Zein nanospheres assisting inorganic and organic drug combination to overcome stent implantation-induced thrombosis and infection.

The complication of stent implantation is the biggest obstacle to the success of its clinical application. In this study, we developed a combination way of 3D printing and the coating technique for preparation of functional polyurethane stents against stent implantation-induced thrombosis and postoperative infection. SEM, XPS, static water contact angle, and XRD demonstrated that the functional polyurethane stent had a 37 µm-thickness membrane composed of zein nanospheres (250-350 nm). Meanwhile, ZnO nanoparticles were encapsulated in zein nanospheres while heparin was adsorbed on the surface, causing 97.1 ± 6.4 % release of heparin in 120 min (first-order kinetic model) and 62.7 ± 5.6 % release of Zn2+ in 9 days (Korsmeyer-Peppas model). The mechanical analysis revealed that the functional polyurethane stents had about 8.61 MPa and 2.5 MPa tensile strength and bending strength, respectively. The in vitro biological analysis showed that the functional polyurethane stents had good EA.hy926 cells compatibility (97.9 ± 3.8 %), anti-coagulation response (comparable plasma protein, platelet adhesion and suppressed clotting) and sustained antibacterial activities by comparison with the bare polyurethane stent. The preliminary evaluation by rabbit ex vivo carotid artery intervention experiment demonstrated that the functional polyurethane stents could maintain blood circulation under the continuous stresses of blood flow. Meanwhile, the detailed data from the simulated implant infection experiment in vivo showed the functional polyurethane stents could effectively reduce microbial infection by 3-6 times lower and improve fibrosis and macrophage infiltration.

Preparation and Evaluation of Upconversion Nanoparticles Based miRNA Delivery Carrier in Colon Cancer Mice Model.

Therapeutic efficacy of solid tumor is often severely hampered by poor penetration of therapeutics into diseased tissues and lack of tumor targeting. In this study, the functionalized upconversion nanoparticles (UCNP)-based delivery vector targeting cancer cells was developed. Firstly, NaYF4:Yb/Tm (UCNP) was prepared with the solvothermal method for the uniform nanoparticle size and brilliant lattice structure. The SiO2 coated UCNP was demonstrated a high upconversion emission and good monodispersity, which was coupled with polyetherimide (PEI) and miR-145 vector. Then, it was further functionalized via hyaluronic acid (HA) (UCNP/PEI/HA Nanocomplex, UCNPs) coating for the targeted delivery and improved biocompatibility. The UCNPs/miR-145 displays an excellent biocompatibility, a high level of cellular uptake and miR-145 expression, which results in a significant cell cycle arrest in G1, and induces CCND1, CDK6 and CCNE2 proteins downregulation. In vivo, the HA-coated UCNPs were enriched at the tumor site by targeting and retention effects, which resulted in a significant inhibition of tumor growth. Histological experiments demonstrated that UCNPs did not show significant toxicity in mice colon cancer model. Taken together, a UCNPs-based delivery platform was successfully constructed and used for miRNA target delivery, which provided a new method and idea for bioengineering and nanotechnology-based tumor therapy.

Bioinspired Drug Delivery Carrier for Enhanced Tumor-Targeting in Melanoma Mice Model.

