Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli.

Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.

A prophylactic effect of aluminium-based adjuvants against respiratory viruses via priming local innate immunity.

Infection caused by respiratory viruses can lead to a severe respiratory disease and even death. Vaccination is the most effective way to prevent the disease, but it cannot be quickly applied when facing an emerging infectious disease. Here, we demonstrated that immunization with an aluminium-zinc hybrid particulate adjuvant (FH-001) alone, bearing great resemblance in morphology with commonly used aluminium-based adjuvants in vaccines, could quickly induce mice to generate a broadly protective immune response to resist the lethal challenge of influenza B viruses. Furthermore, a multi-omics-based analysis revealed that the alveolar macrophage and type I interferon pathway, rather than adaptive immunity and type II interferon pathway, were essential for the observed prophylactic effect of FH-001. More importantly, a similar protective effect was observed against influenza A virus strain A/Shanghai/02/2013(H7N9), A/California/04/2009(H1N1) and respiratory syncytial virus. Therefore, we introduced here a new and promising strategy that can be quickly applied during the outbreak of emerging respiratory viruses.

[Effects of Xuesaitong injection on coagulation function of gastric cancer patients undergoing laparoscopic surgery: a randomized controlled trial].

OBJECTIVE: To study the effects of Xuesaitong Injection, an extract from a Chinese herbal medicine, on coagulation function of gastric cancer patients undergoing laparoscopic surgery. METHODS: Fifty patients undergoing laparoscopic surgery for gastric cancer were randomly divided into control group and Xuesaitong group, with 25 patients in each group. Conventional anti-infection and transfusion treatment was given to the patients in the control group, and intravenous drip infusion of 400 mg Xuesaitong Injection added into 250 mL 5% glucose injection was administered once daily to the patients in the Xuesaitong group for 6 days from the first day after laparoscopic operation besides the conventional anti-infection and transfusion treatment. The values of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and international normalized ratio (INR) were detected by using blood coagulation method, and the values of D-dimer (D-D) before operation, instantly and 3, 5, 7 days after operation were detected by using enzyme-linked immunosorbent assay in the two groups. Furthermore, the development of deep vein thrombosis on the day 7 after operation was observed by vein angiography. RESULTS: There were no significant differences in the values of PT, APTT, INR, FIB and D-D between the two groups before operation, and there were no significant changes in APTT and INR after operation in the two groups. The values of PT decreased in the two groups on day 3 after operation, while there was no significant difference between the two groups. The value of PT in the control group decreased gradually with the extension of postoperation time (P < 0.05), while the PT value in the Xuesaitong group did not change significantly, and there was significant difference between the two groups (P < 0.05). The values of FIB and D-D increased instantly after operation in the two groups as compared with those before operation (P < 0.05), while there was no significant difference between the two groups. With the extension of postoperation time, the values of FIB and D-D increased gradually in the control group (P < 0.05); the D-D value on day 3 after operation increased, but the D-D values on day 5 and day 7 after operation decreased gradually in the Xuesaitong group (P < 0.05). The occurrence rate of deep vein thrombosis on day 7 after operation in the Xuesaitong group was lower than that in the control group. CONCLUSION: Laparoscopic surgery for gastric cancer may cause high agglutination condition, which will increase the risk of deep vein thrombosis, and Xuesaitong Injection can improve this state.

Calcium phosphate nanoparticles as a new generation vaccine adjuvant.

INTRODUCTION: Adjuvants are essential components in vaccine formulations to induce robust immunity against pathogens. The most widely used adjuvants in human vaccines are aluminum salts, that can effectively elicit a T helper type-2 (Th2)-biased humoral immune response for producing a high antibody titer but with a limited cellular immune response. Biocompatible calcium phosphate nanoparticles (CaP-NPs) with tunable characteristics have potentials to function as adjuvants for inducing more balanced T helper type-1 (Th1) and Th2 immune responses. Areas covered: Here we review the preparation procedures and characteristics of CaP-NPs. The process can be well-controlled and readily scaled up. Antigen loading can take place as encapsulation during the particle formation or as passive adsorption post particle formation. Different modalities of immunogens were tested with CaP-NPs as adjuvants. The possible mechanisms of the CaP-NP-based adjuvants are discussed. Expert commentary: With good adjuvant effects and safety profiles, CaP-NPs have the potentials to be a new generation vaccine adjuvant. A more in-depth understanding of the mechanisms of their adjuvanticity could facilitate the process optimization for making adjuvants with preferred characteristics. Interdisciplinary collaborations are essential for testing the biocompatible CaP-NPs in human vaccines for clinical development and eventually for use in marketed vaccines.

Chemical constituents from the fruits of Forsythia suspensa and their antimicrobial activity.

Lignans and phenylethanoid glycosides purified from Forsythia suspensa were reported to display various bioactivities in the previous literature, including the antimicrobial activity. Therefore, the present research is aimed to purify and identify the chemical constituents of the methanol extracts of fruits of F. suspensa. The methanol extracts of fruits of F. suspensa were fractionated and further purified with the assistance of column chromatography to afford totally thirty-four compounds. Among these isolates, 3 ß -acetoxy-20 α -hydroxyursan-28-oic acid (1) was reported from the natural sources for the first time. Some of the purified principles were subjected to the antimicrobial activity examinations against Escherichia coli to explore new natural lead compounds.

[Effect of different position and CO(2) pneumoperitoneum on deep vein hemodynamics of lower limb].

OBJECTIVE: To study the effect of laparoscopic surgery position and CO(2) pneumoperitoneum on deep vein hemodynamics of lower limb. METHODS: Color Doppler ultrasound was adopted to evaluate the diameter, blood velocity and the amount of the blood flow in the lower extremity of 37 gastric cancer patients undergone laparoscopic surgery from November 2007 to August 2008 in our hospital. Three kinds of position(prostration, head-down tilt position and feet-down tilt position) before the establishment of pneumoperitoneum and three different pressure of CO(2) pneumoperitoneum( 8 mm Hg, 12 mm Hg, 16 mm Hg ) after the establishment of pneumoperitoneum were applied during laparoscopic surgery. RESULTS: Compared with the stage of prostration before the pneumoperitoneum, the femoral vein diameter decreased (P<0.05), the velocity and the amount of blood flow increased (P<0.01, P<0.05) in head-down tilt position; the femoral vein diameter increased(P<0.01), the velocity and the amount of blood flow decreased(P<0.01, P<0.05) in feet-down tilt position or the establishment of pneumoperitoneum. Furthermore, with the increase of pneumoperitoneum pressure, the femoral vein diameter increased, the velocity and the amount of blood flow decreased(P<0.05). CONCLUSIONS: The head-down tilt position is conducive for the blood to recirculate, which will degrade the risk of deep vein thrombosis after the laparoscopy. The feet-down tilt position and CO(2) pneumoperitoneum can interfere the recirculation of blood, and increase the risk of deep vein thrombosis after the laparoscopy.