Circular RNA SMARCA5 Promotes a Poor Prognosis and Radiotherapy Resistance for Patients with Hepatocellular Carcinoma.

OBJECTIVE: The involvement of Circular RNA SMARCA5 (cSMARCA5) in several types of cancer has been reported; however, its role in hepatocellular carcinoma (HCC) is unclear. The presented research aimed to explore the expression level of cSMARCA5 in HCC tissues and evaluate the association between cSMARCA5, prognosis, and radiotherapy resistance for patients with HCC. METHODS: This study was designed as a case controlled study and enrolled 106 HCC patients. HCC and paired non-tumor samples were collected from HCC patients. Gene expression was analyzed by RT-qPCR. The association between the expression level of cSMARCA5 and prognosis value and radiotherapy resistance for patients with HCC was analyzed by Kaplan-Meier survival curve and Multivariate Cox analysis. RESULTS: Compared to paracancerous tissue, cSMARCA5 demonstrated higher expression in the tumor tissues (p<0.0001).Higher expression of cSMARCA5 is a risk factor in 3-year overall survival (OS) for HCC patients (HR=1.798, 95%CI: 1.165~3.231, p=0.0321). Multivariate analysis showed that higher expression of cSMARCA5 was related to PVTT (HR=2.136, 95%CI: 1.130~5.218), AFP (>400ng/ml) (HR=2.335, 95%CI: 1.247~5.661), tumor size (>5cm) (HR=3.017, 95%CI: 1.477~5.659), poor histopathologic grading (HR=3.344, 95%CI: 2.175~6.143), and multiple tumor number (HR=2.875, 95%CI: 1.453~3.884). We also found that radiotherapy resistance was related to AFP (>400ng/ml) (OR=2.125, 95%CI: 1.015~3.348), tumor size (>5cm) (OR=2.857, 95%CI: 1.665~4.978), poor histopathologic grading (OR=2.463, 95%CI: 1.389~4.446), multiple tumor number (OR=2.332, 95%CI: 1.538~3.887), and high expression of cSMARCA5 (OR=3.574, 95%CI: 1.663~5.932). CONCLUSION: CircRNA-SMARCA5 is significantly increased in HCC tissues and promotes radiotherapy resistance. More importantly, higher expression level of cSMARCA5 is related to a poorer 3-year OS for patients with HCC.

Silica nanoparticles induce pulmonary damage in rats via VEGFC/D-VEGFR3 signaling-mediated lymphangiogenesis and remodeling.

Silica nanoparticles (SiNPs) are widely used as drug carriers for improving drug delivery and retention. The lungs are highly sensitive to the toxicity of SiNPs entering the respiratory tract. Furthermore, pulmonary lymphangiogenesis, which is the growth of lymphatic vessels observed during multiple pulmonary diseases, plays a vital role in promoting the lymphatic transport of silica in the lungs. However, more research is required on the effects of SiNPs on pulmonary lymphangiogenesis. We investigated the effect of SiNP-induced pulmonary toxicity on lymphatic vessel formation in rats and evaluated the toxicity and possible molecular mechanisms of 20-nm SiNPs. Saline containing 3.0, 6.0, and 12.0 mg/kg of SiNPs was instilled intrathecally into female Wistar rats once a day for five days, then sacrificed on day seven. Lung histopathology, pulmonary permeability, pulmonary lymphatic vessel density changes, and the ultrastructure of the lymph trunk were investigated using light microscopy, spectrophotometry, immunofluorescence, and transmission electron microscopy. CD45 expression in lung tissues was determined using immunohistochemical staining, and protein expression in the lung and lymph trunk was quantified using western blotting. We observed increased pulmonary inflammation and permeability, lymphatic endothelial cell damage, pulmonary lymphangiogenesis, and remodeling with increasing SiNP concentration. Moreover, SiNPs activated the VEGFC/D-VEGFR3 signaling pathway in the lung and lymphatic vessel tissues. SiNPs caused pulmonary damage, increased permeability and resulted in inflammation-associated lymphangiogenesis and remodeling by activating VEGFC/D-VEGFR3 signaling. Our findings provide evidence for SiNP-induced pulmonary damage and a new perspective for the prevention and treatment of occupational exposure to SiNPs.

