Assessment of urban ecosystem health and its influencing factors: a case study of Zibo City, China.

Urban ecosystem health is the foundation of sustainable urban development. It is important to know the health status of urban ecosystem and its influencing factors for formulating scientific urban development planning. Taking Zibo city as the study area, the indicators were selected from five aspects: ecosystem vigor, structure, resilience, service function and population health to establish an assessment index system of urban ecosystem health. The health level of urban ecosystem was assessed, and its changing trend was analyzed from 2006 to 2018 in Zibo. Furthermore, obstacle degree analysis and sensitivity analysis were used to quantitatively analyze the main obstacle factors and sensitivity factors affecting urban ecosystem health, so as to provide references for improving urban ecosystem health. The results showed that the health level of urban ecosystem in Zibo showed an upward trend from 2006 to 2018. The poor structure and ecological environment quality were the main obstacle factors to urban ecosystem health. The impact of changes in a single indicator on urban ecosystem health gradually decreased, but the sensitivity index of indicators had obvious differences. Urban ecosystem health was sensitive to changes in ecosystem structure and resilience. In the future, Zibo should strengthen ecological construction, optimize the industrial structure, and develop green economy to promote urban ecosystem healthy.

Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using in vitro and ex vivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

FNDC5/irisin ameliorates bone loss of type 1 diabetes by suppressing endoplasmic reticulum stress­mediated ferroptosis.

BACKGROUND: Ferroptosis is known to play a crucial role in diabetic osteopathy. However, key genes and molecular mechanisms remain largely unclear. This study aimed to identify a crucial ferroptosis-related differentially expressed gene (FR-DEG) in diabetic osteopathy and investigate its potential mechanism. METHODS: We identified fibronectin type III domain-containing protein 5 (FNDC5)/irisin as an essential FR-DEG in diabetic osteopathy using the Ferroptosis Database (FerrDb) and GSE189112 dataset. Initially, a diabetic mouse model was induced by intraperitoneal injection of streptozotocin (STZ), followed by intraperitoneal injection of irisin. MC3T3-E1 cells treated with high glucose (HG) were used as an in vitro model. FNDC5 overexpression plasmid was used to explore underlying mechanisms in vitro experiments. Femurs were collected for micro-CT scan, histomorphometry, and immunohistochemical analysis. Peripheral serum was collected for ELISA analysis. Cell viability was assessed using a CCK-8 kit. The levels of glutathione (GSH), malondialdehyde (MDA), iron, reactive oxygen species (ROS), and lipid ROS were detected by the corresponding kits. Mitochondria ultrastructure was observed through transmission electron microscopy (TEM). Finally, mRNA and protein expressions were examined by quantitative real-time PCR (qRT-PCR) and western blot analysis. RESULTS: The expression of FNDC5 was found to be significantly decreased in both in vivo and in vitro models. Treatment with irisin significantly suppressed ferroptosis and improved bone loss. This was demonstrated by reduced lipid peroxidation and iron overload, increased antioxidant capability, as well as the inhibition of the ferroptosis pathway in bone tissues. Furthermore, in vitro studies demonstrated that FNDC5 overexpression significantly improved HG-induced ferroptosis and promoted osteogenesis. Mechanistic investigations revealed that FNDC5 overexpression mitigated ferroptosis in osteoblasts by inhibiting the eukaryotic initiation factor 2 alpha (eIF2α)/activated transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) pathway. CONCLUSIONS: Collectively, our study uncovered the important role of FNDC5/irisin in regulating ferroptosis of diabetic osteopathy, which might be a potential therapeutic target.

METTL3 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells through IGF2BP1-Mediated Regulation of Runx2 Stability.

N6-Methyladenosine (m6A) has been reported to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) remains unclear. Here, we found that methyltransferase-like 3 (METTL3) was up-regulated synchronously with m6A during the osteogenic differentiation of hPDLSCs. Functionally, lentivirus-mediated knockdown of METTL3 in hPDLSCs impaired osteogenic potential. Mechanistic analysis further showed that METTL3 knockdown decreased m6A methylation and reduced IGF2BP1-mediated stability of runt-related transcription factor 2 (Runx2) mRNA, which in turn inhibited osteogenic differentiation. Therefore, METTL3-based m6A modification favored osteogenic differentiation of hPDLSCs through IGF2BP1-mediated Runx2 mRNA stability. Our study shed light on the critical roles of m6A on regulation of osteogenic differentiation in hPDLSCs and served novel therapeutic approaches in vital periodontitis therapy.

