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.

Ethyl cellulose/gelatin-carboxymethyl chitosan bilayer films doped with Euryale ferox seed shell polyphenol for cooked meat preservation.

This study evaluated the effects of an edible bilayer containing polyphenols from the Euryale ferox seed shell on ready-to-eat cooked beef products, including the physical, mechanical, antioxidant, and antibacterial capabilities. Here, the bilayer films were prepared by layer-by-layer solution pouring using hydrophobic ethyl cellulose (EC) as the outer layer, and hydrophilic gelatin/carboxymethyl chitosan (GC) as the inner layer. By adjusting the proportion of gelatin to carboxymethyl chitosan, the optical, mechanical, and barrier characteristics of bilayer films were markedly enhanced. Extracted polyphenol (EP) from shell of the Euryale ferox seed performed potent antibacterial property against Listeria monocytogenes (L. monocytogenes). The addition of EP to the inner layer of the optimized bilayer film further improved the mechanical and barrier properties of films, and as expected, the film exhibited antioxidant and antibacterial abilities. Additionally, cooked beef and cooked chicken preservation tests indicated that the active bilayer film showed good inhibition of L. monocytogenes and delayed lipid oxidation in ready-to-eat meat products, and significantly delayed the pH, moisture loss, color and texture changes. This study developed multifunctional bilayer active edible films, which has a great potential in the preservation ready-to-eat cooked meat products.

Evaluating the hypolipidemic effect of garlic essential oil encapsulated in a novel double-layer delivery system.

The limited application of garlic essential oil (GEO) is attributed to its pungent taste, poor water solubility and low bioavailability. Liposomes are nontoxic, biodegradable and biocompatible, and ß-cyclodextrin can inhibit undesirable odors and improve the stability and bioavailability. Thus a promising dual-layer GEO ß-cyclodextrin inclusion compound liposome (GEO-DCL) delivery system with both advantages was designed and prepared in this study. Experimental results indicated that the encapsulation efficiency of GEO-DCLs was 5% higher than that of GEO liposomes (GEO-CLs), reaching more than 88%. In vitro release experiment showed that the release rate of GEO in GEO-DCLs was 40% lower than that of GEO-CLs after incubation in gastric juice for 6-h, indicating that the stability of GEO-DCLs was better than GEO-CLs. Evaluation of the effects of GEO-DCLs on lowering blood lipid levels in hypercholesterolemia mice. GEO-DCLs could reduce the weight and fat deposition in hypercholesterolemia mice. Inhibiting the increase of TC, LDL-C, and decrease of HDL-C in mice. The degree of liver injury was decreased, the number of round lipid droplets in liver cytoplasm was reduced, and the growth of fat cells was inhibited. The lipid-lowering effects of GEO-DCLs were dose-dependent. GEO-DCL can improve the bioavailability of GEO and improve dyslipidemia. Based on GEO's efficacy in lowering blood lipids, this study developed a kind of GEO-DCL compound pomegranate juice beverage with good taste, miscibility and double effect of reducing blood lipids. This study lays a foundation for the application of GEO in the field of functional food.

The impact of financial development on CO2 emissions of global iron and steel industry.

As carbon peaking and carbon neutrality have become a global consensus, more and more countries have introduced relevant policies to adapt to their own countries and formulated corresponding time roadmap. The industrial sector, especially the steel sector, which produces high levels of pollution and carbon emissions, is facing significant pressure to transform its operations to reduce CO2 emissions. Previous studies have shown the importance of financial development (FD) in environmental protection; however, the impact of FD on the CO2 emissions of the steel sector is ignored. This paper examines the impact of FD on the CO2 emissions of the iron and steel industry from a global perspective using comprehensive panel data from a total of 30 countries during the period from 1990 to 2018. Empirical results show that an improved level of FD in a given country reduces the CO2 emissions of the iron and steel industry. The mechanism analysis indicates that FD promotes the upgrading of the structure of the iron and steel industry and the reduction of the CO2 emissions by means of the three-stage least square method. Our results also show that the effect of FD on reducing the CO2 emissions of the iron and steel industry in developing countries is less than its effect in developed countries. Estimation results also show the existence of the environmental Kuznets curve hypothesis in the iron and steel industry. Finally, we discuss the policy implications of achieving carbon neutrality in the steel sector.