Toxic Nephropathy Secondary to Chronic Mercury Poisoning: Clinical Characteristics and Outcomes.

Introduction: Kidney disease secondary to mercury poisoning has not been well documented and is often misdiagnosed and mistreated. Methods: We performed a retrospective analysis of patients diagnosed with having mercury poisoning over a 6-year period between July 2013 and June 2019. Demographics, clinical measures, renal pathologic examinations, treatments, and outcomes were compared between patients with kidney disease and those without kidney disease. Results: Of the 172 patients with mercury poisoning, 46 (26.74%) had renal damage. Among the 46 patients, 41 (89.13%) presented nephrotic syndrome, and 5 (10.87%) showed proteinuria alone. The pathologic abnormality associated with kidney disease caused by mercury poisoning was mainly membranous nephropathy (18 of 35 patients, 51.43%). Among 41 patients with nephrotic syndrome, 25 were treated with chelation therapy alone and 12 with mercury chelation therapy and glucocorticoids. The remaining 4 patients were treated with chelation therapy, glucocorticoids, and immunosuppressive therapies. The overall effective rate was 97.5% (40 patients). There was no significant difference in complete remission rate among the 3 treatment methods (P < 0.05). Conclusion: The main clinical manifestation of kidney disease secondary to chronic mercury poisoning was nephrotic syndrome, which was reflected in pathologic examinations as membranous nephropathy. Kidney disease to chronic mercury poisoning is prone to misdiagnosis and missed diagnosis. Chelation therapy is the main treatment, and the prognosis is good. Patients with severe condition can be supplemented with glucocorticoid.

Broken thermometer in hand: mercury elevation caused by improper debridement.

BACKGROUND: Mercury exposure from broken thermometers is still common in China. CASE REPORT: Here, we report a 2-year-old girl with elevated mercury concentrations in her blood and urine due to improper debridement after pricked by a broken thermometer. She underwent the first debridement at a local hospital, but radiographs showed a dot-like mercury deposit turned into multiple dispersed beads in her wrist tissue. Although the patient had no signs or symptoms of mercury poisoning, her blood and urinary mercury concentrations were significantly elevated. Several radio-opaque densities remained in her hand until a second debridement. At 2 years follow-up, her mercury concentrations in blood and urine and her hand radiograph were normal. CONCLUSIONS: Careful debridement after injury by broken thermometer is important in order to remove mercury in tissues and to prevent its dispersion and further absorption.

Detection of miR-122 by fluorescence real-time PCR in blood from patients with chronic hepatitis B and C infections.

BACKGROUND: This study aims to determine whether relative miR-122 levels in peripheral blood are correlated with chronic hepatitis B (CHB) and chronic hepatitis C (CHC) virus infection and viral replication to determine whether miR-122 can be a new marker for liver injury. METHODS: MicroRNA (miRNA) was extracted from the peripheral blood of 20 CHB patients, 20 CHC patients, and 20 healthy controls. The levels of miR-122 were determined using fluorescence real-time reverse transcription PCR. Then, the associations of miR-122 with CHB and CHC were analyzed, and its correlation with other markers of liver function and viral replication were determined. RESULTS: The expression level of miR-122 in patients with CHB was significantly higher when compared to subjects in the control group (P = 0.007) or CHC patients (P = 0.005). Furthermore, the miR-122 level in patients with CHC was somewhat higher when compared to healthy controls (66% higher), but the difference was not statistically significant (P = 0.229). MiR-122 levels were significantly correlated with ALT (correlation coefficient [R] = 0.7, P < 0.001), AST (R = 0.71, P < 0.001), and HBV NA (R = 0.9, P < 0.001). The regression analysis indicated that the AUC of miR-122 levels in the diagnosis of CHB was 0.87, with a sensitivity of 0.8 and a specificity of 0.8. CONCLUSION: MiR-122 can be used to distinguish healthy persons and patients with CHB infection with high sensitivity and specificity. These present findings presented that the complex and context-specific associations of miR-122 with liver diseases, suggesting that this may be a promising marker for liver injury.