Prediction of risk factors for linezolid-induced thrombocytopenia based on neural network model.

Background: Based on real-world medical data, the artificial neural network model was used to predict the risk factors of linezolid-induced thrombocytopenia to provide a reference for better clinical use of this drug and achieve the timely prevention of adverse reactions. Methods: The artificial neural network algorithm was used to construct the prediction model of the risk factors of linezolid-induced thrombocytopenia and further evaluate the effectiveness of the artificial neural network model compared with the traditional Logistic regression model. Results: A total of 1,837 patients receiving linezolid treatment in a hospital in Xi 'an, Shaanxi Province from 1 January 2011 to 1 January 2021 were recruited. According to the exclusion criteria, 1,273 cases that did not meet the requirements of the study were excluded. A total of 564 valid cases were included in the study, with 89 (15.78%) having thrombocytopenia. The prediction accuracy of the artificial neural network model was 96.32%, and the AUROC was 0.944, which was significantly higher than that of the Logistic regression model, which was 86.14%, and the AUROC was 0.796. In the artificial neural network model, urea, platelet baseline value and serum albumin were among the top three important risk factors. Conclusion: The predictive performance of the artificial neural network model is better than that of the traditional Logistic regression model, and it can well predict the risk factors of linezolid-induced thrombocytopenia.

LncRNA urothelial cancer associated 1 promotes the osteogenic differentiation of human periodontal ligament stem cells by regulating the miR-96-5p/Osx axis.

OBJECTIVES: To investigate the expression of long non-coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) in human periodontal ligament stem cells (hPDLSCs), its effect on osteogenic differentiation of hPDLSCs and its mechanism. DESIGN: The expression of osteogenic genes Osx, Runx2, Ocn and Opn was explored by qPCR. Protein expression in hPDLSCs was estimated by Western blot. The osteogenic differentiation of hPDLSCs was detected by Alizarin red staining assays. The interaction between UCA1 and miR-96-5p was explored by RNA pulldown assay and dual luciferase assay. The interaction between miR-96-5p and Osx 3'-UTR was measured by dual luciferase assay. RESULTS: The expression of UCA1 and miR-96-5p was negatively correlated in hPDLSCs. During the osteogenic differentiation of hPDLSCs, the expression of UCA1 was increased, while the expression of miR-96-5p was decreased. Knockdown of UCA1 in hPDLSCs inhibited osteogenic differentiation but induced upregulation of miR-96-5p expression, and vice versa. In addition, miR-96-5p partially reversed the positive effect of UCA1 on osteogenic differentiation of hPDLSCs. Notably, UCA1 was identified as a miR-96-5p sponge, and miR-96-5p targeted Osx. CONCLUSIONS: Our results demonstrated that the novel UCA1/miR-96-5p/Osx pathway regulates osteogenic differentiation of hPDLSCs and sheds new insights and targets for periodontitis therapeutics.

DAP1 regulates osteoblast autophagy via the ATG16L1-LC3 axis in Graves' disease-induced osteoporosis.

OBJECTIVE: This study aimed to uncover a critical protein and its mechanisms in modulating autophagy in Graves' disease (GD)-induced osteoporosis (OP). METHODS: We discovered the target protein, death-associated protein 1 (DAP1), using bone proteomics analysis. Furthermore, genetic overexpression and knockdown (KD) of DAP1 in bone and MC3T3-E1 cells revealed DAP1 effects on autophagy and osteogenic markers, and autophagic vacuoles in cells were detected using transmission electron microscopy and the microtubule-associated protein 1 light chain 3 alpha (MAP1LC3/LC3) dual fluorescence system. An autophagy polymerase chain reaction (PCR) array kit was used to identify the key molecules associated with DAP1-regulated autophagy. RESULTS: DAP1 levels were significantly higher in the bone tissue of GD mice and MC3T3-E1 cells treated with triiodothyronine (T3). DAP1 overexpression reduced LC3 lipidation, autophagic vacuoles, RUNX family transcription factor 2 (RUNX2), and osteocalcin (OCN) expression in MC3T3-E1 cells, whereas DAP1 KD reversed these changes. In vivo experiments revealed that GD mice with DAP1 KD had greater bone mass than control mice. DAP1-overexpressing (OE) cells had lower levels of phosphorylated autophagy-related 16-like 1 (ATG16L1) and LC3 lipidation, whereas DAP1-KD cells had higher levels. CONCLUSIONS: DAP1 was found to be a critical regulator of autophagy homeostasis in GD mouse bone tissue and T3-treated osteoblasts because it negatively regulated autophagy and osteogenesis in osteoblasts via the ATG16L1-LC3 axis.