Coupling Two Sequential Biocatalysts with Close Proximity into Metal-Organic Frameworks for Enhanced Cascade Catalysis.

The encapsulation of multiple enzyme/nanoenzyme systems within mental-organic frameworks (MOFs) shows great promise for a myriad of practical applications. Herein, two sequential biocatalysts, oxidase and hemin, were coupled together with close proximity using a bifunctional polymer, poly(1-vinylimidazole) (PVI), and encapsulated into MOFs. As a demonstration of the power of such a protocol, glucose oxidase&PVI-hemin encapsulated in ZIF-8 showed significant enhancement of bioactivity for a cascade reaction compared to its counterpart without PVI. For the colorimetric assay of glucose, it showed a low limit of detection of 0.4 µM (S/N = 3), high selectivity, and excellent stability. Because there are numerous biocatalysts that can readily be coupled and encapsulated into MOFs, a myriad of interesting properties can be simply realized by encapsulating different sequential biocatalysts.

In situ terminus-regulated DNA hydrogelation for ultrasensitive on-chip microRNA assay.

In this work, a dynamic terminus-regulated fabric of DNA hydrogel was invented in debt to the reiterative catalysis of terminal deoxynucleotidyl transferase (TdT). It extended free 3'-OH end to an overhang of homopolymeric adenosine base pair, and alternated with branching from the frayed complementary seed oligo T20G5. The cycle of this template-independent and isothermal amplification resulted in a microscale dendritic DNA fractal at first, which then gelatinized into a cohesive and intricate 3D network. Details of the complex were elucidated with gel electrophoresis, confocal and atomic force microscopy. Its well hydrated inner space could further provide plenty of biocompatible chambers for enzymatic transducers fused along the elongation. Taking merits of this neat and flexible setup, an in situ hydrogelation strategy was developed and utilized in the signal cascade of a miRNA biomarker detector on an electrode microarray, thus accomplished an ultrasensitive, selective and high-throughput sensing even for real samples. This collective manipulation of DNA-protein hydrogel ensemble on interface demonstrates its potency as a general scheme of sensitization in bioanalytical applications.

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.

DNA-Based Hybrid Hydrogels Sustain Water-Insoluble Ophthalmic Therapeutic Delivery against Allergic Conjunctivitis.

Clinical need for treating allergic conjunctivitis (AC) is rapidly increasing. However, AC-relevant anti-inflammatory compounds are generally difficult to solubilize in water, thus limiting their therapeutic potential. Solubility-improved eye drop formulations of these compounds have poor bioavailability and a short retention time in ophthalmic tissues. Herein, we report a DNA/poly(lactic-co-glycolicacid) (PLGA) hybrid hydrogel (HDNA) for water-insoluble ophthalmic therapeutic delivery. PLGA pre-encapsulation enables loading of water-insoluble therapeutics. HDNA's porous structure is capable of sustained delivery of therapeutics. Dexamethasone (DEX), with demonstrated activities in attenuating inflammatory symptom in AC, was used as a model system. The designed HDNA hybrid hydrogels significantly improved the DEX accumulation and mediated the gradual DEX release in ophthalmic cells and tissues. Using the HDNA-DEX complexes, potent efficacy in two animal models of AC was acquired. Given this performance, demonstrable biocompatibility, and biodegradability of DNA hydrogel, the HDNA-based ophthalmic therapeutic delivery system enables novel treatment paradigms, which will have widespread applications in the treatment of various eye diseases.

[The effect of different fluoride concentrations on the expression of transforming growth factor-beta1 in ameloblast of rat incisor].

OBJECTIVE: To investigate the effect of different concentrations of fluoride on the expression of transforming growth factor-beta1 (TGF-beta1), and explore the mechanism of dental fluorosis. METHODS: 40 Wistar rats were randomly divided into three groups, and supplied with low dose F(-)(60 mgxL(-1), 13 rats), high dose F(-)(120 mgxL(-1), 13 rats) and distilled water(control group, 14 rats) respectively. After 10 weeks, hematoxylin-eosin (HE) and immunohistochemistry staining were used to observe the changes of the ameloblasts and the expression of TGF-beta1 in rat incisors. RESULTS: The fluoride groups showed typical symptoms of dental fluorosis. The surfaces of the rat teeth fed with fluoride appeared chalky color and cross striation on the enamel surface. The HE staining showed that the morphous of ameloblast were disarranged and cellular derangement, even appeared vacuolar change. TGF-beta1 were expressed both in ameloblast of the secretory and maturation stage, and also in stellate reticulum and stratum intermedium. The expression of TGF-beta1 in rat's ameloblasts in experiment group was significantly lower than that in the control group (P<0.01), yet low dose group and high dose group still had no significant difference (P>0.05). CONCLUSION: By inhibiting the expression of TGF-beta1 in ameloblast, fluoride interferes the normal signal transduction between epithelial and mesenchymal, and affect the differentiation and development of enamel, so leading to the occurrence of dental fluorosis.

