Glucose-induced microRNA-218 suppresses the proliferation and promotes the apoptosis of human retinal pigment epithelium cells by targeting RUNX2.

OBJECTIVE: MicroRNA-218 (miR-218) critical for preventing the progression of numerous diseases, including diseases of the retinal pigment epithelium (RPE). However, the mechanism by which miR-218 regulates the PRE in humans remains largely unknown. Our study investigated the effects of glucose-induced miR-218 expression on human RPE cells (ARPE-19), as well as its targeted regulatory effect. METHODS: The levels of miR-218 and runt-related transcription factor 2 (RUNX2) expression were investigated by RT-qPCR or Western blot assays. Cell viability and apoptosis were assessed by CCK-8 assays, flow cytometry, and Hoechst staining. A luciferase reporter assay was performed to determine whether Runx2 is a target gene of miR-218. RESULTS: Our results showed that glucose up-regulated miR-218 expression, suppressed proliferation, and induced the apoptosis of ARPE-19 cells. We verified that miR-218 could inhibit the proliferation and facilitate the apoptosis of ARPE-19 cells, while inhibition of miR-218 expression produced the opposite effects. In terms of mechanism, we demonstrated that RUNX2 was a direct target of miR-218. Functional experiments showed that Runx2 served as a miR-218 target to help inhibit the proliferation and induction of apoptosis in ARPE-19 cells. CONCLUSION: Our findings suggest the miR-218/Runx2 axis as a potential target for treating diabetic retinopathy (DR).

Heat conjugation of antibacterial agents from amino acids and plant oil.

Antimicrobial peptides are components of the innate immune systems in animals and plants as natural defense against pathogens. Critical issues like manufacturing costs have to be addressed before mass production of these peptides for agriculture or community sterilizations. Here, we report a cost-effective chemical synthesis method to produce antimicrobial cocktails, which was based on the heat conjugation of amino acids in the presence of phosphoric acid and plant oil at 150 °C. The conjugates showed potent biological activities against all tested bacteria including a multi-drug resistant Staphylococcus aureus strain Y5 and ampicillin resistant Pseudomonas aerugenosa ATCC9027 strain, demonstrating potential in agriculture, and prophylactic applications in hospital and community settings.

The selective cysteinyl leukotriene receptor 1 (CysLT1R) antagonist montelukast regulates extracellular matrix remodeling.

Scar formation after filtration surgery of glaucoma is mainly caused by excessive synthesis of new extracellular matrix (ECM) and contraction of subconjunctival tissue mediated by human Tenon fibroblasts (HTFs) and the transforming growth factor (TGF-ß1). Montelukast, a potent and specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist, is a licensed drug clinically used for the treatment of bronchial asthma. In this study, we investigated the effects of montelukast on the contractility of HTFs cultured in a three-dimensional collagen gel. We found that cysLT1R was expressed in HTFs. Interestingly, the expression of cysLT1R was increased in response to TGF-ß1 in a dose dependent manner, suggesting its potential role in TGF-ß1 induced fibrosis. Importantly, we found that montelukast inhibited TGF-ß1-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. In addition, TGF-ß1-induced expression of MMP-1 and MMP-3, generation of fibronectin and type I collagen production, focal adhesion kinase (FAK) and paxillin phosphorylation in HTFs were also ameliorated by montelukast in a dose dependent manner. These results suggested that montelukast might provide therapeutic possibilities for inhibition of scar formation after such surgery.

Generation of sequence variants via accelerated molecular evolution methods.

Directed evolution shortcuts million-year-scale natural evolution in a matter of weeks and generates tens of millions of sequence variants in a single test tube. A team of researchers used random DNA flanked by homologous sequences for in vivo homologous recombination, known as multiplex automated genome engineering (MAGE) to select the most active gene variants. They also adopted this approach to replace hundreds of stop codons in the E. coli genome, showing potential for genome-wide engineering. The blank codon created was harnessed to enlarge the amino acid alphabet, and unnatural amino acid has been incorporated to polypeptides. In phage-assisted continuous evolution (PACE), the target activity was linked to the expression of a protein required for the production of infectious phage, and researchers obtained activities with novel affinities to T3 promoter, ATP, etc. In vitro recombination enables the generation of massive number of artificial lives of potential values. Random combinatorial DNA approach has also been harnessed to construct G-H loop sequences of type O FMDV VP1 gene, and 100 novel radical sequence variants were obtained in a single experiment, which paves the way for the future investigations on the potential development of a polyvalent vaccine to cope with rapid viral variations. The enormous combinatorial diversity of these methods conferred high mutation rates at either full length genes or targeted regions unmatched by natural evolution or previous directed evolution methods. Interactions of mutations or epistasis may have generated beneficial phenotypes from neutral and deleterious mutations. Selection for desired phenotypes may create sequence variants that might never occur in evolution. Accelerated molecular evolution methods, capitalized on random DNA strings, continuous evolution, unnatural amino acids or in vitro recombination, provide infinite opportunities for research, industrial and medical applications.

Recent patents on oligonucleotide synthesis and gene synthesis.

Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular biology. The field is constantly evolving which has led to key advances in oligonucleotide synthesis and gene synthesis technologies, with simplicity, cost effectiveness and high throughput. The miniaturization, multiplexing, microfluidic processing and the integrated microchip engineering will drive down cost and increase productivity without compromising DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA elements and genes as raw material for synthetic biology. This article describes some of the recent patents on oligonucleotide synthesis and gene synthesis.