Nuclear localization of TET2 requires ß-catenin activation and correlates with favourable prognosis in colorectal cancer.

Mutation-induced malfunction of ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is widely reported in haematological malignancies. However, the role of TET2 in solid cancers, including colorectal cancer (CRC), is unclear. Here, we found that TET2 malfunction in CRC is mostly due to decreased nuclear localization and that nuclear localization of TET2 is correlated with better survival of patients. To explore the underlying mechanisms, 14 immortalized solid tumour cell lines and 12 primary CRC cell lines were used. TET2 was mostly detected in the nucleus, and it induced significant DNA demethylation and suppressed cell growth by demethylating RORA and SPARC in cell lines like SW480. While in cell lines like SW620, TET2 was observed in the cytosol and did not affect DNA methylation or cell growth. Further examination with immunoprecipitation-mass spectrometry illustrated that ß-catenin activation was indispensable for the nuclear localization and tumour suppression effects of TET2. In addition, the ß-catenin pathway activator IM12 and the TET2 activator vitamin C were used simultaneously to enhance the effects of TET2 under low-expression conditions, and synergistic inhibitory effects on the growth of cancer were observed both in vitro and in vivo. Collectively, these data suggest that ß-catenin-mediated nuclear localization of TET2 is an important therapeutic target for solid tumours.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosine dioxygenase ten-eleven translocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency.

Both metabolic switch from oxidative phosphorylation to glycolysis (OGS) and epithelial-mesenchymal transition (EMT) promote cellular reprogramming at early stages. However, their connections have not been elucidated. Here, when a chemically defined medium was used to induce early EMT during mouse reprogramming, a facilitated OGS was also observed at the same time. Additional investigations suggested that the two events formed a positive feedback loop via transcriptional activation, cooperated to upregulate epigenetic factors such as Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5, and accelerated pluripotency induction at the early stage. However, at late stages, by over-inducing glycolysis and preventing the necessary mesenchymal-epithelial transition, the two events trapped the cells at a new pluripotency state between naïve and primed states and inhibited further reprogramming toward the naïve state. In addition, the pluripotent stem cells at the new state have high similarity to epiblasts from E4.5 and E5.5 embryos, and have distinct characteristics from the previously reported epiblast-like or formative states. Therefore, the time-dependent cooperation between OGS and EMT in regulating pluripotency should extend our understanding of related fields.

Temperature-Controlled Reversible Exposure and Hiding of Antimicrobial Peptides on an Implant for Killing Bacteria at Room Temperature and Improving Biocompatibility in Vivo.

Modification of implants by antimicrobial peptides (AMPs) can improve the antimicrobial activity of the implants. However, AMPs have some cytotoxicity in vivo when they are exposed at body temperature. To tackle this challenge, we propose to develop a new approach to generating a smart antimicrobial surface through exposure of AMPs on the surface. A polydopamine film was first formed on the substrates, followed by the conjugation of a temperature-sensitive polymer, poly( N-isopropylacrylamide) (pNIPAM), to the film through atom transfer radical polymerization (ATRP). Then, AMPs were conjugated to the NIPAM on the resultant pNIPAM-modified surface through a click chemistry reaction. Because of the temperature-sensitive property of pNIPAM, the AMPs motif was more exposed to the external environment at room temperature (25 °C) than at body temperature (37 °C), making the surface present a higher antimicrobial activity at room temperature than at body temperature. More importantly, such a smart behavior is accompanied with the increased biocompatibility of the surface at body temperature when compared to the substrates unmodified or modified by AMPs or pNIPAM alone. Our in vivo study further verified that pNIPAM-AMP dual modified bone implants showed increased biocompatibility even when they were challenged with the bacteria at room temperature before implantation. These results indicate that the implants are antibacterial at room temperature and can be safely employed during surgery, resulting in no infection after implantations. Our work represents a new promising strategy to fully explore the antimicrobial property of AMPs, while improving their biocompatibility in vivo. The higher exposure of AMPs at room temperature (the temperature for storing the implants before surgery) will help decrease the risk of bacterial infection, and the lower exposure of AMPs at body temperature (the temperature after the implants are placed into the body by surgery) will improve the biocompatibility of AMPs.

Knockout of NCOA5 impairs proliferation and migration of hepatocellular carcinoma cells by suppressing epithelial-to-mesenchymal transition.

