Optimization of production of PLA microbubble ultrasound contrast agents for Hydroxycamptothecin delivery

In this paper, ultrasound contrast agents based on a high molecular polymer poly lactic acid (PLA) and loaded with Hydroxycamptothecin (HCPT) were prepared by combining ultrasound method and a Shirasu Porous Glass (SPG) membrane emulsification technique. A special focus was on the optimization of production of RCPT-PLA microbubbles. Different factors, such as the power and the time of ultrasonic action, the ratio of inner aqueous phase against outer oil phase, and the concentration of PLA were evaluated, and the average size of HCPT-PLA microbubbles, the drug carrying efficiency, as well as the acoustically-triggered drug release at 3kHz ultrasound were determined. The study showed that the HCPT-PLA microbubbles prepared using our optimized conditions, were sphere-like in shape with a mean diameter of 1-7 mu m. The drug loading efficiency reached up to 56.48%. In vitro, the drug release of HCPT-PLA microbubbles increased significantly at 3kHz ultrasound for 30s compared with that of ultrasound free condition. In conclusion, the HCPT-PLA microbubbles has the characteristics desirable for an intravenously administered ultrasound contrast agent for further clinical use

A calibration method of USBL installation error based on attitude determination

The Ultra-short baseline (USBL) positioning system has important application in the positioning of underwater vehicles. The installation error angle of the USBL positioning system has an important influence on the positioning accuracy of USBL system. The traditional calibration methods have limited estimation accuracy for installation error angles and have high route requirements. To solve the above problems, a calibration method of installation error angle based on attitude determination is proposed in this paper. When strapdown inertial navigation system (SINS) and USBL are fixed together in the application process, the installation error angle of USBL is fixed and unchanged. Then the calibration of installation error angle can be accomplished with the idea of attitude determination. The vector observation model based on the installation error angle matrix is established first. Observation vectors are obtained by the relative position of transponders in the USBL coordinate frame. The reference vector is calculated by position of transponder, position and attitude of SINS and lever arm between SINS and USBL. By constructing the observation vectors and the reference vectors, the proposed method can calibrate the installation error angle of SINS and USBL in real time. The advantages of the proposed method are that it has no specific requirements for the calibration route and can calibrate the installation error angle in real time with high accuracy. In order to verify the performance of the proposed algorithm, simulation experiment and field experiment are carried out in this paper. The results of simulation experiment and field experiment show that the proposed method can give the estimated installation error angle of USBL in real time, and the estimated result is the best among several methods. The proposed method can not only achieve the calibration of the installation error angle in circular trajectory, but also in straight trajectory

15-lipoxygenase-1 mediated formation of endogenous GSH- and RNA-adducts and its impact on GSH/GSSG homeostasis in monocytes/macrophages

15-lipoxygenase-1 (15-LOX-1) converts arachidonic acid to 15(S)-hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE), which can decompose to the bifunctional electrophile 4-oxo-2-nonenal (ONE). ONE forms adducts with DNA and proteins, which are potential biomarkers of lipid-peroxidation-mediated oxidative stress. Recently a novel ONE-GSH-adduct was characterized as thiadiazabicyclo-ONE-GSH (TOG). A major objective of the present study was to test whether 15-LOX-1-mediated lipid peroxidation can lead to endogenous TOG formation. The impact of 15-LOX-1 expression on glutathione (GSH) homeostasis was also evaluated. Furthermore, ONE-RNA adducts were also characterized for the first time. 15-LOX-1-mediated formation of TOG was established in RAW267.4 cells that constitutively over-express 15-LOX-1 (R15LO cells). Intracellular TOG levels increased upon treatment with arachidonic acid or calcium ionophore A23187, accompanied by increased lipid peroxidation mediated by 15-LOX-1. The LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) significantly reduced lipid peroxidation and abated TOG formation. Further analysis of membrane lipids suggested translocation of 15-LOX-1 upon calcium mobilization and subsequent oxidation of membrane lipids by 15-LOX-1. The 15-LOX-1-dependent TOG formation was further studied in primary human monocytes. Interleukin-4 (IL-4) treatment differentiated primary human monocytes and induced 15-LOX-1 expression. Arachidonic acid and calcium ionophore A23187 significantly increased TOG levels in IL-4-differentiated primary human monocytes and the increased levels were abolished by CDC. Primary human monocytes had much higher 15-LOX-1 activity than R15LO cells. The increase and decrease of lipid oxidation followed that of TOG levels. In order to study the impact of 15-LOX-1 on GSH/GSH-disulfide (GSSG) homeostasis, a liquid chromatography (LC)/mass spectrometry (MS) method was developed to quantify cellular GSH and GSSG. ONE treatment depleted GSH and compromised the cellular redox state. Interestingly, 15-LOX-1 enhanced the resistance of cells to ONE-induced GSH depletion. Calcium ionophore also depleted GSH. However, 15-LOX-1 exacerbated the effect of calcium ionophore on GSH/GSSG homeostasis. Adducts formed between ONE and the RNA nucleotides guanosine (Guo), adenosine (Ade) and cytidine (Cyd) were characterized to be the heptanone-etheno-adducts. A LC/MS method was developed to quantify these adducts in cells. All three adducts were detected in RAW267.4 cells after ONE treatment. The endogenous levels of these adducts were very low. These results suggest that RNA is highly protected from damage by endogenous ONE

