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Creators/Authors contains: "Chen, Irene A."

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  1. Abstract

    Systems of catalytic RNAs presumably gave rise to important evolutionary innovations, such as the genetic code. Such systems may exhibit particular tolerance to errors (error minimization) as well as coding specificity. While often assumed to result from natural selection, error minimization may instead be an emergent by-product. In an RNA world, a system of self-aminoacylating ribozymes could enforce the mapping of amino acids to anticodons. We measured the activity of thousands of ribozyme mutants on alternative substrates (activated analogs for tryptophan, phenylalanine, leucine, isoleucine, valine, and methionine). Related ribozymes exhibited shared preferences for substrates, indicating that adoption of additional amino acids by existing ribozymes would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate that by-products of ribozyme evolution could lead to adaptive value in specificity and error tolerance.

  2. Various self-cleaving ribozymes appearing in nature catalyze the sequence-specific intramolecular cleavage of RNA and can be engineered to catalyze cleavage of appropriate substrates in an intermolecular fashion, thus acting as true catalysts. The mechanisms of the small, self-cleaving ribozymes have been extensively studied and reviewed previously. Self-cleaving ribozymes can possess high catalytic activity and high substrate specificity; however, substrate specificity is also engineerable within the constraints of the ribozyme structure. While these ribozymes share a common fundamental catalytic mechanism, each ribozyme family has a unique overall architecture and active site organization, indicating that several distinct structures yield this chemical activity. The multitude of catalytic structures, combined with some flexibility in substrate specificity within each family, suggests that such catalytic RNAs, taken together, could access a wide variety of substrates. Here, we give an overview of 10 classes of self-cleaving ribozymes and capture what is understood about their substrate specificity and synthetic applications. Evolution of these ribozymes in an RNA world might be characterized by the emergence of a new ribozyme family followed by rapid adaptation or diversification for specific substrates.
  3. The use of bacteriophages (phages) for antibacterial therapy is under increasing consideration to treat antimicrobial-resistant infections. Phages have evolved multiple mechanisms to target their bacterial hosts, such as high-affinity, environmentally hardy receptor-binding proteins. However, traditional phage therapy suffers from multiple challenges stemming from the use of an exponentially replicating, evolving entity whose biology is not fully characterized (e.g., potential gene transduction). To address this problem, we conjugate the phages to gold nanorods, creating a reagent that can be destroyed upon use (termed “phanorods”). Chimeric phages were engineered to attach specifically to several Gram-negative organisms, including the human pathogens Escherichia coli , Pseudomonas aeruginosa , and Vibrio cholerae , and the plant pathogen Xanthomonas campestris . The bioconjugated phanorods could selectively target and kill specific bacterial cells using photothermal ablation. Following excitation by near-infrared light, gold nanorods release energy through nonradiative decay pathways, locally generating heat that efficiently kills targeted bacterial cells. Specificity was highlighted in the context of a P. aeruginosa biofilm, in which phanorod irradiation killed bacterial cells while causing minimal damage to epithelial cells. Local temperature and viscosity measurements revealed highly localized and selective ablation of the bacteria. Irradiation of the phanorods also destroyed the phages, preventing replicationmore »and reducing potential risks of traditional phage therapy while enabling control over dosing. The phanorod strategy integrates the highly evolved targeting strategies of phages with the photothermal properties of gold nanorods, creating a well-controlled platform for systematic killing of bacterial cells.« less
  4. Abstract

    Chronic wounds represent a large and growing disease burden. Infection and biofilm formation are two of the leading impediments of wound healing, suggesting an important role for the microbiome of these wounds. Debridement is a common and effective treatment for chronic wounds. We analyzed the bacterial content of the wound surface from 20 outpatients with chronic wounds before and immediately after debridement, as well as healthy skin. Given the large variation observed among different wounds, we introduce a Bayesian statistical method that models patient-to-patient variability and identify several genera that were significantly enriched in wounds vs. healthy skin. We found no difference between the microbiome of the original wound surface and that exposed by a single episode of sharp debridement, suggesting that this debridement did not directly alter the wound microbiome. However, we found that aerobes and especially facultative anaerobes were significantly associated with wounds that did not heal within 6 months. The facultative anaerobic genusEnterobacterwas significantly associated with lack of healing. The results suggest that an abundance of facultative anaerobes is a negative prognostic factor in the chronic wound microbiome, possibly due to the increased robustness of such communities to different metabolic environments.