Sustained Delivery of Timolol Using Nanostructured Lipid Carriers-Laden Soft Contact Lenses.

The contact lens prepared by the conventional soaking method using timolol-soaking solution showed poor drug uptake and high burst release with altered critical lens properties. In this study, timolol-loaded nanostructured lipid carriers (NLCs) were prepared and evaluated for enhanced timolol uptake and sustained release for the effective management of glaucoma. The characterization studies indicated that timolol-loaded NLCs were spherical in shape with an average size of 130-138 nm and a zeta potential of -46.6 to 51.3 mV. Critical lens properties such as swelling, optical transmittance, and protein adherence were improved with NLC-laden lenses compared to the conventional soaked lenses (SM-TB). Moreover, SM-TB lens showed low timolol uptake, high burst release, and short release duration up to 24 h compared to timolol-NLC-laden lens that showed high timolol uptake, and the cumulative release was sustained up to 96 h. The ability to sustain timolol release improved proportionally with an increase in the amount of Capmul MCMC8 (liquid lipid) in NLCs. In addition, NLC-laden lens was found to be safe according to the results of ocular irritation and histopathological studies. In the rabbit tear fluid model, NLC-30%-Cap-CL batch showed high timolol concentration at all time points up to 60 h. Further, pharmacodynamic study showed sustained reduction in IOP by NLC-30%-Cap-CL batch for 96 h compared to 48 h and 6 h with SM-TB lens and eye drop solution, respectively. In conclusion, NLCs enhanced timolol uptake in the contact lens from the soaking solution using soaking method with improved in vitro and in vivo results for better clinical outcomes in the patients with glaucoma.

Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine.

Baicalein and scutellarein are bioactive flavones found in the medicinal plant Scutellaria baicalensis Georgi, used in traditional Chinese medicine. Extensive previous work has demonstrated the broad biological activity of these flavonoids, such as antifibrotic, antiviral and anticancer properties. However, their supply from plant material is insufficient to meet demand. Here, to provide an alternative production source and increase production levels of these flavones, we engineered an artificial pathway in an Escherichia coli cell factory for the first time. By first reconstructing the plant flavonoid biosynthetic pathway genes from five different species: phenylalanine ammonia lyase from Rhodotorula toruloides (PAL), 4-coumarate-coenzyme A ligase from Petroselinum crispum (4CL), chalcone synthase from Petunia hybrida (CHS), chalcone isomerase from Medicago sativa (CHI) and an oxidoreductase flavone synthase I from P. crispum (FNSI), production of the intermediates chrysin and apigenin was achieved by feeding phenylalanine and tyrosine as precursors. By comparative analysis of various versions of P450s, a construction expressing 2B1 incorporated with a 22-aa N-terminal truncated flavone C-6 hydroxylase from S. baicalensis (F6H) and partner P450 reductase from Arabidopsis thaliana (AtCPR) was found most effective for production of both baicalein (8.5 mg/L) and scutellarein (47.1 mg/L) upon supplementation with 0.5 g/L phenylalanine and tyrosine in 48 h of fermentation. Finally, optimization of malonyl-CoA availability further increased the production of baicalein to 23.6 mg/L and scutellarein to 106.5 mg/L in a flask culture. This report presents a significant advancement of flavone synthetic production and provides foundation for production of other flavones in microbial hosts.

An integrative database and its application for plant synthetic biology research.

Plant synthetic biology research requires diverse bioparts that facilitate the redesign and construction of new-to-nature biological devices or systems in plants. Limited by few well-characterized bioparts for plant chassis, the development of plant synthetic biology lags behind that of its microbial counterpart. Here, we constructed a web-based Plant Synthetic BioDatabase (PSBD), which currently categorizes 1677 catalytic bioparts and 384 regulatory elements and provides information on 309 species and 850 chemicals. Online bioinformatics tools including local BLAST, chem similarity, phylogenetic analysis, and visual strength are provided to assist with the rational design of genetic circuits for manipulation of gene expression in planta. We demonstrated the utility of the PSBD by functionally characterizing taxadiene synthase 2 and its quantitative regulation in tobacco leaves. More powerful synthetic devices were then assembled to amplify the transcriptional signals, enabling enhanced expression of flavivirus non-structure 1 proteins in plants. The PSBD is expected to be an integrative and user-centered platform that provides a one-stop service for diverse applications in plant synthetic biology research.

