Assay Kits using microplate reader. Activation of NF-κβ signaling after CXCR4 down-regulation in osteosarcoma cells was examined by constructing NF-κβ promoter luciferase reporter plasmid. The expression and activation of NF-κβ Signaling relevant protein were analyzed to investigate the relationship between Akt and NF-κβ signaling after the down-regulation of CXCR4 in osteosarcoma cells. Results: Down-regulation of CXCR4 significantly reduced the cell proliferation, while remarkably increased the cell apoptosis and apoptotic protein expression levels in osteosarcoma cells. Furthermore, down-regulation of CXCR4 induced cell apoptosis was caspase dependent in osteosarcoma cells. This study also showed CXCR4 down-regulation induced apoptosis through inhibiting PI3K/Akt/NF-κβ signaling pathway. In addition, endoplasmic reticulum stress (ERS) activation was involved in cell apoptosis induced down-regulation of CXCR4. Knockdown of partial ERS relevant proteins followed down-regulation of CXCR4 significantly inhibited cell apoptosis and the apoptotic protein expression levels. Conclusions: Taken together, the results demonstrated that down-regulation of CXCR4 could induce apoptosis of human osteosarcoma cells through inhibiting PI3K/Akt/NF-κβ signaling pathway, indicating that CXCR4 could be vital for the clinical therapy of osteosarcoma.

scholarly journals Protective Mechanism of Sulforaphane on Cadmium-Induced Sertoli Cell Injury in Mice Testis via Nrf2/ARE Signaling Pathway

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1774 ◽  
Author(s):  
Shu-Hua Yang ◽  
Li-Hui Yu ◽  
Lin Li ◽  
Yang Guo ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Protective Effect ◽  
Signaling Pathway ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Heme Oxygenase 1 ◽  
Quinone Oxidoreductase ◽  
Expression Levels ◽  
Apoptosis Rate ◽  
Mrna And Protein Expression

The present study evaluated the mechanism underlying the protective effect of sulforaphane (SFN) on cadmium (Cd)-induced Sertoli cell (TM4 cells) injury in mice. The apoptosis rate of cells in each group was detected by flow cytometry. It was determined the effect of SFN on the expression of downstream molecular targets of Nrf2/ARE axis and on the lipid peroxide content. The related genes involved in the nuclear factor E2-related factor 2(Nrf2)/antioxidant response element (ARE) signaling pathway were evaluated by RT-PCR; for example, the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS), while the protein expression levels were assessed by Western blot. Our results showed that the mRNA and protein expression levels of Nrf2, HO-1, NQO1, GSH-Px, and γ-GCS were increased in various degree when the Sertoli cells were to added different concentrations of SFN. Our results also showed that SFN reduced the apoptosis rate, increased the activity of T-SOD, inhibited the increase of the MDA content caused by Cd. Meanwhile, SFN could increase the mRNA and protein expression levels of Nrf2, HO-1 and NQO1 and reduced the mRNA and protein expression levels of GSH-Px and γ-GCS caused by Cd in Sertoli cells (p < 0.01). Taken together, SFN could improve the antioxidant capacity of Sertoli cells, and exert a protective effect on the oxidative damage and apoptosis of Cd-induced Sertoli cells through the activation of Nrf2/ARE signal transduction pathway.

scholarly journals Copper Induces Oxidative Stress and Apoptosis in the Mouse Liver

2020 ◽  
Vol 2020 ◽  
pp. 1-20 ◽  
Author(s):  
Huan Liu ◽  
Hongrui Guo ◽  
Zhijie Jian ◽  
Hengmin Cui ◽  
Jing Fang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Mouse Liver ◽  
B Cell Lymphoma ◽  
Death Domain ◽  
Expression Levels ◽  
Mrna And Protein Expression ◽  
High Doses ◽  
Induced Apoptosis