Mercury Poisoning Presenting With Hypertension: Report of 2 Cases.

BACKGROUND: Mercury poisoning can cause damage to multiple organs. Secondary hypertension, which is usually misdiagnosed and mistreated, has been rarely reported in cases of mercury poisoning. METHODS: We herein present 2 cases of hypertension as the main manifestation of mercury poisoning. RESULTS: Case 1 involved a 42-year-old man with blood pressure of 230/190 mm Hg and urinary mercury level of 131.54 µmol/molCr. The patient had been repeatedly exposed to mercury at his workplace and had been admitted to our department many times. His hypertension quickly normalized after every chelation treatment. Case 2 involved a 10-year-old girl with hypertension (150/110 mm Hg), rash, and convulsions. She was found to have elevated blood levels of renin, angiotensin II, and aldosterone as well as an elevated urinary mercury level. Her hypertension recovered soon after chelation treatment. CONCLUSIONS: Mercury poisoning can cause secondary hypertension as the main clinical manifestation or together with multiorgan damage. Renin-angiotensin system activation may be involved in the occurrence and development of hypertension.

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg∙mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg∙mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Dimethyl fumarate accelerates wound healing under diabetic condition.

Impaired wound healing is a common complication among patients with diabetes mellitus (DM), resulting in high rates of disability and mortality. Recent findings highlighted the critical role of nuclear factor erythroid 2-related factor 2 (NRF2) - a master of cellular antioxidants scavenging excessive DM-induced free radicals - in accelerating diabetic wound healing. Dimethyl fumarate (DMF) is a potent NRF2 activator used for the treatment of multiple sclerosis. However, the effect of DMF on wound healing has not been determined. The present study investigated the effect of DMF on the diabetic and the non-diabetic wound healing in streptozotocin-induced diabetic mice and non-diabetic control mice. DMF activated NRF2 signaling under both conditions. Interestingly, DMF attenuated oxidative damage and inflammation, and accelerated wound closure in the diabetic mice. However, this effect was not observed in non-diabetic mice. Keratinocytes were treated with normal glucose (NG), high glucose (HG), or hydrogen peroxide (H2O2), in the presence or absence of DMF to assess the role of reactive oxygen species (ROS) - inducible in DM - in mediating DMF-induced protection. Both HG and H2O2 elevated ROS, oxidative damage, and inflammation, the effects of which were similarly blunted by DMF. However, in spite of the activation of NRF2, DMF lost this capability under the NG condition. The findings of this study demonstrate that ROS activate the protective effect of DMF on the diabetic wound healing.

SP600125 suppresses Keap1 expression and results in NRF2-mediated prevention of diabetic nephropathy.

c-Jun N-terminal kinase (JNK) contributes to the pathogenesis of diabetic nephropathy (DN). The JNK inhibitor SP600125 was reported to ameliorate DN. However, the mechanism remained unclear. We previously reported that SP600125 activated nuclear factor erythroid 2-related factor 2 (NRF2), a governor of the cellular antioxidant defense system, in the aortas of the diabetic mice. Given the critical role of NRF2 in preventing DN, the present study aimed to test whether or not NRF2 is required for SP600125's protection against DN. To test the role of NRF2 in SP600125's effect, streptozotocin-induced C57BL/6 wild-type (WT) and Nrf2-knockout (KO) diabetic mice were treated in the presence or absence of SP600125, for 24 weeks. To explore the mechanism by which SP600125 activates NRF2, mouse mesangial cells (MMCs) were treated with high glucose (HG), in the presence or absence of either SP600125 or JNK siRNA. SP600125 significantly attenuated the diabetes-induced renal oxidative stress, inflammation, fibrosis, pathological change and dysfunction in the WT, but not the Nrf2 KO mice. SP600125 inactivated JNK, inhibited kelch-like ECH-associated protein 1 expression, preserved NRF2 protein and facilitated its nuclear translocation in the kidneys of the WT mice, the effects of which were similarly produced by either SP600125 or JNK siRNA in HG-treated MMCs. Further, both SP600125 and JNK siRNA alleviated HG-induced mesangial oxidative stress and expression of inflammatory and fibrotic genes. The present study demonstrates that NRF2 is required for SP600125's protection against DN. SP600125 activates NRF2 possibly via inhibition of JNK-induced Keap1 expression.