Sodium-glucose cotransporter 2 inhibitor canagliflozin alleviates vascular calcification through suppression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome.

AIMS: Vascular calcification (VC) is prevalent in pathological processes such as diabetes, chronic kidney disease (CKD), and atherosclerosis, but effective therapies are still lacking by far. Canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor, has been approved for the treatment of type 2 diabetes mellitus and exhibits beneficial effects against cardiovascular disease. However, the effect of CANA on VC remains unknown. In this study, we hypothesize that CANA protects against VC. METHODS AND RESULTS: Micro-computed tomography analysis and alizarin red staining revealed that CANA treatment prevented aortic calcification in CKD rats and in VitD3-overloaded mice. Moreover, CANA alleviated the calcification of rat and human arterial rings. Alizarin red staining revealed that calcification of rat and human vascular smooth muscle cells (VSMCs) was attenuated by CANA treatment and this phenomenon was confirmed by calcium content assay. In addition, CANA downregulated the expression of osteogenic differentiation markers Runx2 and BMP2. Of interest, qPCR and western blot analysis revealed that CANA downregulated the expression of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), and the downstream signalling molecules Caspase-1 and IL-1ß in VSMCs as well. Both NLRP3 inhibitor MCC950 and knockdown of NLRP3 by siRNA independently resulted in decreased calcification of VSMCs. By contrast, activation of NLRP3 exacerbated VSMC calcification, and this effect was prevented by the addition of CANA. CONCLUSIONS: Our study for the first time demonstrates that CANA exerts a protective effect on VC at least partially via suppressing the NLRP3 signalling pathway. Therefore, supplementation of CANA as well as inhibition of NLRP3 inflammasome presents a potential therapy for VC.

Long non­coding RNA SNHG5 promotes osteogenic differentiation of human periodontal ligament stem cells via mediating miR­23b­3p/Runx2 axis.

The treatment of bone loss due to periodontitis has posed a great challenge for physicians for decades. Therefore, it is of extraordinary significance to identify an effective regeneration scheme for alveolar bone. This study aimed to investigate long non-coding RNA (lncRNA) small nucleolar RNA host gene 5 (SNHG5) whether sponges microRNA-23b-3p (miR-23b-3p) to achieve the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Results revealed that the expression of SNHG5 was upregulated whereas that of miR-23b-3p was downregulated in osteogenic hPDLSCs. Alizarin red staining assays and qRT-PCR demonstrated that SNHG5 silencing or miR-23b-3p overexpression inhibits hPDLSCs osteogenic differentiation and vice versa. In addition, miR-23b-3p partially abolished the promotive effect of SNHG5 on osteogenic differentiation of hPDLSCs. Dual luciferase report and RNA pulldown assay verified that miR-23b-3p is a regulatory target of SNHG5 and that Runx2 is a gene target of miR-23b-3p. In brief, the results demonstrate that SNHG5 promotes the osteogenic differentiation of hPDLSCs by regulating the miR-23b-3p/Runx2 axis. Our study provides novel mechanistic insights into the critical role of lncRNA SNHG5 as a miR-23b-3p sponge to regulate Runx2 expression in hPDLSCs and may serve as a potential therapeutics target for periodontitis.

A 3D-printed SERS bionic taster for dynamic tumor metabolites detection.

The variation of tumor-associated metabolites in extracellular microenvironment timely reflects the development, the progression and the treatment of cancers. Conventional methods for metabolite detection lack the efficiency to grasp the dynamic metabolic alterations. Herein, we developed a SERS bionic taster which enabled real-time analysis of extracellular metabolites. The instant information of cell metabolism was provided by the responsive Raman reporters, which experienced SERS spectral changes upon metabolite activation. Such a SERS sensor was integrated into a 3D-printed fixture which fits the commercial-standard cell culture dishes, allowing in-situ acquisition of the vibrational spectrum. The SERS taster can not only accomplish simultaneous and quantitative analysis of multiple tumor-associated metabolites, but also fulfill the dynamic monitoring of cellular metabolic reprogramming, which is expected to become a promising tool for investigating cancer biology and therapeutics.

Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is one of the most serious soil-borne diseases of wheat. Among 58 bacterial isolates from the rhizosphere soil of winter wheat seedlings, strain YB-1631 was found to have the highest in vitro antagonism to F. pseudograminearum growth. LB cell-free culture filtrates inhibited mycelial growth and conidia germination of F. pseudograminearum by 84.14% and 92.23%, respectively. The culture filtrate caused distortion and disruption of the cells. Using a face-to-face plate assay, volatile substances produced by YB-1631 inhibited F. pseudograminearum growth by 68.16%. In the greenhouse, YB-1631 reduced the incidence of FCR on wheat seedlings by 84.02% and increased root and shoot fresh weights by 20.94% and 9.63%, respectively. YB-1631 was identified as Bacillus siamensis based on the gyrB sequence and average nucleotide identity of the complete genome. The complete genome was 4,090,312 bp with 4357 genes and 45.92% GC content. In the genome, genes were identified for root colonization, including those for chemotaxis and biofilm production, genes for plant growth promotion, including those for phytohormones and nutrient assimilation, and genes for biocontrol activity, including those for siderophores, extracellular hydrolase, volatiles, nonribosomal peptides, polyketide antibiotics, and elicitors of induced systemic resistance. In vitro production of siderophore, ß-1, 3-glucanase, amylase, protease, cellulase, phosphorus solubilization, and indole acetic acid were detected. Bacillus siamensis YB-1631 appears to have significant potential in promoting wheat growth and controlling wheat FCR caused by F. pseudograminearum.

In vitro construction of liver organoids with biomimetic lobule structure by a multicellular 3D bioprinting strategy.

Liver disease is one of the serious threats to human life and health. Three-dimensional (3D) liver models, which simulate the structure and function of natural liver tissue in vitro, have become a common demand in medical, scientific and pharmaceutical fields nowadays. However, the complex cellular composition and multi-scale spatial arrangement of liver tissue make it extremely challenging to construct liver models in vitro. According to HepaRG preference and printing strategy, the formulation of bioink system with opposite charge is optimized. The sodium alginate-based bioink 1 and dipeptide-based bioink 2 are used to ensure structural integrity and provide flexible designability, respectively. The HepaRG/HUVECs/LX-2-laden liver organoids with biomimetic lobule structure are fabricated by a multicellular 3D droplet-based bioprinting strategy, to mimic the cell heterogeneity, spatial structure and extracellular matrix (ECM) features. The liver organoids can maintain structural integrity and multicellular distribution within the printed lobule-like structure after 7 days of culture. Compared with the 2D monolayer culture, the constructed 3D organoids show high cell viability, ALB secretion and urea synthesis levels. This study provides a droplet-based and layer-by-layer 3D bioprinting strategy for in vitro construction of liver organoids with biomimetic lobule structure, giving meaningful insights in the fields of new drugs, disease modelling, and tissue regeneration.

Biomass partitioning and ionomics of Macadamia with high manganese and low phosphorus concentrations.

Knowledge of the ionome of plant organs helps us understand a plant's nutritional status. However, the ionome of Macadamia (Proteaceae), which is an important nut-producing tree, remains unknown. We aimed to characterise the allocation of biomass and nutrient-partitioning patterns in three macadamia genotypes. We excavated 15 productive trees (three cultivars at 21years of age; two cultivars at 16years of age) in an orchard. Biomass, nutrient concentrations, and contents of roots, stems, branches, and leaves were analysed. Dry weight of roots, stems, branches and leaves accounted for 14-20%, 19-30%, 36-52%, and 12-18% of total plant weight, respectively. No significant difference was found in the total biomass among the cultivars at the same age. Compared with most crop plants, macadamia had low phosphorus (P) concentrations in all organs (<1gkg-1 ), and low leaf zinc (Zn) concentration (8mgkg-1 ). In contrast, macadamia accumulated large amounts of manganese (Mn), with a 20-fold higher leaf Mn concentration than what is considered sufficient for crop plants. Leaves exhibited the highest nutrient concentrations, except for iron and Zn, which exhibited the highest concentrations in roots. The organ-specific ionomics of Macadamia is characterised by low P and high Mn concentrations, associated with adaptation to P-impoverished habitats.