Nuclear receptor coactivator 5 (NCOA5) plays important roles in the development of a variety of malignancies. However, the underlying mechanisms remain obscure. In this study, we successfully generated the NCOA5 knockout hepatocellular carcinoma (HCC) cells by CRISPR/Cas9 - mediated genome editing and found that knockout of NCOA5 inhibited the proliferation and tumor microsphere formation of HCC cells significantly. Moreover, the migration ability of NCOA5 knockout HCC cells declined. Mechanistic analyses indicated that knockout of NCOA5 can suppress the epithelial - mesenchymal transition (EMT) in HCC cells. In conclusion, our findings provide a mechanistic insight into the role of NCOA5 in HCC progression.

Aggregation-Induced Emission Probe for Study of the Bactericidal Mechanism of Antimicrobial Peptides.

Multidrug resistant bacterial infection has become one of the most serious threats to human health. Antimicrobial peptides (AMPs) have been identified as potential alternatives to antibiotics owing to their excellent bactericidal activity. However, the complicated bactericidal mechanism of AMPs is still poorly understood. Fluorescence imaging has many advantages in terms of dynamic monitoring, easy operation, and high sensitivity. In this study, we developed an aggregation-induced emission (AIE)-active probe AMP-2HBT by decorating the antimicrobial peptide HHC36 (KRWWKWWRR) with an AIEgen of 2-(2-hydroxyphenyl)benzothiazole (HBT). This AIE-active probe exhibited an excellent light-up fluorescence after binding with bacteria, enabling a real-time monitoring of the binding process. Moreover, a similar time-dependent bactericidal kinetics was observed for the AIE-active probe and HHC36 peptide, which indicated that the bactericidal activity of the peptide was not compromised by decorating with the AIEgen. The bactericidal mechanism of HHC36 peptide was further investigated by super-resolution fluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM), which suggested that the probe tended to accumulate on the bacterial membrane and efficiently disrupt the membrane structure to kill both Gram-positive and -negative bacteria. This AIE-active probe thus provided a convenient tool to investigate the bactericidal mechanism of AMPs.

Immobilization of an antimicrobial peptide on silicon surface with stable activity by click chemistry.

Infections associated with biomedical implants and devices pose a serious clinical challenge in hospitals worldwide. Antimicrobial peptides (AMPs) have become a great prospect to inhibit this type of infection due to their broad-spectrum antimicrobial activity and low cytotoxicity. However, it is still a challenge to apply AMPs on the biomaterial surface as the activity of AMPs is sensitive to salt or enzyme. In the present study, we prepared a spacer molecule, poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (polySBMA), on a model silicon surface via surface-initiated atom transfer radical polymerization (SI-ATRP). We then modified the antimicrobial peptide HHC36 (KRWWKWWRR) with l-propargylglycine (PraAMP) to improve its salt-tolerant activity and integrated PraAMP onto the spacer molecule using click chemistry. We employed X-ray photoelectron spectroscopy (XPS), contact angle goniometry, and atomic force microscopy (AFM) to confirm the success of the immobilization process. We also characterized the antimicrobial activity and stability of the surface with an antimicrobial assay. The results reveal that the modified surface exhibits good antimicrobial activity to inhibit 98.26% of E. coli, 83.72% of S. aureus, and 81.59% of P. aeruginosa. Furthermore, as compared to the control group without the polySBMA spacer, the modified surface improved its resistance to enzymolysis. An in vitro CCK-8 assay also illustrated that this surface showed negligible cytotoxicity to mouse bone mesenchymal stem cells.

Influence and mechanism of 5-aminolevulinic acid-photodynamic therapy on the metastasis of esophageal carcinoma.

BACKGROUD: Photodynamic therapy (PDT) for the treatment of esophageal cancer was more and more popularly used since it was approved for the treatment of advanced esophageal cancer in 1996. It has been reported to influence the tumor growth and metastasis via a variety of signaling pathways, but its mechanism remains to be further studied. This research studied the effects of ALA-PDT on esophageal carcinoma in vitro and in vivo, discovering its molecular regulating mechanism and the way to enhence the PDT effect. METHODS: Eca-109 cells were incubated with a medium containing EGFR tyrphostin AG1478 or PI3K inhibitor LY294002, then with ALA, and the cells were irradiated with the laser 6h later. The cell viability was measured with MTT assay, and the migration ability was detected by transwell experiments 24h post-ALA-PDT. The gene and protein expression on EGFR/PI3K/AKT signaling pathway was analyzed by realtime PCR and Western blotting respectively. Then, RFP-Eca-109 burdened nude mice model was constructed, and were treated with ALA-PDT when the tumor volume reached 150-350mm3. The gene and protein expression were analyzed 24h and 50days post-ALA-PDT. RESULTS: Our study showed that ALA-PDT respectively combined with AG1478, LY294002 could synergistically reduce the growth and migration ability of the Eca-109 cells in vitro and significantly down-regulate the protein expression of EGFR/PI3K and PI3K/AKT, meanwhile, significantly down-regulate the gene expression of EGFR when combining with AG1478. Forthermore, ALA-PDT could significantly decrease the tumor growth and metastasis and down-regulate the gene expression of EGFR and the protein expression of EGFR and PI3K in the tumor of mice. CONCLUSION: This study revealed a molecular mechanism of ALA-PDT and developed a new modality application of therapy, by combining ALA-PDT with small molecular inhibitors, for better effect in the clinical practice of esophageal carcinoma.