Quantitative Analysis of Hepatic Toxicity in Rats Induced by Inhalable Silica Nanoparticles Using Acoustic Radiation Force Imaging.

OBJECTIVES: The purposes of this study were to verify whether inhalable silicon dioxide (SiO2 ) nanoparticles could induce hepatic injury and to investigate the relationship between the exposure time and SiO2 nanoparticle dosage by using acoustic radiation force impulse imaging (ARFI). METHODS: A total of 72 rats were randomly separated into 9 groups with 8 in each: blank control group, 0.9% normal saline group, polyacrylate (PPE) group, 25%, 50%, and 100% SiO2 groups, and 25%, 50%, and 100% SiO2 /PPE groups with inhaled SiO2 nanoparticle concentrations similar to the SiO2 groups. After successful modeling and design, the hepatic shear wave velocity (SWV) values of the 9 groups were obtained on days 3, 7, 14, 21, and 28 by using ARFI, and the intragroup and intergroups differences in the SWVs were compared. The serum alanine aminotransferase (ALT) and aspartate aminotransferase were tested and compared on day 28. Hepatic tissues were collected for histologic observation on day 28. RESULTS: The pathologic results verified that inhalable SiO2 nanoparticles could induce hepatic injury. Compared with the control group, the hepatic SWV and serum ALT values in the SiO2 groups and SiO2 /PPE groups were elevated (P < .05). The dosage and exposure time of SiO2 played a key role in the elevation of the SWV in the SiO2 and SiO2 /PPE groups. The correlation between the ALT level and SWV was significant on day 28 (P < .05). CONCLUSIONS: Inhalable SiO2 nanoparticles and SiO2 /PPE were able to induce hepatic injury in rats. Using ARFI to evaluate hepatic toxicity induced by SiO2 nanoparticles was effective in this study.

Polyacrylate/nanosilica causes pleural and pericardial effusion, and pulmonary fibrosis and granuloma in rats similar to those observed in exposed workers.

Nanomaterials offer great benefit as well as potential damage to humans. Workers exposed to polyacrylate coatings have pleural effusion, pericardial effusion, and pulmonary fibrosis and granuloma, which are thought to be related to the high exposure to nanomaterials in the coatings. The study aimed to determine whether polyacrylate/silica nanoparticles cause similar toxicity in rats, as observed in exposed workers. Ninety male Wistar rats were randomly divided into five groups with 18 rats in each group. The groups included the saline control group, another control group of polyacrylate only, and low-, intermediate-, and high-dose groups of polyacrylate/nanosilica with concentrations of 3.125, 6.25, and 12.5 mg/kg. Seventy-five rats for the 1-week study were terminated for scheduled necropsy at 24 hours, 3 days, and 7 days postintratracheal instillation. The remaining 15 rats (three males/group) had repeated ultrasound and chest computed tomography examinations in a 2-week study to observe the pleural and pericardial effusion and pulmonary toxicity. We found that polyacrylate/nanosilica resulted in pleural and pericardial effusions, where nanosilica was isolated and detected. Effusion occurred on day 3 and day 5 post-administration of nanocomposites in the 6.25 and 12.5 mg/kg groups, it gradually rose to a maximum on days 7-10 and then slowly decreased and disappeared on day 14. With an increase in polyacrylate/nanosilica concentrations, pleural effusion increased, as shown by ultrasonographic qualitative observations. Pulmonary fibrosis and granuloma were also observed in the high-dose polyacrylate/nanosilica group. Our study shows that polyacrylate/nanosilica results in specific toxicity presenting as pleural and pericardial effusion, as well as pulmonary fibrosis and granuloma, which are almost identical to results in reported patients. These results indicate the urgent need and importance of nanosafety and awareness of toxicity of polyacrylate/nanosilica.