Exercise protects aged mice against coronary endothelial senescence via FUNDC1-dependent mitophagy.

Vascular aging contributes to adverse changes in organ function and is a significant indicator of major cardiac events. Endothelial cells (ECs) participate in aging-provoked coronary vascular pathology. Regular exercise is associated with preservation of arterial function with aging in humans. However, the molecular basis is not well understood. The present study was aimed to determine the effects of exercise on coronary endothelial senescence and whether mitochondrial clearance regulator FUN14 domain containing 1 (FUNDC1)-related mitophagy and mitochondrial homeostasis were involved. In mouse coronary arteries, FUNDC1 levels showed gradually decrease with age. Both FUNDC1 and mitophagy levels in cardiac microvascular endothelial cells (CMECs) were significantly reduced in aged mice and were rescued by exercise training. Exercise also alleviated CMECs senescence as evidenced by senescence associated ß-galactosidase activity and aging markers, prevented endothelial abnormal cell migration, proliferation, and eNOS activation in CMECs from aged mice, and improved endothelium-dependent vasodilation of coronary artery, reduced myocardial neutrophil infiltration and inflammatory cytokines evoked by MI/R, restored angiogenesis and consequently alleviated MI/R injury in aging. Importantly, FUNDC1 deletion abolished the protective roles of exercise and FUNDC1 overexpression in ECs with adeno-associated virus (AAV) reversed endothelial senescence and prevented MI/R injury. Mechanistically, PPARγ played an important role in regulating FUNDC1 expressions in endothelium under exercise-induced laminar shear stress. In conclusion, exercise prevents endothelial senescence in coronary arteries via increasing FUNDC1 in a PPARγ-dependent manner, and subsequently protects aged mice against MI/R injury. These findings highlight FUNDC1-mediated mitophagy as potential therapeutic target that prevents endothelial senescence and myocardial vulnerability.

Deletion of SIRT6 in vascular smooth muscle cells facilitates vascular calcification via suppression of DNA damage repair.

Vascular calcification is an important risk factor for cardiovascular events, accompanied by DNA damage during the process. The sirtuin 6 (SIRT6) has been reported to alleviate atherosclerosis, which is related to the reduction of DNA damage. However, whether smooth muscle cell SIRT6 mediates vascular calcification involving DNA damage remains unclear. Western blot and immunofluorescence revealed that SIRT6 expression was decreased in human vascular smooth muscle cells (HVSMCs), human and mouse arteries during vascular calcification. Alizarin red staining and calcium content assay showed that knockdown or deletion of SIRT6 significantly promoted HVSMC calcification induced by high phosphorus and calcium, accompanied by upregulation of osteogenic differentiation markers including Runx2 and BMP2. By contrast, adenovirus-mediated SIRT6 overexpression attenuated osteogenic differentiation and calcification of HVSMCs. Moreover, ex vivo study revealed that SIRT6 overexpression inhibited calcification of mouse and human arterial rings. Of note, smooth muscle cell-specific knockout of SIRT6 markedly aggravated Vitamin D3-induced aortic calcification in mice. Mechanistically, overexpression of SIRT6 reduced DNA damage and upregulated p-ATM during HVSMCs calcification, whereas knockdown of SIRT6 showed the opposite effects. Knockdown of ATM in HVSMCs abrogated the inhibitory effect of SIRT6 overexpression on calcification and DNA damage. This study for the first time demonstrates that vascular smooth muscle cell-specific deletion of SIRT6 facilitates vascular calcification via suppression of DNA damage repair. Therefore, modulation of SIRT6 and DNA damage repair may represent a therapeutic strategy for vascular calcification.

Activin receptor-like kinase 7 promotes apoptosis of vascular smooth muscle cells via activating Smad2/3 signaling in diabetic atherosclerosis.

Vascular smooth muscle cells (VSMCs) is a vital accelerator in the late phase of diabetic atherosclerosis, but the underlying mechanism remains unclear. The aim of our study was to investigate whether activin receptor-like kinase 7 (ALK7)-Smad2/3 pathway plays an important role in VSMC apoptosis of diabetic atherosclerosis. It was shown that ALK7 expression was obviously elevated in the aorta of ApoE-/- mice with type 2 diabetes mellitus. Inhibition of ALK7 expression significantly improved the stability of atherosclerotic plaques and reduced cell apoptosis. Further experiments showed that ALK7 knockdown stabilized atherosclerotic plaques by reducing VSMC apoptosis via activating Smad2/3. Our study uncovered the important role of ALK7-Smad2/3 signaling in VSMCs apoptosis, which might be a potential therapeutic target in diabetic atherosclerosis.

Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification.

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD). Cell death such as apoptosis plays a critical role in vascular calcification. Ferroptosis is a type of iron-catalyzed and regulated cell death resulting from excessive iron-dependent reactive oxygen species and lipid peroxidation. However, it is unclear whether ferroptosis of vascular smooth muscle cells (VSMCs) regulates vascular calcification in CKD. Our results showed that high calcium and phosphate concentrations induced ferroptosis in rat VSMCs in vitro. Inhibition of ferroptosis by ferrostatin-1 dose-dependently reduced mineral deposition in rat VSMCs under pro-osteogenic conditions, as indicated by alizarin red staining and quantification of calcium content. In addition, gene expression analysis revealed that ferrostatin-1 inhibited osteogenic differentiation of rat VSMCs. Similarly, ferrostatin-1 remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in vitamin D3-overloaded mice in vivo. Moreover, inhibition of ferroptosis by either ferrostatin-1 or deferoxamine attenuated aortic calcification in rats with CKD. Mechanistically, high calcium and phosphate downregulated expression of SLC7A11 (a cystine-glutamate antiporter), and reduced glutathione (GSH) content in VSMCs. Additionally, GSH depletion induced by erastin (a small molecule initiating ferroptotic cell death) significantly promoted calcification of VSMCs under pro-osteogenic conditions, whereas GSH supplement by N-acetylcysteine reduced calcification of VSMCs. Consistently, knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification. Furthermore, high calcium and phosphate downregulated glutathione peroxidase 4 (GPX4) expression, and reduced glutathione peroxidase activity. Inhibition of GPX4 by RSL3 promoted VSMC calcification. Thus, repression of the SLC7A11/GSH/GPX4 axis triggers ferroptosis of VSMCs to promote vascular calcification under CKD conditions, providing a novel targeting strategy for vascular calcification.

Warfarin Adherence Among Patients with Atrial Fibrillation in Rural Area of Dongyang, China: A Questionnaire-Based Study.

Introduction: Adherence to warfarin is associated with improved outcome in patients with atrial fibrillation (AF), but the adherence status of patients in rural areas of China is not known. Methods: A questionnaire-based study evaluating warfarin adherence of rural residents with AF was carried out in Dongyang, China. Potentially eligible patients were screened and contacted by telephone, and their demographic characteristics were collected. Illness perception was assessed using the Brief Illness Perception Questionnaire (BIPQ), and warfarin adherence was assessed using a Chinese-version adherence scale. Univariate and multivariate analyses were conducted to identify factors associated with unsatisfactory adherence. Results: A total of 201 patients (male, n=99; mean age, 70.3±8.12 years) were included, among whom 95 (47.3%) patients showed good adherence and 63 (31.3%) poor adherence. Number of co-dispensed drugs (multivariate analysis: odds ratio [OR]=3.64, 95% confidence interval [CI] 1.35-9.81, p=0.011) and BIPQ score (OR=1.25, 95% CI 1.17-1.33, p<0.001) were identified as factors associated with good adherence. Conclusion: Medical adherence to warfarin needs to improve in rural patients with AF. Efforts that can reduce the number of co-dispensed drugs and increase illness perception may improve warfarin adherence. This study may benefit future management of warfarin administration to rural patients with AF.

Downregulation of HDAC9 by the ketone metabolite ß-hydroxybutyrate suppresses vascular calcification.

