Investigating the Dewatering Efficiency of Sewage Sludge with Optimized Ratios of Electrolytic Manganese Residue Components.

As an industrial waste residue, Electrolytic Manganese Residue (EMR) can greatly promote sludge dewatering and further particle-size optimization can significantly strengthen sludge dewaterability. In this study, the effects of ammonium sulfate, calcium sulphate dihydrate, and manganese carbonate in EMR on sludge dewatering performance were investigated using the response surface optimization method. It was found that the optimized ratio of three components in EMR was 1.0:1.6:2.2 based on capillary suction time (CST), specific resistance of filtration (SRF), and zeta potential of dewatered sludge. The composition ratio of particle-size optimized EMR was modified based on the above optimization, resulting in a significant increase in sludge dewatering performance (CST and SRF reduced by 8.7% and 11.2%, respectively). Compared with those in original sludge, the content of bound extracellular polymeric substances in the conditioned sludge with optimized ratio was drastically reduced while that of soluble extracellular polymeric substances was slightly increased, which was in accordance with the decline of fluorescence intensity. These findings indicated the disintegration of extracellular polymeric substances, the enhancement of hydrophobicity, and dewatering properties of the sludge. In summary, optimized EMR can effectively intensify the dewaterability of sludge, providing a competitive solution for dewatering and further disposal of sludge.

Astragaloside IV Inhibits Bleomycin-Induced Ferroptosis in Human Umbilical Vein Endothelial Cells by Mediating LPC.

Ferroptosis, as an iron-dependent programmed cell death pathway, can induce a variety of cardiovascular diseases. Astragaloside IV (AS-IV), which is purified from Astragalus membranaceus, can protect endothelial function and promote vascular regeneration. However, the role played by AS-IV in ferroptosis remains unknown. In this study, the lipid metabolomics in HUVECs treated with/without bleomycin and/or AS-IV were explored using LC/MS. The most differential metabolite between groups was further identified via GO and pathway enrichment analyses. The effects of lysophosphatidylcholine (LPC), AS-IV, and FIN56 on cell viability were explored using the CCK-8 assay, their effects on cell senescence were examined by ß-galactosidase staining, and their effects on ferroptosis were detected by a flow cytometric analysis of lipid ROS levels, transmission electron microscopy, and an assay for cellular iron levels. The related mechanisms were investigated by real-time PCR and Western blot assays. Our results showed that LPC, as the most differential metabolite, inhibited cell viability but promoted cell apoptosis and senescence as its concentration increased. Also, the decreased cell activity, increased iron ion and lipid ROS levels, and the enhanced cell senescence induced by LPC treatment were all significantly reversed by AS-IV but further enhanced by FIN56 treatment. The changes in mitochondrial morphology caused by the LPC treatment were significantly alleviated by the AS-IV treatment, while treatment with FIN56 reversed those phenomena. Moreover, AS-IV partially upregulated the levels of SLC7A11 and GPX4 expression which were reduced by LPC. However, those changes were prevented by FIN56 treatment. In conclusion, our data suggested that AS-IV could serve as a novel drug for treating ferroptosis-related diseases.

Targeting Mps1 in combination with paclitaxel inhibits osteosarcoma progression by modulating spindle assembly checkpoint and Akt/mTOR signaling.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents and is characterized by early metastasis and frequent recurrence, which greatly affects patient prognosis and survival rates. However, the treatment of OS, its recurrence and subsequent metastasis is now at a clinical bottleneck. To explore new OS chemotherapeutic targets, investigate new therapeutic strategies and improve patient prognosis and survival rates, the roles of paclitaxel (PTX) and monopolar spindle kinase 1 (Mps1) in OS were investigated using in vivo and in vitro models. Mps1 expression was upregulated in OS samples and associated with patient survival times. Moreover, spindle assembly checkpoint (SAC) activation and upregulation of Akt/mTOR signaling were both positively associated with OS progression. PTX treatment significantly inhibited Mps1 expression, as well as migration of OS cells both in vitro. In addition, the combination of Mps1 knockdown and PTX treatment inhibited OS progression in vivo. Mps1 overexpression inhibited the expression of SAC markers and upregulated Akt and mTOR expression, while Mps1 knockdown had the opposite effect. Cells subjected to combined Mps1 knockdown and PTX treatment exhibited activation of SAC and inhibition of Akt/mTOR signaling compared with Mps1 knockdown or PTX treatment alone. Based on these observations, Mps1 inhibition combined with PTX treatment may represent a potentially effective strategy for the treatment of OS.