Nanosilica and Polyacrylate/Nanosilica: A Comparative Study of Acute Toxicity.

We compared the acute toxicity of nanosilica and polyacrylate/nanosilica instillation in Wistar rats (n = 60). Exposure to nanosilica and polyacrylate/nanosilica showed a 30% mortality rate. When compared with saline-treated rats, animals in both exposure groups exhibited a significant reduction of PO2 (P < 0.05) at both 24 and 72 hr. after exposure. Both exposure groups exhibited a significant reduction of neutrophils in arterial blood compared to saline controls (P < 0.05) 24 hr. after exposure. The levels of blood ALT and LDH in exposed groups were found to be significantly increased (P < 0.05) 24 hr. following exposure. The exposed groups exhibited various degrees of pleural effusion and pericardial effusion. Our findings indicated respiratory exposure to polyacrylate/nanosilica and nanosilica is likely to cause multiple organ toxicity.

Protective effects of penehyclidine hydrochloride on acute lung injury caused by severe dichlorvos poisoning in swine.

BACKGROUND: Organophosphate poisoning is an important health problem in developing countries which causes death mainly by inducing acute lung injury. In this study, we examined the effects of penehyclidine hydrochloride (PHC), a selective M-receptor inhibitor, on dichlorvos-induced acute lung injury in swine. METHODS: Twenty-two female swines were randomly divided into control (n = 5), dichlorvos (n = 6), atropine (n = 6), and PHC (n = 5) groups. Hemodynamic data, extravascular lung water index (EVLWI), and pulmonary vascular permeability index (PVPI) were monitored; blood gas analysis and acetylcholinesterase (AchE) levels were measured. PaO2/FiO2, cardiac index (CI), and pulmonary vascular resistance indices (PVRI) were calculated. At termination of the study, pulmonary tissue was collected for ATPase activity determination and wet to dry weight ratio (W/D) testing 6 hours post-poisoning. TUNEL assay, and Bax, Bcl-2, and caspase-3 expression were applied to pulmonary tissue, and histopathology was observed. RESULTS: After poisoning, PHC markedly decreased PVRI, increased CI more effectively than atropine. Anticholinergic treatment reduced W/D, apoptosis index (AI), and mitigated injury to the structure of lung; however, PHC reduced AI and caspase-3 expression and improved Bcl-2/Bax more effectively than atropine. Atropine and PHC improved ATPase activities; a significant difference between groups was observed in Ca(2+)-ATPase activity, but not Na(+)-K(+)-ATPase activity. CONCLUSIONS: The PHC group showed mild impairment in pathology, less apoptotic cells, and little impact on cardiac function compared with the atropine group in dichlorvos-induced acute lung injury.

Dual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagy.

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling has been found in several types of human cancer, including hepatocellular carcinoma (HCC). NVP-BEZ235 is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against HCC in preclinical models. Autophagy is a cellular lysosomal degradation pathway essential for the regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mTOR signaling and often counteracts the efficacy of certain cancer therapeutic agents. In this study, we explored the role of autophagy in apoptosis induced by NVP-BEZ235 in two HCC cell lines, Hep3B and PLC/PRF/5, and identified the mechanism of combinatorial treatment. NVP-BEZ235 was effective in inhibiting the growth of the two HCC cell lines possibly though induction of apoptosis. NVP-BEZ235 also potently increased the expression of LC3-II and decreased the expression of p62, indicating induction of autophagy. When NVP-BEZ235 was used in combination with Atg5 siRNA or the autophagy inhibitor 3-methyladenine (3-MA), enhancement of the inhibitory effects on the growth of HCC cells was detected. In addition, enhanced induction of apoptosis was observed in cells exposed to the combination of NVP-BEZ235 and Atg5 siRNA or 3-MA. Thus, induction of autophagy by NVP-BEZ235 may be a survival mechanism that counteracts its anticancer effects. Based on these data, we suggest a strategy to enhance the anticancer efficacy of BEZ235 by blockade of autophagy. Thus, our study provides a rationale for the clinical development of combinations of NVP-BEZ235 and autophagy inhibitors for the treatment of HCC and other malignancies.