Preparation of an antimicrobial surface by direct assembly of antimicrobial peptide with its surface binding activity.

Antimicrobial peptides (AMPs) are a broad prospect for clinical application against bacterial infections of biomaterials. However, a bottleneck exists as there is a lack of simple technology to prepare AMPs on biomaterials with sufficient activity, as the activity of AMP is dependent on the correct orientation on the biomaterial. In the present study, based on the conventional AMP (Tet213: KRWWKWWRRC) and surface binding peptide (SKHKGGKHKGGKHKG), we designed an Anchor-AMP that could be directly assembled onto the surface of the biomaterial and also showed excellent antimicrobial activity. By characterizing the surface using a quartz crystal microbalance with dissipation (QCM-D), contact angle, atom force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS), we found that Anchor-AMP could adsorb onto the titanium surface with a strong affinity. Different from Tet213 peptide, Anchor-AMP exhibits excellent antimicrobial activity on the titanium surface being able to inhibit 95.33% of Escherichia coli and 96.67% of Staphylococcus aureus after 2.5 h. The improved antimicrobial activity is a result of improved orientation of Anchor-AMP on the biomaterial compared to that of the Tet213 peptide. In addition, the antimicrobial activity of Anchor-AMP was active for more than 24 h. The CCK-8 assay illustrated that the modified titanium surface showed negligible cytotoxicity to bone marrow mesenchymal stem cells. The in vivo results showed that it exhibited excellent antimicrobial activity after 5 and 7 days, inhibiting 89.32% and 99.78% of S. aureus, respectively. We also demonstrated that Anchor-AMP could be applied on a variety of surfaces including gold (Au), polymethyl methacrylate (PMMA) and hydroxyapatite (HA) with strong affinity and good antimicrobial activity.

Thermoresponsive Self-Assembled ß-Cyclodextrin-Modified Surface for Blood Purification.

For patients with liver failure, bilirubin (BR) is one of the endogenous toxins in their blood. Although blood purification can remove the bilirubin from the body in clinics, the detoxification system needs to be improved, and the cost needs to be decreased. In the present study, we developed a recyclable model surface that can strongly remove bilirubin. We first prepared adamantane (Ad) on a model gold surface by self-assembly. Then, we integrated the ß-cyclodextrin dimer (CDD) onto the surface with host-guest interactions between one of the CD cavities in the CDD and Ad. We characterized the surface with XPS, static contact angle measurements, and AFM. In addition, we employed QCM-D to characterize the preparation process as well as the detoxification of the surface. We demonstrated that this modified surface could strongly adsorb bilirubin through host-guest interactions between the CD cavities in the CDD and bilirubin and that the detoxification was improved 1.7 times (compared to the surface only with Ad). Interestingly, after characterization with QCM-D, this surface could be recycled due to the thermoresponsive property of the host-guest interaction between the CDD and Ad. After adsorbing the toxin and increasing the temperature to 45 °C, the CDD with bilirubin could be removed from the surface. Then, the refreshed surface with CDD could be prepared again at room temperature. This cycle could be repeated at least 3 times. Additionally, during each cycle, the modified surface exhibited good detoxification to bilirubin. This modified surface also showed strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg). An in vitro platelet adhesion assay showed that the adhesion of the platelets on the modified surface decreased and that the platelets were in an inactivated state. The hemolysis assay showed that this surface exhibited no hemolysis activity in the samples to red blood cells (RBCs). The CCK-8 assay showed that this surface had negligible cytotoxicity to L929 cells. This work has taken advantage of the host-guest self-assembly between ß-CD and BR/Ad for special recognizing adsorption, as well as the thermoresponse of ß-CD-Ad inclusion for recyclable application, and these results demonstrate that this technology has great potential for removing bilirubin and decreasing clinic costs.