HA15 alleviates bone loss in ovariectomy-induced osteoporosis by targeting HSPA5.

The imbalance between osteogenesis and adipogenesis in the bone marrow is the main characteristic of osteoporosis (OP). Thus, exploring regulation of the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts and adipocytes is important to identify novel targets for the treatment of OP. In the present study, the master regulator of endoplasmic reticulum (ER) stress, heat shock protein family A (Hsp70) member 5 (HSPA5) was shown to significantly accumulate in osteoblasts and adipocytes, but not in osteoclasts in bone sections from aged and postmenopausal OP mice. In vitro study revealed that HSPA5 negatively modulated osteogenic differentiation and positively promoted adipogenic differentiation, and that targeting HSPA5 with its inhibitor HA15 enhanced osteogenic differentiation and inhibited adipogenic differentiation. Also, HA15 treatment induces ER stress and autophagy, and decreases apoptosis in cells. We constructed a postmenopausal OP model in mice with ovariectomy surgery, and treated the mice with HA15. The results showed that HA15 treatment induced appropriate ER stress, activated autophagy and decreased apoptosis in osteoblasts, thereby alleviating bone loss in vivo. Our results indicated that HSPA5 participated in OP pathogenesis by regulating the differentiation of BMSCs. HSPA5 may serve as a new target for the treatment of OP, and targeting HSPA5 with HA15 prevents the progression of OP and provides a candidate therapeutic molecule for postmenopausal OP.

MiR-665 Regulates Vascular Smooth Muscle Cell Senescence by Interacting With LncRNA GAS5/SDC1.

Background: Vascular aging is considered a special risk factor for cardiovascular diseases, and vascular smooth muscle cells (VSMCs) play a major role in aging-related vascular remodeling and in the pathological process of atherosclerosis. Recent research has reported that long non-coding RNA/microRNA (lncRNA/miRNA) is a critical regulator of cellular senescence. However, the role and mechanism of lncRNA GAS5/miR-665 axis in VSMC senescence remain incompletely understood. Methods: Cellular senescence was evaluated using senescence-associated ß-gal activity, the NAD+/NADH ratio, and by immunofluorescence staining of γH2AX immunofluorescence. Differentially expressed miRNAs (DEMs) were identified by miRNA microarray assays and subsequently validated by quantitative real-time PCR (qRT-PCR). A dual luciferase reporter assay was conducted to confirm the binding of lncRNA GAS5 and miR-665 as well as miR-665 and syndecan 1 (SDC1). Serum levels of miR-665, lncRNA GAS5, and SDC1 in 93 subjects were detected by qRT-PCR. The participants were subdivided into control, aging, and early vascular aging (EVA) groups, and their brachial-ankle pulse wave velocity (baPWV) was measured. Results: A total of 20 overlapping DEMs were identified in young and old VSMCs via microarray analysis. MiR-665 showed a significant alteration and, therefore, was selected for further analysis. Upregulation of miR-665 was found in aging VSMCs, and downregulation of miR-665 caused an inhibition of VSMCs senescence. Subsequently, the dual luciferase reporter assay determined the binding site of miR-665 with the 3'-UTR of lncRNA GAS5 and SDC1. Increased expression of lncRNA GAS5 expression inhibited the miR-665 level and VSMC senescence. However, as shown in rescue experiment results, either miR-665 overexpression or SDC1 knockdown significantly reversed the effects of lncRNA GAS5 on VSMC senescence. Finally, compared with that of the control group, miR-665 was highly expressed in serum samples in the aging and EVA groups, especially in the EVA groups. On the contrary, serum levels of lncRNA GAS5 and SDC1 were lower in these two groups. Collectively, in the aging and EVA groups, miR-665 expression was negatively correlated with lncRNA GAS5 and SDC1 expression. Conclusion: miR-665 inhibition functions as a vital modulator of VSMC senescence by negatively regulating SDC1, which is achieved by lncRNA GAS5 that sponges miR-665. Our findings may provide a new treatment strategy for aging-related cardiovascular diseases.

