Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.     

NSun2 promotes cell migration through methylating autotaxin mRNA

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.     

Loganetin and 5‐fluorouracil synergistically inhibit the carcinogenesis of gastric cancer cells via down‐regulation of the Wnt/β‐catenin pathway

Although most gastrointestinal tumours are sensitive to 5‐fluorouracil (5FU), drug resistance is commonly occurred after 5FU therapy in gastric cancer (GC). Loganetin is the primary active compound in Cornus officinali. However, the synergetic effects of loganetin and 5FU on GC remain unknown. Here, we investigated the synergetic effects and the underlying mechanism of loganetin and 5FU on proliferation, stem‐like properties, migration, and invasion of GC both in vitro and in vivo. We found that loganetin alone inhibited the proliferation, stem‐like properties, migration and invasion of GC cells in vitro. Importantly, the loganetin remarkably enhanced the anti‐cancer effect of 5FU on GC cells and the Wnt/β‐catenin pathway might be involved in this process. Animal experiments further confirmed the synergistic effects of 5FU and loganetin on inhibiting cell growth and metastasis of GC. These results suggested that loganetin could synergistically increase the effect of 5FU against GC, which sheds light on effective combinational drug strategies for GC treatment.     

An In Vitro and In Vivo Evaluation of a Reporter Gene/Probe System hERL/18F-FES

Purpose

To evaluate the feasibility of a reporter gene/probe system, namely the human estrogen receptor ligand binding domain (hERL)/16α-[¹⁸F] fluoro-17β-estradiol (¹⁸F-FES), for monitoring gene and cell therapy.

Methods

The recombinant adenovirus vector Ad5-hERL-IRES-VEGF (Ad-EIV), carrying a reporter gene (hERL) and a therapeutic gene (vascular endothelial growth factor, VEGF165) through an internal ribosome entry site (IRES), was constructed. After transfection of Ad-EIV into bone marrow mesenchymal stem cells (Ad-EIV-MSCs), hERL and VEGF165 mRNA and protein expressions were identified using Real-Time qRT-PCR and immunofluorescence. The uptake of ¹⁸F-FES was measured in both Ad-EIV-MSCs and nontransfected MSCs after different incubation time. Micro-PET/CT images were obtained at 1 day after injection of Ad-EIV-MSCs into the left foreleg of the rat. The right foreleg was injected with nontransfected MSCs, which served as self-control.

Results

After transfection with Ad-EIV, the mRNA and protein expression of hERL and VEGF165 were successfully detected in MSCs, and correlated well with each other (R² = 0.9840, P<0.05). This indicated the reporter gene could reflect the therapeutic gene indirectly. Ad-EIV-MSCs uptake of ¹⁸F-FES increased with incubation time with a peak value of 9.13%±0.33% at 150 min, which was significantly higher than that of the control group. A far higher level of radioactivity could be seen in the left foreleg on the micro-PET/CT image than in the opposite foreleg.

Conclusion

These preliminary in vitro and in vivo studies confirmed that hERL/¹⁸F-FES might be used as a novel reporter gene/probe system for monitoring gene and cell therapy. This imaging platform may have broad applications for basic research and clinical studies.     

Environmental impact assessment of galvanized sheet production: a case study in Shandong Province,China

Purpose

Galvanized sheet is the most widely used coated steel plate globally in the industry of construction, automobile, electronics manufacturing, etc. Large amounts of resources and energy are used in galvanized sheet production, which likewise generates vast amounts of pollutant emissions. In the face of the rapid growth of the production and demand of galvanized sheet in China, it is very important to find out the key factors of the environment impact in the production of galvanized sheet. An evaluation of the environmental impact of galvanized sheet production in China was conducted by using the framework of life cycle assessment to improve resource saving and environmental protection in the galvanized sheet industry, and update the life cycle inventory database of galvanized sheet production.

Methods

The environmental impact assessment was carried out based on the life cycle assessment framework by the use of ReCiPe 2016 method which was applicable on a global scale to evaluate the environmental impact of galvanized sheet production. Methods of uncertainty analysis and sensitivity analysis were adopted to provide credible support.

