COX-2/sEH Dual Inhibitor PTUPB Inhibits Epithelial-Mesenchymal Transformation of Alveolar Epithelial Cells by Suppressing TGF-β1-Smad2/3 Signaling Pathway

Background: Arachidonic acid (ARA) metabolites are involved in the pathogenesis of epithelial-mesenchymal transformation (EMT). However, the role of ARA metabolism in the progression of EMT in pulmonary fibrosis (PF) has not been fully elucidated. The purpose of this study was to investigate the role of cytochrome P450 oxidase (CYP)/ soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) metabolic disorders of ARA in EMT during PF.Methods: A signal intratracheal injection of bleomycin (BLM) was given to induce PF in C57BL/6J mice. A COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation to EMT in PF mice. In vitro experiments, murine alveolar epithelial cells (MLE12) and human alveolar epithelial cells (A549) were used to explore the roles and mechanisms of PTUPB on transforming growth factor (TGF)-β1-induced EMT. Results: PTUPB treatment reversed the increase of mesenchymal marker molecule α-smooth muscle actin (α-SMA) and the loss of epithelial marker molecule E-Cadherin in lung tissue of PF mice. In vitro, COX-2 and sEH protein levels were increased in TGF-β1-treated alveolar epithelial cells (AECs). PTUPB decreased the expression of α-SMA and restored the expression of E-cadherin in TGF-β1-treated AECs, accompanied by reduced migration and collagen synthesis. Moreover, PTUPB alleviated the activation of the TGF-β1-Smad2/3 pathway induced by TGF-β1 in AECs.Conclusion: PTUPB inhibits TGF-β1-induced EMT via inhibition of the TGF-β1-Smad2/3 pathway, which holds great promise for the clinical treatment of PF.

Toll-like Receptor 2 as a Marker Molecule of Advanced Ovarian Cancer

Ovarian cancer is a global problem that affects women of all ages. Due to the lack of effective screening tests and the usually asymptomatic course of the disease in the early stages, the diagnosis is too late, with the result that less than half of the patients diagnosed with ovarian cancer (OC) survive more than five years after their diagnosis. In this study, we examined the expression of TLR2 in the peripheral blood of 50 previously untreated patients with newly diagnosed OC at various stages of the disease using flow cytometry. The studies aimed at demonstrating the usefulness of TLR2 as a biomarker in the advanced stage of ovarian cancer. In this study, we showed that TLR2 expression levels were significantly higher in women with more advanced OC than in women in the control group. Our research sheds light on the prognostic potential of TLR2 in developing new diagnostic approaches and thus in increasing survival in patients with confirmed ovarian cancer.

miR-34c inhibits PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway

The purpose of this study was to explore the effect of miR-34c on PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway, so as to find a new method for early diagnosis and treatment of cardiovascular disease. HA-VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) at 0 h, 12 h, 24 h, 48 h or 36 h to explore the optimal time for phenotypic transformation of VSMCs. And then, PDGF-BB-induced HA-VSMCs were transfected with miR-34c mimics/mimics NC and pcDNA3.1-PDGFR-β/pcDNA3.1-NC to observe cell biological behaviour. CCK8 was used to detect cell proliferation activity. Transwell chamber assay was used to detect cell invasion. Early apoptosis was analyzed by flow cytometry. The expression of α-SMA and Smemb was detected by immunofluorescence staining. The expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 were analyzed by Western blot analysis. The expression of miR-34a, miR-34b and miR-34c was detected by RT-PCR, and the targeting relationship between miR-34c and PDGFR-β was detected by luciferase reporting assay. The results indicated the proliferation and migration of PDGF-BB-induced HA-VSMCs significantly increased, and apoptosis significantly decreased. Besides, α-SMA decreased significantly, while Smemb increased significantly. Furthermore, expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 increased significantly, and SIRT1 decreased significantly. Experimental results showed that, miR-34c mimics significantly inhibited cell proliferation and migration, and promoted cell apoptosis, and miR-34c inhibitor had the opposite effects. MiR-34c mimics significantly increased α-SMA expression and decreased Smemb expression, while the opposite effects were reflected after transfection with miR-34c inhibitor. Moreover, miR-34c mimics significantly decreased the expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1, and significantly increased the expression of SIRT1, while miR-34c inhibitor had the opposite effects. Luciferase assay confirmed that PDGFR-β was a potential target of miR-34c. Subsequently, PDGF-BB-induced HA-VSMCs were co-transfected with miR-34c mimics and pcDNA3.1-PDGFR-β. The results indicated that PDGFR-β reversed the biological function of miR-34c mimic. The results revealed the potential application value of miR-34c as a marker molecule of phenotypic transformation, providing a potential target for improving phenotypic transformation.