Targeting DKK1 prevents development of alcohol-induced osteonecrosis of the femoral head in rats.

Osteonecrosis of the femoral head (ONFH) is a devastating bone disease characterized by avascular or aseptic necrosis of the femoral head, and alcohol consumption is reported one of the leading risks to this disease. Previous studies have linked Dickkopf-1 (DKK1) to the occurrence of ONFH, but the role of DKK1 in alcohol-induced ONFH (AONFH) has not been fully discussed. In this study, we found that the expression level of DKK1 was dramatically increased in serum and bone samples from AONFH patients, experimental AONFH rats, and cultured bone marrow mesenchymal stem cells (BMMSCs) with ethanol stimulation. Elevated DKK1 inhibited Wnt/ß-catenin signaling in vivo and in vitro, while knockdown of DKK1 enhanced the nuclear translocation of ß-catenin and promoted osteogenesis and inhibited adipogenesis in the BMMSC cell line C3H10T1/2. Local injection of DKK1 knockout lentivirus into the femoral head of rats alleviated the progression of AONFH, with activated Wnt/ß-catenin signaling, increased bone formation, reduced number of empty adipose lacunae and restored blood supply. In conclusion, our findings confirmed the important role of DKK1 and canonical Wnt/ß-catenin pathway in AONFH. We propose that DKK1 may be a prognostic marker of AONFH and targeting DKK1 to activate the canonical Wnt/ß-catenin pathway and restore osteogenic potential could be a promising therapeutic strategy to prevent AONFH.

Olmesartan alleviates bleomycin-mediated vascular smooth muscle cell senescence via the miR-665/SDC1 axis.

Olmesartan (OMST) is a new angiotensin II receptor antagonist recently approved by the FDA to treat cardiovascular diseases. We investigated the molecular mechanisms by which OMST regulates vascular senescence. In the present study, bleomycin (BLM) was used to induce senescence in vascular smooth muscle cells (VSMCs); after which, the cells were treated with OMST. The effects of OMST on BLM-mediated cell senescence were evaluated using cell adhesion, NAD+/NADH, and Annevin V/PI double staining assays, as well as by immunofluorescence staining of γH2AX, Edu flow cytometry, and evaluations of senescence-associated ß-gal activity. Differentially expressed microRNAs (DEMs) were identified by miRNA microarray assays, and subsequently validated by quantitative real time PCR. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the miR-665 promoter. The target genes of miR-665 were predicted and confirmed using luciferase reporter assays. We found that miR-665 was upregulated in VSMCs in response to BLM-induced cellular senescence. BSP studies revealed that CpG sites in the promoter region of the miR-665 gene underwent extensive demethylation during BLM-induced cellular senescence, and there was a concomitant up-regulation of miR-665 expression. SDC1 mRNA was identified as a direct target of miR-665. Either miR-665 overexpression or SDC1 knockdown significantly reversed the effects of OMST on BLM-induced VSMC senescence. Moreover, SDC1 overexpression partially reversed the changes that occurred in cells with BLM-induced senescence caused by miR-665 overexpression. Our findings suggest that the miR-665/SDC1 axis functions as a vital modulator of VSMC senescence, and may represent a novel biological target for treating atherosclerosis.

[Olmesartan inhibits age-associated migration and invasion of human aortic vascular smooth muscle cells by upregulating miR-3133 axis].