Copper (Cu) is an essential trace element involved in the normal physiological processes of animals. However, excessive exposure to Cu can produce numerous detrimental impacts. The aim of this study was to investigate the effects of Cu on oxidative stress and apoptosis as well as their relationship in the mouse liver. Four-week-old ICR mice (n=240) were randomly assigned to different Cu (Cu2+-CuSO4) treatment groups (0, 4, 8, and 16 mg/kg) for periods of 21 and 42 days. The high doses of Cu exposure could induce oxidative stress, by increasing the levels of reactive oxygen species (ROS) and protein carbonyls (PC) and decreasing the activities of antisuperoxide anion (ASA) and antihydroxyl radical (AHR) and content of glutathione (GSH), as well as activities and mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Moreover, high doses of Cu exposure induced hepatic apoptosis via the mitochondrial apoptotic pathway, as characterized by the depolarization of mitochondrial membrane potential (MMP); significantly increased mRNA and protein expression levels of cytosolic cytochrome (Cyt c), apoptosis-inducing factor (AIF), endonuclease G (Endo G), apoptosis protease-activating factor-1 (Apaf-1), cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bcl-2 antagonist killer (Bak), Bcl-2-associated X protein (Bax), and Bcl-2-interacting mediator of cell death (Bim); and decreased mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL). Furthermore, the activation of the tumor necrosis factor receptor-1 (TNF-R1) signaling pathway was involved in Cu-induced apoptosis, as characterized by the significantly increased mRNA and protein expression levels of TNF-R1, Fas-associated death domain (FADD), TNFR-associated death domain (TRADD), and cleaved caspase-8. These results indicated that exposure to excess Cu could cause oxidative stress triggered by ROS overproduction and diminished antioxidant function, which in turn promoted hepatic apoptosis via mitochondrial apoptosis and that the TNF-R1 signaling pathway was also involved in the Cu-induced apoptosis.

scholarly journals IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2174
Author(s):  
Jin-Young Kim ◽  
Garam Kim ◽  
Sung-Chul Lim ◽  
Hong-Seok Choi
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Breast Cancer Cells ◽  
Regulatory Mechanism ◽  
Mammary Tumorigenesis ◽  
Underlying Mechanism ◽  
Breast Tumorigenesis ◽  
Mrna And Protein Expression ◽  
First Time

Phospholipids are crucial materials that are not only required for cell membrane construction but also play significant roles as signaling molecules. LPIN1 is an enzyme that displays phosphatidate phosphatase activity in the triglyceride and phospholipid synthesis pathway. Recent studies have shown that overexpression of LPIN1 is involved in breast tumorigenesis, but the underlying mechanism regulating LPIN1 expression has not been elucidated yet. In the present study, we showed that the IL-33-induced COT-JNK1/2 signaling pathway regulates LPIN1 mRNA and protein expression by recruiting c-Jun to the LPIN1 promoter in breast cancer cells. IL-33 dose-dependently and time-dependently increased LPIN1 mRNA and protein expression. Moreover, IL-33 promoted colony formation and mammary tumorigenesis via induction of LPIN1 expression, while inhibition of LPIN1 disturbed IL-33-induced cell proliferation and mammary tumorigenesis. IL-33-driven LPIN1 expression was mediated by the COT-JNK1/2 signaling pathway, and inhibition of COT or JNK1/2 reduced LPIN1 expression. COT-JNK1/2-mediated IL-33 signaling activated c-Jun and promoted its binding to the promoter region of LPIN1 to induce LPIN1 expression. These findings demonstrated the regulatory mechanism of LPIN1 transcription by the IL-33-induced COT/JNK1/2 pathway for the first time, providing a potential mechanism underlying the upregulation of LPIN1 in cancer.

scholarly journals High expression of an unknown long noncoding RNA RP11-290L1.3 from GDM macrosomia and its effect on preadipocyte differentiation

2021 ◽  
Author(s):  
Yu Lin ◽  
Yingying Zhang ◽  
Lei Xu ◽  
Wei Long ◽  
Chunjian Shan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Pathway Analysis ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Adipocyte Differentiation ◽  
Preadipocyte Differentiation ◽  
Expression Levels ◽  
Mrna And Protein Expression