Research on Brain Signals Classification Based on Deep Learning.

With the continuous expansion of brain-computer communication, the precise identification of brain signals has become an essential task for brain-computer equipment. However, existing classification methods are primarily concentrated on the extraction features of brain signals and obtain unacceptable performance when directly used the model to a new brain signals data, which is caused by the different people has extraordinary brain signals. In this work, we utilize the deep learning methods not only extract the features of brain signals but also learn the order information of brain signals, which can satisfy the universal brain signals. Indeed, we utilize the classification features dimension distance loss function to optimize the proposed model and enhance the classification accuracy and we compare our model with existing classification methods to evaluate proposed model. From our extensive experimental results and analysis, we can conclude that our model can achieve the classification of brain signals with the reasonable accuracy and acceptable costs.

Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research.

The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 µg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.

Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in Escherichia coli.

5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues. In this study, GmCHS7 (chalcone synthase from Glycine max) and GuCHR (chalcone reductase from Glycyrrhizza uralensis) were preliminarily utilized to improve the CHR ratio (CHR product to total CHS product). The use of this enzyme combination improved the final CHR ratio from 39.7% to 50.3%. For further optimization, a protein-protein interaction strategy was employed, basing on the spatial adhesion of GmCHS7:PDZ and GuCHR:PDZlig. This strategy further increased the ratio towards the CHR-derived product (54.7%), suggesting partial success of redirecting metabolic flux towards the reduced branch. To further increase the total carbon metabolic flux, 15 protein scaffolds were programmed with stoichiometric arrangement of the three sequential catalysts GmCHS7, GuCHR and MsCHI (chalcone isomerase from Medicago sativa), resulting in a 1.4-fold increase in total flavanone production, from 69.4 mg/L to 97.0 mg/L in shake flasks. The protein self-assembly strategy also improved the production and direction of the lineage-specific compounds 7,4'-dihydroxyflavone and daidzein in Escherichia coli. This study presents a significant advancement of 5-deoxy(iso)flavonoid production and provides the foundation for production of value-added 5-deoxy(iso)flavonoids in microbial hosts.

Exosome-mediated small RNA delivery for gene therapy.

Small RNAs, including small interfering RNAs (siRNA) and microRNAs (miRNA), are emerging as promising therapeutic drugs against a wide array of diseases. The key obstacle for the successful clinical application of small RNAs is to develop a safe delivery system directed at the target tissues only. Current small RNA transfer techniques use viruses or synthetic agents as delivery vehicles. The replacement of these delivery vehicles with a low toxicity and high target-specific approach is essential for making small RNA therapy feasible. Because exosomes have the intrinsic ability to traverse biological barriers and to naturally transport functional small RNAs between cells, they represent a novel and exciting delivery vehicle for the field of small RNA therapy. As therapeutic delivery agents, exosomes will potentially be better tolerated by the immune system because they are natural nanocarriers derived from endogenous cells. Furthermore, exosomes derived from genetically engineered cells can deliver small RNAs to target tissues and cells. Thus, exosome-based delivery of small RNAs may provide an untapped, effective delivery strategy to overcome impediments such as inefficiency, nonspecificity, and immunogenic reactions. In this review, we briefly describe how exosomal small RNAs function in recipient cells. Furthermore, we provide an update and overview of new findings that reveal the potential applications of exosome-based small RNA delivery as therapeutics in clinical settings. WIREs RNA 2016, 7:758-771. doi: 10.1002/wrna.1363 For further resources related to this article, please visit the WIREs website.

Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse.

Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system.