OBJECTIVE: To explore the effects of olmesartan on age-associated migration and invasion capacities and microRNA (miRAN) axis in human aortic vascular smooth muscle cells (HA-VSMCs). METHODS: Cultured HA-VSMCs were divided into control group, bleomycin-mediated senescence (BLM) group and bleomycin + olmesartan treatment group. Wound-healing assay and Boyden chambers invasion assay were used to assess the changes in migration and invasion of the cells, gelatin zymography was used to analyze matrix metalloproteinase-2 (MMP-2) activation in the cells. The differentially expressed miRNAs were identified by miRNA microarray assay and validated by quantitative real-time PCR. MiR-3133 inhibitor was used to examine the effects of molecular manipulation of olmesartan on age-associated migration and invasion and MMP-2 activation in the cells. RESULTS: Compared with those of the control group, the percentage of the repopulated cells and the number of cells crossing the basement membrane increased significantly in BLM group [(78.43±12.76)% vs (42.47±7.22)%, P < 0.05; 33.33±5.51 vs 13.00±4.36, P < 0.05]. A significant increase of MMP-2 activation was found in BLM group as compared with the control group (1.66 ± 0.27 vs 0.87 ± 0.13, P < 0.05). Olmesartan significantly inhibited BLM-induced enhancement of cell migration and invasion and MMP-2 secretion in the cells. MiR-3133 was significantly downregulated in BLM group and upregulated in olmesartan group. Transfection with miR-3133 inhibitor significantly reversed the effects of olmesartan on age-associated migration and invasion of the cells [(85.87±7.39)% vs (49.77±3.05)%; 34.67±2.31 vs 20.00±4.58, P < 0.05] and MMP-2 activation in the cells (1.76±0.19 vs 0.94±0.10, P < 0.05). CONCLUSIONS: Olmesartan inhibits the migration and invasion of ageassociated HA-VSMCs probably by upregulating of the miR-3133 axis.

Fibronectin 1 activates WNT/ß-catenin signaling to induce osteogenic differentiation via integrin ß1 interaction.

Osteoporosis (OP) is a systemic skeletal disease leading to fragility fractures and is a major health issue globally. WNT/ß-catenin signaling regulates bone-remodeling processes and plays vital roles in OP development. However, the underlying regulatory mechanisms behind WNT/ß-catenin signaling in OP requires clarification, as further studies are required to identify novel alternate therapeutic agents to improve OP. Here we report that fibronectin 1 (FN-1) promoted differentiation and mineralization of osteoblasts by activating WNT/ß-catenin pathway, in cultured pre-osteoblasts. With isobaric tags for relative and absolute quantitation labeling proteomics analysis, we investigated protein changes in bone samples from OP patients and normal controls. FN-1 accumulated in osteoblasts in bone samples from OP patients and age-related OP mice compared to control group. In addition, we observed that integrin ß1 (ITGB1) acts as an indispensable signaling molecule for the interplay between FN-1 and ß-catenin, and that FN-1 expression increased, but ITGB1 expression decreased in osteoblasts during OP progression. Therefore, our study reveals a novel explanation for WNT/ß-catenin pathway inactivation in OP pathology. Supplying of FN-1 and ITGB1 may provide a potential therapeutic strategy in improving bone formation during OP.

Nutrient release from fish cage aquaculture and mitigation strategies in Daya Bay, southern China.

Finfish cage culture is the most predominant form of mariculture. The rapid expansion of fish cage culture systems has raised concerns about their environmental impact, such as nutrient release. In this study, for the first time, we estimated the release of nitrogen (N) and phosphorus (P) from fish cage culture in Daya Bay, southern China, by constructing N and P budget models based on a mass balance principle. In addition, the contribution and importance of nutrients from fish culture and other nutrient sources, including submarine groundwater discharge, benthic sediments, local rivers, and atmospheric deposition were assessed. The annual amount of N and P released from fish cage culture was 205.6 metric tons (hereafter tons) of N and 39.2 tons of P, including 142.7 tons of dissolved inorganic nitrogen (DIN) and 15.1 tons of dissolved inorganic phosphorus (DIP). Among the analyzed nutrient sources, the contributions of DIN and DIP from fish culture were 7.0% and 2.7%, respectively. For cages consuming conventional trash fish, 142 kg of N and 26 kg of P were released into the environment per ton of fish products, much higher than the values (72 kg N and 17.3 kg P) for cages using formulated feed. In fish culture, the dissolved nutrients were more N rich, but the particulate nutrients were more P rich. The ratio of cage-derived N and P was 21.1, higher than the ratio of coastal seawater (27.1), indicating that cage culture may also impact the local nutrient forms around farming regions. Oyster cultivation and harvest removed 126.3 tons of N and 35.1 tons of P from of the bay. Replacing trash fish with formulated feed and co-culturing of nutrient extractive species (e.g., bivalves, macroalgae) and deposit-feeding species (e.g., sea cucumber) in fish culture zones can be efficient nutrient mitigation strategies.