Identification of cholest-4-ene-3,6-dione as a Novel Neuroprotectant in Ischemic Stroke and Its Lipidomics.

Purpose: Stroke is a leading cause of disability and death globally. However, there are few clinical drugs for stroke therapy. Novel and effective neuroprotectants are called on the way. Methods: In this study, 93 steroids from a constructed steroidal library were randomly numbered and blindly evaluated in an L-glutamate-induced HT-22 oxidative stress model. The neuroprotective effects of 5 candidates were further investigated in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs), D-glutamate-induced excitotoxicity of CGNs, and cortical neuron (CN) models. Results: Interestingly, unblinding revealed that cholest-4-ene-3,6-dione (78), a cholesterol derivative, was first found to have comprehensive neuroprotective effects in all cell models. 78 administration also decreased the infarction volume and improved motor function in middle cerebral artery occlusion (MCAO) model rats. Additionally, 78 treatment decreased intercellular reactive oxygen species (ROS) and NO production in the HT-22 cell model. Finally, lipidomics and molecular docking results showed that 78 may exert its neuroprotective effects by increasing platelet-activating factor (PAF) analog 1-(9Z-pentadecenoyl)-glycero-3-phosphocholine production. Conclusion: This study indicates that 78, a novel neuroprotectant, is a promising therapeutic candidate with comprehensive neuroprotective effects for the treatment of ischemic stroke by decreasing ROS/reactive nitrogen species (RNS) levels and increasing 1-(9Z-pentadecenoyl)-glycero-3-phospho-choline production.

Epigenetic and post-translational modifications in autophagy: biological functions and therapeutic targets.

Autophagy is a conserved lysosomal degradation pathway where cellular components are dynamically degraded and re-processed to maintain physical homeostasis. However, the physiological effect of autophagy appears to be multifaced. On the one hand, autophagy functions as a cytoprotective mechanism, protecting against multiple diseases, especially tumor, cardiovascular disorders, and neurodegenerative and infectious disease. Conversely, autophagy may also play a detrimental role via pro-survival effects on cancer cells or cell-killing effects on normal body cells. During disorder onset and progression, the expression levels of autophagy-related regulators and proteins encoded by autophagy-related genes (ATGs) are abnormally regulated, giving rise to imbalanced autophagy flux. However, the detailed mechanisms and molecular events of this process are quite complex. Epigenetic, including DNA methylation, histone modifications and miRNAs, and post-translational modifications, including ubiquitination, phosphorylation and acetylation, precisely manipulate gene expression and protein function, and are strongly correlated with the occurrence and development of multiple diseases. There is substantial evidence that autophagy-relevant regulators and machineries are subjected to epigenetic and post-translational modulation, resulting in alterations in autophagy levels, which subsequently induces disease or affects the therapeutic effectiveness to agents. In this review, we focus on the regulatory mechanisms mediated by epigenetic and post-translational modifications in disease-related autophagy to unveil potential therapeutic targets. In addition, the effect of autophagy on the therapeutic effectiveness of epigenetic drugs or drugs targeting post-translational modification have also been discussed, providing insights into the combination with autophagy activators or inhibitors in the treatment of clinical diseases.

Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins.

Tumor-suppressive microRNA-449a induces growth arrest and senescence by targeting E2F3 in human lung cancer cells.

MicroRNA-449a (miR-449a) was significantly downregulated in 156 lung cancer tissues (p<0.001). We found that the low expression of miR-449a was highly correlated with cancer recurrence and survival of lung cancer patients. The transient introduction of miR-449a caused cell cycle arrest and cell senescence in A549 and 95D cells. Further studies revealed that E2F3 was a direct target of miR-449a in lung cancer cells. miR-449a also suppressed tumor formation in vivo in nude mice. These results suggest that miR-449a plays an important role in lung cancer tumorigenesis and that miR-449a might predict cancer recurrence and survival of lung cancer patients.

Immobilized heparin and its anti-coagulation effect on polysulfone membrane surface.

Polysulfone has been widely used as hemodialysis membrane material because of its excellent physiochemical performance. There is still a need to further improve its anti-coagulation property in clinical practice. In this work, we covalently immobilized heparin onto polysulfone membrane to improve its anti-coagulation performance. Low temperature plasma technique with environmentally friendly nitrogen as the gas source, as well as N-ethyl-N'-[3-dimethylaminopropy] carbodiimide hydrochloride/hydroxy-2,5-dioxopyrolidine-3-sulfonicacid sodium chemistry were utilized to immobilize heparin onto the surface of polysulfone membrane. X-ray photoelectron spectroscopy, attenuated total reflectance Fourier-transform infrared spectroscopy, as well as water contact angle results confirmed successful binding of heparin to the membrane surface. Only slight permeability differences were observed between the immobilized surface and the unmodified surface, while the polysulfone membrane had become more hydrophilic after immobilization. The blood coagulation time was greatly prolonged after modification and less platelets adhesion was observed on the heparin immobilized surface. Also, compared with heparin injection doses in clinical, the heparinized process in our work consumed less heparin. Our study suggests that the immobilized heparin has local anti-coagulation effect, while reducing the doses.

Fibronectin and bone morphogenetic protein-2-decorated poly(OEGMA-r-HEMA) brushes promote osseointegration of titanium surfaces.

To be better used as medical implants in orthopedic and dental clinical applications, titanium and titanium-based alloys need to be capable of inducing osteogenesis. Here we describe a method that allows the facile decoration of titanium surfaces to impart an osteogenesis capacity. A Ti surface was first deposited on a poly(OEGMA-r-HEMA) film using surface-initiated atom-transfer radical polymerization (SI-ATRP) with the further step of carboxylation. The modified surfaces were resistant to cell adhesion. Fibronectin (FN) and recombinant human bone morphogenetic protein-2 (rhBMP-2) were further immobilized onto p(OEGMA-r-HEMA) matrices. Our results demonstrate that the FN- and rhBMP-2-conjugated polymer surfaces could induce the adhesion of MC3T3 cells on Ti surfaces. Moreover, the protein-tethered surface exhibited enhanced cell differentiation in terms of alkaline phosphatase activity compared to that of the pristine Ti surface at similar cell proliferation rates. This research establishes a simple modification method of Ti surfaces via Ti-thiolate self-assembled monolayers (SAMs) and SI-ATRP and identifies a dual-functional Ti surface that combines antifouling and osseointegration promotion.

5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells.

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [(3)H]thymidine or 5-bromo-2'-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via "click" chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2'-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells.

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.

[Methods of BMP immobilization and evaluation for Ti-based dental implant surface modification].

Ti-based biomaterial has been widely used as dental and bone implant material although its bioactivity still needs improvement especially for dental implant. The bone morphogenetic proteins(BMPs) bound to Ti-based materials will attract the mesenchymal stem cells to differentiate into osteoblast cells, which benefits the response to the protein-material surface, and finally leads to new bone formation. Several methods including physically mixing, coating, plasma immobilization and cross-linking were used to investigate how BMPs bind to Ti-based biomaterials. The latest research papers are focused on the structure and function of BMPs, and the methods to bind BMPs to Ti-based biomaterial and the evaluation methods after protein immobilization are reviewed in this paper.