A major goal of synthetic biology is to reprogram cells to perform complex tasks. Here we show how a combination of in vitro and in vivo selection rapidly identifies a synthetic riboswitch that activates protein translation in response to the herbicide atrazine. We further demonstrate that this riboswitch can reprogram bacteria to migrate in the presence of atrazine. Finally, we show that incorporating a gene from an atrazine catabolic pathway allows these cells to seek and destroy atrazine.
Reactions of glycine, alanine, and phenylalanine with acetic anhydride and ammonium thiocyanate give the 1-acetyl-2-thiohydantoins 2a-c. These results appear to contradict prior literature reports pertaining to this reaction.
The synthesis and antibiofilm activities of sulfonamide, urea, and thiourea oroidin analogues are described. The most active derivative was able to selectively inhibit P. aeruginosa biofilm development and is also shown to be non-toxic upwards of 1 mM to the development of C. elegans in comparison to other similar isosteric analogues and the natural product oroidin.Bacterial biofilm formation is often described as a developmental process initiated when free floating (planktonic) bacteria adhere to a surface suitable for growth and initiate the formation of a microcolony. 1 Occupation of a biofilm growth state confers to the bacteria a unique set of phenotypic traits which include resistance to microbicides and antibiotics that would often lead to eradication. [2][3][4] In a medical setting, biofilms pose a serious threat to individuals who suffer from a myriad of diseases. Recent estimates have attributed biofilm-associated infections as being responsible for upwards of 75% of microbial infections in the human body. 5 This problem is further exacerbated by the increased spread of antibiotic resistance. Additionally, biofilms are known to infect patients with indwelling medical devices (IMDs a ) such as catheters and heart stents. 6-8 Remediation of biofilm infected IMDs is traditionally accomplished by device removal due to the lack of antibiotic efficacy.As the medical community works towards new approaches aimed at combating the deleterious effects of biofilms, one area that has garnered significant attention is the identification of nonmicrobicidal modulators of biofilm growth and maintenance (Figure 1). 9-13 By not directly killing bacteria, it is postulated that development of resistance to these molecules would be mitigated or significantly impaired. Implementation of remediation therapies which focus on the co-dosing of an antibiofilm modulator with an antibiotic also provides an attractive avenue for treatment. One of the few naturally occurring scaffolds shown to possess non-microbicidal antibiofilm properties are compounds derived from the oroidin class of natural products. Oroidin 5 has been reported to be a moderate inhibitor of Pseudomonas aeruginosa PAO1/ PA14 biofilm growth (PAO1 IC 50 = 190 μM, PA14 IC 50 = 166 μM). 14 *To whom correspondence should be addressed. We have recently begun to focus on the development of methodologies to access oroidin analogues for antibiofilm screening. [15][16][17] Of these approaches, development of conditions for a generic reductive acylation reaction has allowed access to previously unattainable oroidin derivatives through the use of acid chlorides, anhydrides, succinimide esters, and trichloromethylketone pyrroles. 18 In a continued effort, we sought to further apply this approach in generating isosteric analogues possessing sulfonamide, urea and thiourea functionalities to further probe the structure-activity relationships (SAR) of the oroidin family in the context of antibiofilm activity and preliminary toxicity.The Gram-negative γ-proteobacteria Pseu...
Methodology is presented for assembling fluorescently labeled bivalent molecules from monovalent constituents, without side chain protection or coupling agents. To illustrate the procedure, a series of bivalent peptidomimetics directed toward the Trk receptors were prepared and screened via fluorescent activated cell sorting scan assays.
A pilot library of 2-aminoimidazole triazoles (2-AITs) was synthesized and assayed against Acinetobacter baumannii and methicillin-resistant Staphylococus aureus (MRSA). Results from these studies show that these new derivatives have improved biofilm dispersal activities as well as antibacterial properties against A. baumannii. With MRSA biofilms they are found to possess biofilm inhibition capabilities at low micromolar concentrations.
Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.
Libraries of monovalent compounds can be reacted with each other to give libraries of bivalent ones. If those reactions are efficient, and if the products do not need to be purified, large numbers of bivalent compounds can be produced rapidly, and one might say there is a "combinatorial advantage" to doing so. However, selective formation of heterobivalent products must be possible otherwise statistical mixtures will form. This tutorial review describes methods that will give heterobivalent compounds almost exclusively. Although there are relatively few methods that will give that desired selectivity, such methods are becoming increasingly important as the potential applications of bi- and multivalent compounds emerge.
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