Recognition of Bimolecular Logic Operation Pattern Based on a Solid-State Nanopore

Nanopores have a unique advantage for detecting biomolecules in a label-free fashion, such as DNA that can be synthesized into specific structures to perform computations. This method has been considered for the detection of diseased molecules. Here, we propose a novel marker molecule detection method based on DNA logic gate by deciphering a variable DNA tetrahedron structure using a nanopore. We designed two types of probes containing a tetrahedron and a single-strand DNA tail which paired with different parts of the target molecule. In the presence of the target, the two probes formed a double tetrahedron structure. As translocation of the single and the double tetrahedron structures under bias voltage produced different blockage signals, the events could be assigned into four different operations, i.e., (0, 0), (0, 1), (1, 0), (1, 1), according to the predefined structure by logic gate. The pattern signal produced by the AND operation is obviously different from the signal of the other three operations. This pattern recognition method has been differentiated from simple detection methods based on DNA self-assembly and nanopore technologies.

Understanding the Toxin Effects of β-Zearalenol and HT-2 on Bovine Granulosa Cells Using iTRAQ-Based Proteomics

Zearalenone (ZEA) and T-2 are the most common mycotoxins in grains and can enter the animal and human food-chain and cause many health disorders. To elucidate the toxic response profile, we stimulated bovine granulosa cells (GCs) with β-zearalenol or HT-2. Using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic, 178 and 291 differentially expressed proteins (DEPs, fold change ≥ 1.3 and p-value < 0.05) in β-zearalenol and HT-2 groups were identified, respectively. Among these DEPs, there were 66 common DEPs between β-zearalenol and HT-2 groups. These 66 DEPs were associated with 23 biological processes terms, 14 molecular functions terms, and 19 cellular components terms. Most heat shock proteins (HSPs) were involved in the toxic response. Reactive oxygen species accumulation, the endoplasmic reticulum (ER)-stress related marker molecule (GRP78), and apoptosis were activated. β-zearalenol and HT-2 inhibited oestradiol (E2) production. These results emphasized the important function of HSPs, clarified oxidative stress, and demonstrated the caspase-3 signaling cascade involved in mycotoxin-treated toxic response, along with decreased E2 production. This study offers new insights into the toxicity of β-zearalenol and HT-2 on ovarian granulosa cells.

Quantification of Internal Filtration in Hollow Fiber Hemodialyzers with Medium Cut-Off Membrane

Background: Inadequate removal of molecules between 5 and 50 KDa may cause long-term complication in chronic hemodialysis. Medium cut-off (MCO) is a new class of membranes with enhanced sieving properties and negligible albumin loss. MCO membrane makes it possible to perform expanded hemodialysis (HDx), a technique based on high internal filtration (IF).The present study is designed to quantify IF in 2 MCO dialyzers (Theranova 400 and 500, Baxter, Deerfield, USA) using a nuclear imaging technique previously validated. Methods: Blood and dialysate compartment pressure drop along with transmembrane pressure; they were measured in a closed in vitro circuit with human blood (blood flow [QB] = 300 and 400 mL/min; dialysate flow 500 mL/min; net ultrafiltration rate 0 mL/min). A non-diffusible marker molecule (albumin macro-aggregates labeled with 99Tc metastable) was injected in the blood compartment and nuclear emission was recorded by a gamma camera. Relative variations in the concentration of the marker molecule along the length of the filter were used to calculate local cross filtration. Results: Based on marker concentration profiles, IF was estimated. For Theranova 400, IF were 29.7 and 41.6 mL/min for QB of 300 and 400 mL/min. For Theranova 500, IF were 31.6 and 53.1 mL/min for QB of 300 and 400 mL/min respectively. Conclusions: MCO membrane provides significant amounts of IF due to the particular combination between hydraulic permeability of the membrane and reduced inner diameter of the fibers. High IF combined with enhanced sieving profile of MCO membrane leads to improved removal of a wider spectrum of uremia retention molecules in HDx, without requiring complex equipment.

An in situ hybridization study of perlecan, DMP1, and MEPE in developing condylar cartilage of the fetal mouse mandible and limb bud cartilage

<p>The main purpose of this <em>in situ</em> hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (<em>perlecan, DMP1</em>, and <em>MEPE)</em> in developing primary (tibial) and secondary (condylar) cartilage. <em>Perlecan</em> mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, <em>perlecan</em> mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. <em>DMP1</em> mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, <em>DMP1</em> mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 <em>may</em> be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. <em>MEPE</em> mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of <em>in situ</em> hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.</p>