As a widely used first-line chemotherapy drug for tumor, Doxorubicin (DOX) can induce various side effects on normal tissues because of its non-specific distribution in the body. Emerging evidence has shown that platelets have the capability to recognize and interact with tumor cells. Inspired by this, the platelet-based drug delivery system was constructed by loading of DOX in platelet cytoplasm and modification of transferrin on the surface of platelet (Tf-P-DOX). The encapsulation efficiency of DOX in platelet was the highest at the DOX concentration of 0.05 mM, and reached to 64.9%. Fluorescence microscopy showed that the Tf-P-DOX facilitated cell uptakes and enhanced intracellular drug accumulation in B16F10 cells. Compared with free DOX, Tf-P-DOX exhibited an enhanced effect on cell apoptosis at the same concentration of DOX. In vivo imaging system showed that the near-infrared fluorescence of B16F10 tumor-bearing mice was mainly accumulated in the tumor site, which caused the inhibition of tumor growth in mice. The morphological changes of tumor tissue in Tf-P-DOX group was significant in comparison with those of the control group, including the small nucleus, the insufficiency of cancerous nest, and the infiltration of inflammatory cells, while Tf-P-DOX did not show significant adverse effects on normal tissues. Compared with the control group, the levels of caspase 9 and caspase 3 protein expressions were increased significantly in Tf-P-DOX group. Our studies suggest platelets can be repurposed as promising carriers for efficient targeting and treatment of solid tumors.

Relationship between Maternal PBMC HBV cccDNA and HBV Serological Markers and its Effect on HBV Intrauterine Transmission.

OBJECTIVE: To investigate the relationship between maternal peripheral blood mononuclear cells (PBMC) hepatitis B virus (HBV) covalenty closed circular deoxyribonucleic acid (cccDNA) and other HBV serological markers and its effects on HBV intrauterine transmission. METHODS: We enrolled 290 newborns and their hepatitis B surface antigen (HBsAg) positive mothers. HBV cccDNA in PBMC and HBV DNA in serum were detected by a real-time PCR-TaqMan probe while HBV serological markers were detected with an electrochemiluminescence immunoassay. RESULTS: There was a positive correlation between the levels of PBMC HBV cccDNA and serum HBV DNA and HBeAg (r = 0.436 and 0.403, P < 0.001). The detection rate of pattern A ['HBsAg (+), HBeAg (+), and anti-HBc (+)'] was significantly higher in the PBMC HBV cccDNA positive group than in the control group (χ2 = 48.48, P < 0.001). There was a significant association between HBV intrauterine transmission and PBMC HBV cccDNA (χ2 = 9.28, P = 0.002). In the presence of serum HBV DNA, HBeAg, and PBMC HBV cccDNA, the risk of HBV intrauterine transmission was three times higher (OR = 3.69, 95% CI: 1.30-10.42) than that observed in their absence. The risk of HBV intrauterine transmission was the greatest (OR = 5.89, 95% CI: 2.35-14.72) when both PBMC HBV cccDNA and pattern A were present. A Bayesian network model showed that maternal PBMC HBV cccDNA was directly related to HBV intrauterine transmission. CONCLUSION: PBMC HBV cccDNA may be a direct risk factor for HBV intrauterine transmission. Our study suggests that serological markers could be combined with PBMC-related markers in prenatal testing.

Meta-analysis of antiviral protection of white spot syndrome virus vaccine to the shrimp.

Currently, white spot syndrome virus (WSSV) is one of the most serious pathogens that impacts shrimp farming around the world. A WSSV vaccine provides a significant protective benefit to the host shrimp. Although various types of vaccines against WSSV have emerged, the immune effects among them were not compared, and it remains unclear which type of vaccine has the strongest protective effect. Meanwhile, due to the lack of effective routes of administration and immunization programs, WSSV vaccines have been greatly limited in the actual shrimp farming. To answer these questions, this study conducted a comprehensive meta-analysis over dozens of studies and compared all types WSSV vaccines, which include sub-unit protein vaccines, whole virus inactivated vaccines, DNA vaccines and RNA-based vaccines. The results showed that the RNA-based vaccine had the highest protection rate over the other three types of vaccines. Among the various sub-unit protein vaccines, VP26 vaccine had the best protective effects than other sub-unit protein vaccines. Moreover, this study demonstrated that vaccines expressed in eukaryotic hosts had higher protection rates than that of prokaryotic systems. Among the three immunization modes (oral administration, immersion and injection) used in monovalent protein vaccines, oral administration had the highest protection rate. In natural conditions, shrimp are mostly infected by the virus orally. These results provide a guide for exploration of a novel WSSV vaccine and help facilitate the application of WSSV vaccines in shrimp farming.

Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity.

Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis) vaccine against Helicobacter pylori (H. pylori). Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

Detecting protein-DNA interactions using a modified yeast one-hybrid system.

The yeast one-hybrid (Y1H) system has been among the methods of choice to detect protein-DNA interactions. However, conventional Y1H systems with a single auxotrophic reporter gene often suffer from high incidence of false positives to demonstrate a limited power in large-scale screenings. Here we describe a refined Y1H system that uses two independent bait sequences, each controlling a distinct reporter gene integrated in the host genome. With these modifications and a method of targeted DNA methylation, we succeeded in efficient isolation of clones for methylated DNA-binding proteins from mammalian cDNA libraries.

Camptothecin induces apoptosis in cancer cells via microRNA-125b-mediated mitochondrial pathways.

Camptothecin (CPT) is an effective chemotherapeutic agent for treatment of patients with cancer. The mechanisms underlying CPT-mediated responses in cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug sensitivity. However, the interaction between camptothecin and miRNA has not been previously explored. In this study, we verified that miR-125b was down-regulated in CPT-induced apoptosis in cancer cells and that ectopic expression of miR-125b partially restored cell viability and inhibited cell apoptosis that was induced by CPT. In addition, we demonstrated that CPT induced apoptosis in cancer cells by miR-125b-mediated mitochondrial pathways via targeting to the 3'-untranslated (UTR) regions of Bak1, Mcl1, and p53. A significant increase in Bak1, Mcl1, and p53 protein levels was detected in response to the treatments of CPT. It is noteworthy that the expression levels of Bak1, Mcl1, and p53 increased in a time-dependent manner and negatively correlated with miR-125b expression. It is noteworthy that we revealed that miR-125b directly targeted the 3'UTR regions of multiple genes in a CPT-induced mitochondrial pathway. In addition, most targets of miR-125b were proapoptotic genes, whereas some of the targets were antiapoptotic genes. We hypothesized that miR-125b may mediate the activity of chemotherapeutic agents to induce apoptosis by regulating multiple targets. This is the first report to show that camptothecin induces cancer cell apoptosis via miRNA-mediated mitochondrial pathways. The results suggest that suppression of miR-125b may be a novel approach for the treatment of cancer.

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance.

OBJECTIVE: Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). METHODS: A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. RESULTS: The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan-Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). CONCLUSIONS: The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC.

Clinical Significance of CENP-H Expression in Uterine Cervical Cancer.

OBJECTIVE: This work aims to investigate the expression pattern and clinicopathologic significance of centromere protein H (CENP-H) in uterine cervical cancer (UCC). METHODS: The level of CENP-H expression in the paraffin sections of 62 UCC cases was determined by the SP immunohistochemical method, with complete clinicopathologic data in all cases. Statistical analysis was conducted to evaluate the prognostic and diagnostic significance of CENP-H using SPSS13.0 software package. RESULTS: Immunohistochemical assay showed strong CENP-H expression in 61.29% (38/62) of the paraffin-embedded cervical cancer tissues. Statistical analysis revealed a strong correlation between the CENP-H expression and the clinical classification (P=0.038) of the cervical carcinoma. The expression increased with rise of the stages. The analysis of Cox proportional hazards regression model suggested that CENP-H expression (P=0.002) and tumor stage (P=0.001) were independent prognostic markers for the survival of UCC patients. The survival analysis showed that the survival rate was significantly lower in patients with high expression of CENP-H than in those with low expression of CENP-H (P=0.001). CONCLUSIONS: CENP-H is likely to be a valuable marker for carcinogenesis and progression of UCC. It might be used as the important diagnostic and prognostic marker for cervical carcinoma patients, especially for those at early stage.