Vascular calcification is an actively regulated process resembling bone formation and contributes to the cardiovascular morbidity and mortality of chronic kidney disease (CKD). However, an effective therapy for vascular calcification is still lacking. The ketone body ß-hydroxybutyrate (BHB) has been demonstrated to have health-promoting effects including anti-inflammation and cardiovascular protective effects. However, whether BHB protects against vascular calcification in CKD remains unclear. In this study, Alizarin Red staining and calcium content assay showed that BHB reduced calcification of vascular smooth muscle cells (VSMCs) and arterial rings. Of note, compared with CKD patients without thoracic calcification, serum BHB levels were lower in CKD patients with thoracic calcification. Supplementation with 1,3-butanediol (1,3-B), the precursor of BHB, attenuated aortic calcification in CKD rats and VitD3-overloaded mice. Furthermore, RNA-seq analysis revealed that BHB downregulated HDAC9, which was further confirmed by RT-qPCR and western blot analysis. Both pharmacological inhibition and knockdown of HDAC9 attenuated calcification of human VSMCs, while overexpression of HDAC9 exacerbated calcification of VSMCs and aortic rings, indicating that HDAC9 promotes vascular calcification under CKD conditions. Of note, BHB treatment antagonized HDAC9-induced vascular calcification. In addition, HDAC9 overexpression activated the NF-κB signaling pathway and inhibition of NF-κB attenuated HDAC9-induced VSMC calcification, suggesting that HDAC9 promotes vascular calcification via activation of NF-κB. In conclusion, our study demonstrates that BHB supplementation inhibits vascular calcification in CKD via modulation of the HDAC9-dependent NF-κB signaling pathway. Moreover, we unveil a crucial mechanistic role of HDAC9 in vascular calcification under CKD conditions; thus, nutritional intervention or pharmacological approaches to enhance BHB levels could act as promising therapeutic strategies to target HDAC9 for the treatment of vascular calcification in CKD. © 2022 The Pathological Society of Great Britain and Ireland.

Identification of Serum Exosome-Derived circRNA-miRNA-TF-mRNA Regulatory Network in Postmenopausal Osteoporosis Using Bioinformatics Analysis and Validation in Peripheral Blood-Derived Mononuclear Cells.

Background: Osteoporosis is one of the most common systemic metabolic bone diseases, especially in postmenopausal women. Circular RNA (circRNA) has been implicated in various human diseases. However, the potential role of circRNAs in postmenopausal osteoporosis (PMOP) remains largely unknown. The study aims to identify potential biomarkers and further understand the mechanism of PMOP by constructing a circRNA-associated ceRNA network. Methods: The PMOP-related datasets GSE161361, GSE64433, and GSE56116 were downloaded from the Gene Expression Omnibus (GEO) database and were used to obtain differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to determine possible relevant functions of differentially expressed messenger RNAs (mRNAs). The TRRUST database was used to predict differential transcription factor (TF)-mRNA regulatory pairs. Afterwards, combined CircBank and miRTarBase, circRNA-miRNA as well as miRNA-TF pairs were constructed. Then, a circRNA-miRNA-TF-mRNA network was established. Next, the correlation of mRNAs, TFs, and PMOP was verified by the Comparative Toxicogenomics Database. And expression levels of key genes, including circRNAs, miRNAs, TFs, and mRNAs in the ceRNA network were further validated by quantitative real-time PCR (qRT-PCR). Furthermore, to screen out signaling pathways related to key mRNAs of the ceRNA network, Gene Set Enrichment Analysis (GSEA) was performed. Results: A total of 1201 DE mRNAs, 44 DE miRNAs, and 1613 DE circRNAs associated with PMOP were obtained. GO function annotation showed DE mRNAs were mainly related to inflammatory responses. KEGG analysis revealed DE mRNAs were mainly enriched in osteoclast differentiation, rheumatoid arthritis, hematopoietic cell lineage, and cytokine-cytokine receptor interaction pathways. We first identified 26 TFs and their target mRNAs. Combining DE miRNAs, miRNA-TF/mRNA pairs were obtained. Combining DE circRNAs, we constructed the ceRNA network contained 6 circRNAs, 4 miRNAs, 4 TFs, and 12 mRNAs. The expression levels of most genes detected by qRT-PCR were generally consistent with the microarray results. Combined with the qRT-PCR validation results, we eventually identified the ceRNA network that contained 4 circRNAs, 3 miRNAs, 3 TFs, and 9 mRNAs. The GSEA revealed that 9 mRNAs participate in many important signaling pathways, such as "olfactory transduction", "T cell receptor signaling pathway", and "neuroactive ligand-receptor interaction". These pathways have been reported to the occurrence and development of PMOP. To sum up, key mRNAs in the ceRNA network may participate in the development of osteoporosis by regulating related signal pathways. Conclusions: A circRNA-associated ceRNA network containing TFs was established for PMOP. The study may help further explore the molecular mechanisms and may serve as potential biomarkers or therapeutic targets for PMOP.