15-lipoxygenase-1 mediated formation of endogenous GSH- and RNA-adducts and its impact on GSH/GSSG homeostasis in monocytes/macrophages

15-lipoxygenase-1 (15-LOX-1) converts arachidonic acid to 15(S)-hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE), which can decompose to the bifunctional electrophile 4-oxo-2-nonenal (ONE). ONE forms adducts with DNA and proteins, which are potential biomarkers of lipid-peroxidation-mediated oxidative stress. Recently a novel ONE-GSH-adduct was characterized as thiadiazabicyclo-ONE-GSH (TOG). A major objective of the present study was to test whether 15-LOX-1-mediated lipid peroxidation can lead to endogenous TOG formation. The impact of 15-LOX-1 expression on glutathione (GSH) homeostasis was also evaluated. Furthermore, ONE-RNA adducts were also characterized for the first time. 15-LOX-1-mediated formation of TOG was established in RAW267.4 cells that constitutively over-express 15-LOX-1 (R15LO cells). Intracellular TOG levels increased upon treatment with arachidonic acid or calcium ionophore A23187, accompanied by increased lipid peroxidation mediated by 15-LOX-1. The LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) significantly reduced lipid peroxidation and abated TOG formation. Further analysis of membrane lipids suggested translocation of 15-LOX-1 upon calcium mobilization and subsequent oxidation of membrane lipids by 15-LOX-1. The 15-LOX-1-dependent TOG formation was further studied in primary human monocytes. Interleukin-4 (IL-4) treatment differentiated primary human monocytes and induced 15-LOX-1 expression. Arachidonic acid and calcium ionophore A23187 significantly increased TOG levels in IL-4-differentiated primary human monocytes and the increased levels were abolished by CDC. Primary human monocytes had much higher 15-LOX-1 activity than R15LO cells. The increase and decrease of lipid oxidation followed that of TOG levels. In order to study the impact of 15-LOX-1 on GSH/GSH-disulfide (GSSG) homeostasis, a liquid chromatography (LC)/mass spectrometry (MS) method was developed to quantify cellular GSH and GSSG. ONE treatment depleted GSH and compromised the cellular redox state. Interestingly, 15-LOX-1 enhanced the resistance of cells to ONE-induced GSH depletion. Calcium ionophore also depleted GSH. However, 15-LOX-1 exacerbated the effect of calcium ionophore on GSH/GSSG homeostasis. Adducts formed between ONE and the RNA nucleotides guanosine (Guo), adenosine (Ade) and cytidine (Cyd) were characterized to be the heptanone-etheno-adducts. A LC/MS method was developed to quantify these adducts in cells. All three adducts were detected in RAW267.4 cells after ONE treatment. The endogenous levels of these adducts were very low. These results suggest that RNA is highly protected from damage by endogenous ONE

Bushen Huoxue Recipe inhibits endometrial epithelial-mesenchymal transition through the transforming growth factor-β/nuclear factor kappa-B pathway to improve polycystic ovary syndrome-mediated infertility