Nanoexposure, unusual diseases, and new health and safety concerns.

Accumulating studies in animals have shown that nanoparticles could cause unusual rapid lung injury and extrapulmonary toxicity. Whether exposure of workers to nanoparticles may result in some unexpected damage as seen in animals is still a big concern. We previously reported findings regarding a group of patients exposed to nanoparticles and presenting with an unusual disease. The reported disease was characterized by bilateral chest fluid, pulmonary fibrosis, pleural granuloma, and multiorgan damage and was highly associated with the nanoparticle exposure. To strengthen this association, further information on exposure and the disease was collected and discussed. Our studies show that some kinds of nanomaterials, such as silica nanoparticles and nanosilicates, may be very toxic and even fatal to occupational workers exposed to them without any effective personal protective equipment. More research and collaborative efforts on nanosafety are required in order to prevent and minimize the potential hazards of nanomaterials to humans and the environment.

Nanomaterials in humans: identification, characteristics, and potential damage.

Nanomaterials are increasingly being used for commercial purposes. However, concerns about the potential risks of exposure to humans have been raised. We previously reported unusual pulmonary disease and death in a group of patients with occupational exposure to spray paint. However, the nanoparticle and chemical composition of the exposure was not fully described. The present study aimed to isolate and identify the nanoparticles observed in the patients' biopsies and report the potential deleterious effects to human lungs using electron microscopy. Using electron microscopy and energy dispersive x-ray analysis, silica nanoparticles were identified and characterized mainly in macrophages, pulmonary microvessels, vascular endothelial cells, microlymphatic vessels, pleural effusions, and a few in alveolar epithelial cells and pulmonary interstitial tissue (with no microscale particles present). Notably, damage to alveolar epithelial cells, macrophages, vascular endothelial cells, and the blood-gas barrier was observed. Given the well-documented toxicity of microscale silica, it is possible that these silica nanoparticles may have contributed in part to the illness reported in these workers. Such a possibility supports the adoption of controls and prevention strategies to minimize inhalation of nanoparticles by workers, and it highlights the urgent need and the importance of the nanosafety study in humans.

Immunosuppressive therapy of cyclosporin A for severe benzene-induced haematopoietic disorders and a 6-month follow-up.

Long-term exposure to benzene can potentially result in severe haematotoxicities, including pancytopaenia, aplastic anaemia and myelodysplastic syndrome, which are often accompanied by life-threatening symptoms and high mortality. Previous studies demonstrate that benzene-induced haematotoxicities are immune-mediated and that cyclosporin A is a prominent treatment in acquired aplastic anaemia. This study aims to evaluate the potential role of cyclosporin A immunosuppressive therapy for severe benzene-induced haematotoxicity. Between January 2002 and December 2008, 41 patients with severe benzene-induced haematopoietic disorders from five hospitals were enrolled in the study, 22 patients received cyclosporin A, supportive treatments and/or oral testosterone undecanoate, 19 patients were treated with supportive treatments and/or oral testosterone undecanoate as the control group, and a 6-month follow-up was conducted. The results showed that in the cyclosporin A group, 19 of 22 patients (86.36%) had responded to the treatments completely or partially with increased platelets, white blood cells and hemoglobulin counts by the fourth week (P=0.005), the sixth week (P=0.001) and the third month post-treatment (P=0.034), respectively. However, in the control group treated by supportive methods, only 5 of 19 patients (26.32%) responded to the treatments partially (P<0.001). Cyclosporin A in conjunction with supportive treatments may be an effective treatment modality for patients with severe benzene-induced haematopoietic disorders, which in turn implies that these haematotoxicities are immune-mediated.