Evolution of Organic Solvent-Resistant DNA Polymerases.

The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.

Discovery of novel compounds as potent activators of Sirt3.

Among the sirtuin enzymes, Sirt3 is one of the most important deacetylases as it regulates acetylation levels in mitochondria, which are linked to the metabolism of multiple organs and therefore involved in many types of age-related human diseases such as cancer, heart diseases and metabolic diseases. Given the dearth of direct activators of Sirt3, the identification of new modulators could be a key step in the development of new therapeutics. Here we report the discovery of Sirt3 modulators, including activators, through the use of DNA encoded library technology (DEL) and computational high-throughput screening methodologies. Top hits from both screenings against Sirt3 were evaluated according to their activity and affinity. Our best activator is more potent than the previously reported activator Honokiol. Docking studies suggest that our activators identified from virtual screening interact with Sirt3 at a site similar to Honokiol, whereas the activators identified from DEL selection interact with Sirt3 at an atypical site. Our results establish the attractiveness of these high-throughput screening technologies in identifying novel and potent Sirt3 activators and, therefore, in associated therapeutic applications.

Olive leaves extract alleviates inflammation and modifies the intrinsic apoptotic signal in the leukemic bone marrow.

Introduction: Current anti-leukemic chemotherapies with multiple targets suffer from side effects. Synthetic drugs with huge off-target effects are detrimental to leukemic patients. Therefore, natural plant-based products are being increasingly tested for new anti-leukemic therapy with fewer or no side effects. Herein, we report the effect of ethanolic olive leaves extract (EOLE) on the K562 cell line and on the bone marrow (BM) of N-ethyl-N-nitrosourea (ENU)-induced leukemic mice. Methods: Using standard methodologies, we assessed viability, chromatin condensation, and induction of apoptosis in EOLE-treated K562 cells in-vitro. The anti-leukemic activity of EOLE was assayed by measuring ROS, levels of various cytokines, expression of iNOS and COX-2 gene, and changes in the level of important apoptosis regulatory and cell signaling proteins in-vivo. Result: K562 cells underwent apoptotic induction after exposure to EOLE. In the BM of leukemic mice, EOLE therapy decreased the number of blast cells, ROS generation, and expression of NF-κB and ERK1/2. IL-6, IL-1ß, TNF-α, iNOS, and COX-2 were among the inflammatory molecules that were down-regulated by EOLE therapy. Additionally, it decreased the expression of anti-apoptotic proteins BCL2A1, BCL-xL, and MCL-1 in the BM of leukemic mice. Discussion: Chronic inflammation and anomalous apoptotic mechanism both critically contribute to the malignant transformation of cells. Inflammation in the tumor microenvironment promotes the growth, survival, and migration of cancer cells, accelerating the disease. The current investigation showed that EOLE treatment reduces inflammation and alters the expression of apoptosis regulatory protein in the BM of leukemic mice, which may halt the progression of the disease.

Combined Application of Essential Oil Compounds and Bacteriophage to Inhibit Growth of Staphylococcus aureus In Vitro.

Staphylococcus aureus is considered an important human pathogen. This study aimed to investigate the combination of essential oil compounds (EOCs) and bacteriophage as alternative antimicrobials to control S. aureus in vitro. Here, four EOCs (alpha-pinene, 3-carene, (+)-limonene, (1S)-(-)-ß-pinene) were evaluated by disc diffusion assay (DDA) and growth inhibition assay (GIA) to determine inhibitory effects against five strains of S. aureus. Phage adsorption assays were performed with phage K up to 120 h at 6, 13, and 37 °C to determine lytic activity. Combinations of phage K and EOCs against S. aureus were also evaluated at 37 °C. Alpha-pinene exhibited significantly greater inhibition towards S. aureus strains when compared to other EOCs tested by the DDA. GIAs indicate that all S. aureus strains exhibited significantly reduced growth (P < 0.006) over a 48-h period when exposed to EOCs. Phage adsorption assays indicate that phage K has high lytic activity at 37 °C with at least a 1.5-log increase in the number of plaque-forming units (PFU) over 6 h when compared to 6 and 13 °C. S. aureus strains showed significantly reduced growth (P < 0.05) when treated with combined phage K and EOCs. Results from the combined effect of EOC and phage indicate that phage alone inhibits S. aureus in vitro at 37 °C as effectively as EOCs alone or in combination with phage with variability between strains. The results from DDA, GIA, and phage adsorption assays indicate that select EOCs and phage K can be used as antimicrobials against S. aureus in vitro with potential application in situ.