AbstractObjective To investigate the target and mechanism of action of Bushen Huoxue Recipe (BSHX) for the treatment of infertility in polycystic ovary syndrome (PCOS), to provide a basis for the development and clinical application of herbal compounds.Methods Prediction and validation of active ingredients and targets of BSHX for the treatment of PCOS by using network pharmacology-molecular docking technology. In an animal experiment, the rats were randomly divided into four groups (control group, model group, BSHX group, metformin group, n = 16 in each group), and letrozole combined with high-fat emulsion gavage was used to establish a PCOS rat model. Body weight, vaginal smears, and number of embryos were recorded for each group of rats. Hematoxylin-eosin (HE) staining was used to observe the morphological changes of ovarian and endometrial tissues, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the serum inflammatory factor levels. Expression levels of transforming growth factor-β (TGF-β), transforming growth factor beta activated kinase 1 (TAK1), nuclear factor kappa-B (NF-κB), Vimentin, and E-cadherin proteins were measured by western blot (WB).Results Ninety active pharmaceutical ingredients were obtained from BSHX, involving 201 protein targets, of which 160 were potential therapeutic targets. The active ingredients of BSHX exhibited lower binding energy with tumor necrosis factor-α (TNF-α), TGF-β, TAK1, and NF-κB protein receptors (< −5.0 kcal/mol). BSHX significantly reduced serum TNF-α levels in PCOS rats (p < .01), effectively regulated the estrous cycle, restored the pathological changes in the ovary and endometrium, improved the pregnancy rate, and increased the number of embryos. The results of WB suggested that BSHX can down-regulate protein expression levels of TGF-β and NF-κB in endometrial tissue (p < .05), promote the expression level of E-cadherin protein (p < .001), intervene in the endometrial epithelial-mesenchymal transition (EMT) process.Conclusions TGF-β, TAK1, NF-κB, and TNF-α are important targets of BSHX for treating infertility in PCOS. BSHX improves the inflammatory state of PCOS, intervenes in the endometrial EMT process through the TGF-β/NF-κB pathway, and restores endometrial pathological changes, further improving the pregnancy outcome in PCOS

Multi-Scenario Simulation of Land Use and Landscape Ecological Risk Response Based on Planning Control

This study applied territorial spatial planning control to a land use multi-scenario simulation in Changde, China, and measured the landscape ecological risk response. It embedded five planning control schemes, respectively, involving inertial development, urban expansion size quantity control, ecological spatial structure control, land use zoning control, and comprehensive control. Findings show that: (1) Woodland and arable land in Changde occupy 31.10% and 43.35% of land use, respectively, and constitute the main functional space of the research area. The scale of construction land in Changde has enlarged continuously, with ecological space represented by woodland and water constantly squeezed and occupied. (2) Comprehensive control has the most remarkable restraining effect on the disordered spread of construction land, while ecological space structure control is the most effective way to control ecological land shrinkage. (3) The overall landscape ecological risk index expanded over 2009&ndash;2018, presenting an S-type time evolution curve of &ldquo;sharp increase&ndash;mitigation&rdquo;. Landscape ecological risk presents a single-core, double-layer circle structure with the north and east regions as the core, attenuating to the periphery. (4) Landscape ecological risk under land use zoning control increased significantly more than in other scenarios. Comprehensive control best prevented landscape ecological risk and restrained the disorderly expansion of construction land

15-oxo-Eicosatetraenoic Acid, a Metabolite of Macrophage 15-Hydroxyprostaglandin Dehydrogenase That Inhibits Endothelial Cell Proliferation

The formation of 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) as a product from rabbit lung 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid was first reported more than 30 years ago. However, the pharmacological significance of 15-oxo-ETE formation has never been established. We have now evaluated 15-lipoxygenase (LO)-1-mediated arachidonic acid (AA) metabolism to 15-oxo-ETE in human monocytes and mouse RAW macrophages that stably express human 15-LO-1 (R15L cells). A targeted lipidomics approach was used to identify and quantify the oxidized lipids that were formed. 15-oxo-ETE was found to be a major AA-derived LO metabolite when AA was given exogenously or released from endogenous esterified lipid stores by calcium ionophore (CI) calcimycin (A-23187). This established the R15L cells as a useful in vitro model system. Pretreatment of the R15L cells with cinnamyl-3,4-dihydroxycyanocinnamate significantly inhibited AA- or CI-mediated production of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid [15(S)-HETE] and 15-oxo-ETE, confirming the role of 15-LO-1 in mediating AA metabolite formation. Furthermore, 15(S)-HETE was metabolized primarily to 15-oxo-ETE. Pretreatment of the R15L cells with the 15-hydroxyprostaglandin dehydrogenase (PGDH) inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) reduced AA- and 15(S)-HETE-mediated formation of 15-oxo-ETE in a dose-dependent manner. This confirmed that macrophage-derived 15-PGDH was responsible for catalyzing the conversion of 15(S)-HETE to 15-oxo-ETE. Finally, 15-oxo-ETE was shown to inhibit the proliferation of human vascular vein endothelial cells by suppressing DNA synthesis, implicating a potential antiangiogenic role. This is the first report describing the biosynthesis of 15-oxo-ETE by macrophage/monocytes and its ability to inhibit endothelial cell proliferation