Engineering Circularized mRNAs for the Production of Spider Silk Proteins.

Biomaterials offer unique properties that make them irreplaceable for next-generation applications. Fibrous proteins, such as various caterpillar silks and especially spider silk, have strength and toughness not found in human-made materials. In early studies, proteins containing long tandem repeats, such as major ampullate spidroin 1 (MaSp1) and flagelliform silk protein (FSLP), were produced using a large DNA template composed of many tandem repeats. The hierarchical DNA assembly of the DNA template is very time-consuming and labor-intensive, which makes the fibrous proteins difficult to study and engineer. In this study, we designed a circularized mRNA (cmRNA) employing the RNA cyclase ribozyme mechanism. cmRNAs encoding spider silk protein MaSp1 and FSLP were designed based on only one unit of the template sequence but provide ribosomes with a circular and infinite translation template for production of long peptides containing tandem repeats. Using this technique, cmRNAs of MaSp1 and FSLP were successfully generated with circularization efficiencies of 8.5% and 36.7%, respectively, which supported the production of recombinant MaSp1 and FSLP larger than 110 and 88 kDa, containing tens of repeat units. Western blot analysis and mass spectrometry confirmed the authenticity of MaSp1 and FSLP, which were produced at titers of 22.1 and 81.5 mg · liter-1, respectively. IMPORTANCE Spider silk is a biomaterial with superior properties. However, its heterologous expression template is hard to construct. The cmRNA technique simplifies the construction and expression strategy by proving the ribosome a circular translation template for expression of long peptides containing tandem repeats. This revolutionary technique will allow researchers to easily build, study, and experiment with any fiber proteins with sequences either from natural genes or artificial designs. We expect a significantly accelerated development of fibrous protein-based biomaterials with the cmRNA technique.

Self-Assembled Nanosized Vehicles from Amino Acid-Based Amphiphilic Polymers with Pendent Carboxyl Groups for Efficient Drug Delivery.

Developing safe and efficient delivery vehicles for chemotherapeutic drugs has been a long-standing demanding. Amino acid-based polymers are promising candidates to address this challenge due to their excellent biocompatibility and biodegradation. Herein, a series of well-defined amphiphilic block copolymers were prepared by PET-RAFT polymerization of N-acryloyl amino acid monomers. By altering monomer types and the block ratio of the copolymers, the copolymers self-assembled into nanostructures with various morphologies, including spheres, rod-like, fibers, and lamellae via hydrophobic and hydrogen bonding interactions. Significantly, the nanoparticles (NPs) assembled from amphiphilic block copolymers poly(N-acryloyl-valine)-b-poly(N-acryloyl-aspartic acid) (PV-b-PD) displayed an appealing cargo loading efficiency (21.8-32.6%) for a broad range of drugs (paclitaxel, doxorubicin (DOX), cisplatin, etc.) due to strong interactions. The DOX-loaded PV-b-PD NPs exhibited rapid cellular uptake (within 1 min) and a great therapeutic performance. These drug delivery systems provide new insights for regulating the controlled morphologies and improving the efficiency of drug delivery.

The prevalence of molar incisor hypomineralization: evidence from 70 studies.

OBJECTIVE: A growing number of studies have investigated the prevalence of Molar Incisor Hypomineralization (MIH) around the world. The aim of this study was to systematically estimate the pooled prevalence of MIH. METHODS: A comprehensive literature research was completed in English and Chinese databases. Random effect models were used to calculate the pooled prevalence. To address the heterogeneity, meta-regression, and sensitivity analyzes were conducted. Publication bias was estimated by trim and fill method. RESULTS: Seventy eligible studies were included. The pooled prevalence of MIH was 14.2% globally. In subgroup analysis, South America (18.0%, 95% CI: 13.8-22.2) and Spain (21.1%, 95% CI: 17.7-24.6) had the highest prevalence. There was no significant difference between males (14.3%, 95% CI: 12.0-16.6) and females (14.4%, 95% CI: 12.8-15.9). The prevalence of MIH among children 10 years of age or younger (15.1%, 95% CI: 12.1-18.2) was much higher than the prevalence of MIH among older children (12.1%, 95% CI: 8.0-16.3). Sample size explained 15.7% heterogeneity. CONCLUSION: MIH has a high incidence globally, especially among children <10 years old. It is, therefore, imperative to develop more appropriate dental healthcare strategies to care for these children and to identify the etiology of MIH to prevent it occurring.