Aims: Gestational diabetes mellitus (GDM)-induced macrosomia is predominantly characterized by fat accumulation, which is closely related to adipocyte differentiation. An unknown long noncoding RNA RP11-290L1.3, referred to as RP11, was identified to be dramatically upregulated in the umbilical cord blood of women with GDM-induced macrosomia in our previous study. We conducted this study to identify the function of RP11 in GDM-induced macrosomia. Methods: The effects of RP11 gain- and loss-of-function on HPA-v (human preadipocytes-visceral) adipogenesis were determined with lentivirus mediated cell transduction. The mRNA and protein expression levels of adipogenesis makers were evaluated by qPCR/western blot. Then, we performed the Microarray and pathway analysis to explore the possible mechanisms by which RP11 regulates adipogenesis. Results: Overexpression of RP11 significantly enhanced adipocyte differentiation and increased the mRNA and protein expression levels of adipogenesis makers, such as PPAR-γ, SREBP1c, and FASN by qPCR/western blot. Knockdown of RP11 showed opposite effects. Microarray and pathway analysis showed, after RP11 knockdown, 1,612 genes were upregulated and 583 genes were downregulated which were found to be mainly involved in metabolic pathways, insulin signaling pathway and MAPK signaling pathway. Conclusion: In conclusion, the unknown lncRNA RP11 serves a positive factor on preadipocyte differentiation which could shed light on fetal fat accumulation in GDM.

scholarly journals Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Bashan Zhang ◽  
Rong Li ◽  
Wenfeng Wang ◽  
Xueming Zhou ◽  
Beijing Luo ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Osteoblast Differentiation ◽  
Missense Mutations ◽  
Wild Type ◽  
Expression Levels ◽  
Type Group ◽  
In The Wild ◽  
Mrna And Protein Expression ◽  
Gsk 3Β

Abstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

scholarly journals Protective Effects of Microrna-22 Against Endothelial Cell Injury by Targeting NLRP3 Through Suppression of the Inflammasome Signaling Pathway in a Rat Model of Coronary Heart Disease

2017 ◽  
Vol 43 (4) ◽  
pp. 1346-1358 ◽  
Author(s):  
Wei-Qiang Huang ◽  
Peng Wei ◽  
Ri-Qi Lin ◽  
Feng Huang
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Endothelial Cell ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lumen Formation ◽  
Caspase 1 ◽  
Mrna And Protein Expression

Background/Aims: This study aimed to identify the role of microRNA-22 (miR-22) in endothelial cell (EC) injury in coronary heart disease (CHD) by targeting NLRP3 through the inflammasome signaling pathway. Methods: A total of 24 healthy male Sprague-Dawley (SD) rats were divided into normal and atherosclerosis groups. The atherosclerosis rats were assigned into blank, negative control (NC), miR-22 mimic, miR-22 inhibitor and miR-22 inhibitor + siNLRP3 groups. A luciferase reporter gene assay was used to detect the relationship between miR-22 and NLRP3. MiR-22 expression as well as NLRP3 and caspase-1 mRNA and protein expression were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The activity and apoptosis of coronary arterial endothelial cells (CAECs) were determined by MTT and Hoechst 33258. CAEC lumen formation was detected by a lumen formation assay. An enzyme-linked immunosorbent assay (ELISA) was used to detect IL-1β, IL-6, IL-10 and IL-18 levels. Results: The results indicated that the atherosclerosis group significantly decreased miR-22 expression but increased NLRP3 and caspase-1 mRNA and protein expression. The cell survival rate was significantly increased in the miR-22 mimic group and significantly reduced in the miR-22 inhibitor group. The miR-22 mimic group displayed a lower apoptosis rate and more cells with obvious lumen walls and numerous tubular structures, while cells in the miR-22 inhibitor group were unable to form lumen walls and had a scattered distribution compared to the blank group. The ELISA showed that IL-1β, IL-6 and IL-18 levels were markedly decreased, while IL-10 was clearly increased in the miR-22 mimic group. In contrast, in the miR-22 inhibitor group, IL-1β, IL-6 and IL-18 levels were significantly increased, and IL-10 levels were decreased. Conclusion: Our findings indicated that miR-22 could lower the levels of pro-inflammatory cytokines by inhibiting the NLRP3 inflammasome pathway, which suppresses CAEC apoptosis and protects CAECs in rats with CHD.

scholarly journals miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiong Ma ◽  
Chunxia Zhou ◽  
Xuejun Chen
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Cell Proliferation And Migration ◽  
Hh Signaling ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

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