[The effect of different concentrations of fluoride on the expression of Smad2/3 in ameloblast of rat incisor].

PURPOSE: To investigate the effect of different concentrations of fluoride on the expression of Smad2/3 which is a specific intracellular signal transduction molecule of TGF-ß, and to explore the mechanism of dental fluorosis in rat. METHODS: Forty Wistar rats were randomly divided into 3 groups.HE and immunohistochemistry were used to detect the changes of the ameloblasts and the expression of Smad2/3 in rat incisors. MetaMorph microscope images analysis system and SPSS12.0 software package were used to analyze the images and data. RESULTS: Typical symptoms of dental fluorosis were found in the fluoride group. The expression of Smad2/3 in the ameloblasts in the experimental group was significantly lower than that in the control group (P<0.01); but the difference was not significant between the low-dose group and high-dose group(P>0.05). CONCLUSIONS: By inhibiting the expression of Smad2/3 in ameloblasts, fluoride affects the differentiation and development of enamel,leading to the occurrence of dental fluorosis in rat.

[Effect of fluoride on the expression of MMP-20/TIMP-2 in ameloblast of rat incisor and the antagonistic effect of melatonin against fluorosis].

PURPOSE: To study the effect of concentration of fluoride on the expression of matrix metalloproteinase-20(MMP-20) and tissue inhibitors of metalloproteinase-2 (TIMP-2) in the ameloblast of rat incisor,and explore the formation mechanism of dental fluorosis. By comparing the different expression of MMP-20,TIMP-2 between fluoride group and the melatonin group,to decide whether melatonin has antagonitic effect on dental fluorosis. METHODS: Forty Wistar rats were randomly divided into 6 groups. The groups were as follows: control group,low-dose group, high-dose group,normal saline group and melatonin group. The animals were sacrificed 10 weeks after treatment. HE and immunohistochemical staining were used to observe the changes of ameloblasts and the expression of MMP-20 and TIMP-2 in rat incisors. MetaMorph microscope images analysis system was used to analyze the images, and SPSS12.0 software package was used for data analysis. RESULTS: The surface of rat incisors fed with fluoride had chalky color change and cross stritations could be seen on the enamel surface.In the fluoride group,the ameloblasts were disarranged, cells arranged in multi-layer,even showing vacuolar change.The changes in the high-dose group was severer than the low-dose group. MMP-20, TIMP-2 were expressed both in the secretory ameloblasts, and in the odontoblasts.The expression of MMP-20 in rat's ameloblasts in the experimental group was significantly lower than that in the control group (P < 0.01); and no significant difference was found between the low-dose and high-dose groups(P > 0.05). The difference of expression of TIMP-2 was not significant among all the groups. The difference of expression of MMP-20 and TIMP-2 was not significant between the melatonin and the fluoride groups. CONCLUSIONS: The excessive fluoride can inhibit the secretion of MMP-20 and disturb the balance between MMP-20 and TIMP-2,which lead to the delay of amelogenin removal and enamel demineralization. Melatonin has no antagonistic effect on the dental fluorosis. Supported by National Natural Science Foundation of China (30600509) and Natural Science Foundation of Liaoning Province (20102278).

[Preliminary study of BMSCs seeded into collagen I-glycosaminoglycan matrices induced toward cartilage].

OBJECTIVE: To investigate the possibility of BMSCs seeded into collagen I -glycosaminoglycan (CG) matrices to form the tissue engineered cartilage through chondrocyte inducing culture. METHODS: Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT) cross-linked CG matrices at 1 x 10(6) cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen II immunohistochemical staining. RESULTS: The examinations under the inverted phase contrast microscope indicated the 2nd and 3rd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-like spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen II staining appeared around the cells in the experimental group. There was no positive collagen II staining appeared in the control group. CONCLUSION: It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.