Results and discussion

The midpoint categories of global warming and fossil resource scarcity, as well as the endpoint categories of human health contributed most to environmental burden, which were mainly caused by carbon dioxide emissions and coal consumption. Environmental impact was dominated by the key process of continuous casting billet production, followed by electrolytic zinc production and electricity generation.

Conclusions

Additional CO₂-reducing measures should be implemented in galvanized sheet production to slow the effect of global warming. Moreover, biomass char reducing agents, rather than coal-based reducing agents, should be utilized in steelmaking to reduce fossil resource consumption. Furthermore, renewable energy, rather than coal-based electricity, should be used in galvanized sheet production to reduce carbon emissions and fossil resource consumption. Increasing the recycling rate of scrap steel and zinc waste can save resources and reduce environmental burden. The results of this study can provide guidance in the reduction of resource consumption and environmental burden of galvanized sheet production to the maximum extent.

     

2,6-Disubstituted pyrazines and related analogs as NR2B site antagonists of the NMDA receptor with anti-depressant activity

Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (sc 60 mg/kg).     

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019     

Silencing SOX2 Expression by RNA Interference Inhibits Proliferation,Invasion and Metastasis,and Induces Apoptosis through MAP4K4/JNK Signaling Pathway in Human Laryngeal Cancer TU212 Cells

SRY (sex determining region Y)-box 2 (SOX2) plays an important role in tumor cell metastasis and apoptosis. Laryngeal squamous cell carcinoma (LSCC), responsible for 1.5% of all cancers, is one of the most common head and neck malignancies. Accumulating evidence shows that SOX2 is overexpressed in several human tumors, including lung cancer, esophageal carcinoma, pancreatic carcinoma, breast cancer, ovarian carcinoma and glioma. Our study aimed to investigate the silencing effects of SOX2 expression using RNA interference (RNAi) on various biological processes in laryngeal cancer TU212 cells, including proliferation, apoptosis, invasion and metastasis. We also studied the involvement of the MAPK/JNK signaling pathway in the biological effects of SOX2 siRNA in TU212 cells. We found that silencing SOX2 decreased the proliferation, migration, and invasion of TU212 cells, and induced apoptosis. This effect of silencing SOX2 could be reversed by silencing MAP4K4. Therefore, we consider SOX2 as a key regulator of the upstream MAP4K4/JNK signaling pathways that could be a potential therapeutic target in the treatment of patients with or prevention of laryngeal cancer.     

Lymphocyte-derived microparticles induce apoptosis of airway epithelial cells through activation of p38 MAPK and production of arachidonic acid

The airway epithelium is critical for the normal integrity and function of the respiratory system. Excessive epithelial cell apoptosis contributes to cell damage and airway inflammation. We previously demonstrated that lymphocyte-derived microparticles (LMPs) induce apoptosis of human bronchial epithelial cells. However, the underlying mechanisms contributing to LMPs-evoked epithelial cell death are largely unknown. Here we used bronchial and lung tissue cultures to confirm the pro-apoptotic effects of LMPs. In cell culture experiments, we found that LMPs induced human airway epithelial cell apoptosis with associated increases in caspase-3 activity. In addition, LMPs treatment triggered oxidative stress in epithelial cells by enhancing production of malondialdehyde, superoxide, and reactive oxygen species (ROS), and by inhibiting production of the antioxidant glutathione. Moreover, decreasing cellular ROS with the antioxidant N-acetylcysteine rescued epithelial cell viability. Together, these results demonstrate an important role for oxidative stress in LMPs-induced cell death. In epithelial cells, LMPs treatment induced phosphorylation of p38 MAPK and arachidonic acid accumulation. Moreover, arachidonic acid was significantly cytotoxic towards LMPs-treated epithelial cells, whereas inhibition of p38 MAPK was protective against these cytotoxic effects. Similarly, inhibition of arachidonic acid production led to decreased caspase-3 activity, thus rescuing airway epithelial cells from LMPs-induced cell death. In conclusion, our results show that LMPs induce airway epithelial cell apoptosis by activating p38 MAPK signaling and stimulating production of arachidonic acid, with consequent increases in oxidative stress and caspase-3 activity. As such, LMPs may be regarded as deleterious markers of epithelial cell damage in respiratory diseases.