Heparinized polyvinyl alcohol to specifically adsorb low-density lipoprotein from plasma.

INTRODUCTION: A medical adsorbent for blood purification was developed to specifically adsorb low-density lipoprotein (LDL) from hypercholesterolemia patient's plasma by covalently immobilizing heparin onto the surface of polyvinyl alcohol (PVA) with the couplant toluence-2,4-diisocyanate (TDI). METHODS: We used IR to demonstrate the success of covalently immobilizing heparin onto the surface, and investigated its adsorption of LDL, and primarily evaluated its hemo-compatibility using tests for platelet adhesion, the degree of platelet activation and a hemolysis test. RESULTS: (1) Heparin was successfully covalently immobilized onto the surface, the maximum amount of heparin immobilized on the surface of 1g PVA-1799 granules was about 5 µg; (2) one optimal condition for adsorption of LDL from hyperlipidemia plasma was a pH within the range of 7.2∼9.5, accordingly the adsorptive ratio (adsorbent/g: plasma/L=1:2) for LDL was about 70%; (3) it exhibited good hemo-compatibility. CONCLUSION: The adsorbent results in satisfactory adsorption of LDL with good hemo-compatibility; it could potentially be used as a blood purification material, and immobilization of heparin onto medical materials may be a way to develop an LDL-specific adsorbent for blood purification.

A yeast one-hybrid system to screen for methylated DNA-binding proteins.

We had previously exploited a method for targeted DNA methylation in budding yeast to succeed in one-hybrid detection of methylation-dependent DNA-protein interactions. Based on this finding, we developed a yeast one-hybrid system to screen cDNA libraries for clones encoding methylated DNA-binding proteins. Concurrent use of two independent bait sequences in the same cell, or dual-bait system, effectively reduced false positive clones, which were derived from methylation-insensitive sequence-specific DNA-binding proteins. We applied the dual-bait system to screen cDNA libraries and demonstrated efficient isolation of clones for methylated DNA-binding proteins. This system would serve as a unique research tool for epigenetics.

Toxic effects of T-2 toxin on reproductive system in male mice.

The present study was conducted to evaluate the effects of T-2 toxin on semen quality, fertility and serum testosterone concentration in mice. Adult male mice were mated with sexually mature untreated female mice after being exposed to intraperitoneal injection of T-2 toxin at 0, 5, 10 or 15 mg/kg body weight daily for 7 successive days. Semen quality, serum testosterone concentration and fertility of treated mice were assessed. The results showed that the number of abnormal spermatozoa increased significantly and a significant decrease in spermatozoa with integrated acrosome was observed in males treated with T-2 toxin at all doses, As well, the amount of live spermatozoa decreased significantly in mice treated with 10 and 15 mg/kg body weight T-2 toxin. Low pregnancy rate and high fetal resorption rate were observed when females were mated with T-2 toxin-exposed males. Testicular and cauda epididymal sperm counts, efficiency of sperm production and serum testosterone concentration were significantly reduced in mice treated with T-2 toxin at all doses in a dose-dependent manner. In conclusion, these findings indicated that T-2 toxin presented toxic effects on reproductive system of adult male mice.

[Transformation of Dunaliella salina by using glass beads--a novel transformation method].

A novel transformation method was firstly established using glass beads in Dunaliella salina (D. salina). The results showed that the GUS gene, a reporter gene, was successfully expressed in D. salina. Cells of D. salina presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized including the transformation consecutive time, rotate speed, concentration of the plasmid and PEG 6000. The experiment indicated that this fit together can obtain the best results for D. salina transformation: adding 150 microL PEG and 90 microL plasmid DNA to 800 microL culture of D. salina (10(6) cells/mL) containing 300 mg glass beads, swirling 12 seconds under the rotate speed 2400r/min. This newly method can be used as a potential tool in the research of D. salina gene engineering with the advantage of more simpleness, convenience, quickness and less expense.

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions.

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.