Engineering an Artificial Membrane Vesicle Trafficking System (AMVTS) for the Excretion of ß-Carotene in Escherichia coli.

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E. coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted ß-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted ß-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted ß-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of ß-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a ß-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted ß-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.

Pegylated Interferon-α Plus Ribavirin Therapy Improves Left Ventricular Diastolic Dysfunction in Patients With Chronic Hepatitis C Attaining Sustained Virological Response.

BACKGROUND: Pegylated interferon (pegIFN) in combination with ribavirin (RBV) has successfully improved the rate of sustained virological response (SVR) in chronic hepatitis C virus (HCV) infected individuals, which reduces the progression of the chronic liver disease. However, the influence of combination therapy (pegIFN/RBV) on cardiac function has yielded ambiguous results. The present study aimed to evaluate the effects of combination therapy with pegIFN/RBV on cardiac function of HCV-infected individuals with SVR. MATERIALS AND METHODS: Cardiac function was assessed and correlated in 142 treatment-naïve patients with HCV infections by determining cardiac biomarkers and echocardiography before treatment and for 24 weeks post-treatment. RESULTS: An SVR was achieved by 50.7% of all patients. Serum N-terminal pro-B-type natriuretic peptide levels were significantly higher in all patients before treatment and decreased significantly 24 weeks post-treatment in the SVR group (62.84 [36.98-102.73] versus 22.87 [15.64-56.92] pg/mL, P < 0.01). Peak early diastolic annular velocity (E') was significantly lower (7.69 ± 2.48 versus 9.74 ± 2.68cm/s, P < 0.001) and E/E' was higher (10.04 ± 2.51 versus 8.18 ± 2.31, P < 0.001) in all patients with SVR. However, there were no statistically significant differences in biomarkers and echocardiographic parameters for patients without SVR. In addition, multivariate analysis identified age (odds ratio [OR] = 1.076; 95% CI: 1.031-1.125; P < 0.001), NT-proBNP (OR = 1.122; 95% CI: 1.002-1.248; P = 0.015), and SVR (OR = 0.532; 95% CI: 0.214-0.895; P = 0.023) as statistically significant independent variables associated with left ventricular diastolic dysfunction. CONCLUSIONS: The present study showed no adverse effects of combination therapy on cardiac function of HCV-infected individuals with SVR. Subsequent viral eradication resulted in improvement of left ventricular diastolic dysfunction.

[Inhibitory effect of cationic liposome-mediated antisense c-myb oligonucleotide on the growth of glioma].

OBJECTIVE: To aim at demonstrating whether cationic liposome-mediated antisense c-myb oligonucleotide(LipoAON) can inhibit the growth of C6 glioma by intravenous injection. METHODS: Intracerebral C6 glioma cells were implanted into the left caudal nucleus of forty-eight male Wistar rats. There were four groups: LipoAON(n = 12), antisense c-myb oligonucleotide (AON; n = 12), cationic liposome (Lipo; n = 12), and normal saline (NS; n = 12). Six days after tumor implantation, the above-mentioned drugs were injected into the right femoral veins of the rats respectively. Two days later, the same drugs were injected into the left femoral veins. The appetite, motor and weight of every animal were closely observed during the whole experiment. Six rats of each group were respectively killed 4 days and 10 days after the end of administration. The weight change, pathologic examination and immunohistochemical analysis of c-myb expression of the tumor were completed. RESULTS: In LipoAON group, the growth of the tumors was significantly inhibited in a short time after treatment and c-myb expression was down-regulated. But in the AON group and Lipo group, the growth of the tumors was not inhibited and c-myb expression was not down-regulated, compared with that in NS group. The inhibitory effect of LipoAON on the tumors rapidly declined with time and c-myb expression was again up-regulated. CONCLUSION: 1. Cationic liposome (LipofectAMINE) as transfection vehicle makes c-myb easily penetrate BBTB and enter the tumor. The technique is simple, safe, highly effective for the transfection of c-mybAON; 2. LipoAON has marked inhibitory effect on the growth of C6 glioma. The AON technical method for inhibiting the expression of c-myb oncogene has a research perspective in the treatment of glioma; 3. The inhibitory effect of LipoAON on the growth of glioma declines with time. The question about how to make c-myb AON have highly effective, sustained and stable expression in the tumor still requires further research.