Recent Progress Toward the Templated Synthesis and Directed Evolution of Sequence-Defined Synthetic Polymers

Biological polymers such as nucleic acids and proteins are ubiquitous in living systems, but their ability to address problems beyond those found in nature is constrained by factors such as chemical or biological instability, limited building-block functionality, bioavailability, and immunogenicity. In principle, sequence-defined synthetic polymers based on nonbiological monomers and backbones might overcome these constraints; however, identifying the sequence of a synthetic polymer that possesses a specific desired functional property remains a major challenge. Molecular evolution can rapidly generate functional polymers but requires a means of translating amplifiable templates such as nucleic acids into the polymer being evolved. This review covers recent advances in the enzymatic and nonenzymatic templated polymerization of nonnatural polymers and their potential applications in the directed evolution of sequence-defined synthetic polymers.Chemistry and Chemical Biolog

In Vivo Targeting through Click Chemistry

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.Engineering and Applied Science

Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1

DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.Chemistry and Chemical Biolog

An in vitro translation, selection and amplification system for peptide nucleic acids

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.Chemistry and Chemical Biolog

Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors

Engineering and Applied Science

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine

The Art of Machine Use Subversion in Digital Poetry

Howard Becker (1982) terms a cooperative network of people organized through and around joint knowledge and conventions of practice as an “art world”. A new “art world is born when it brings together people who never cooperated before to produce art based on and using conventions previously unknown or not exploited in that way” (310). This essay examines digital poetry art practice as an example of Becker’s type of novel, networked, and collaborative cultural activity. The diffusion of Internet technologies and the ubiquity of computing has allowed for the creation of many new art worlds, digital poetry being just one example. Furthermore, digital poetry art practice demonstrates a long history of machine use subversion, as we see technologies initially designed for other uses being repurposed to create digital poetry. In these cases, most consequentially to our thinking regarding digital poetry, what occurs is the process described by Becker: “the people who develop new art worlds participate in the broad currents of intellectual and expressive interest growing out of extant tradition and practice” (314). Although the digital poem is a distinct and unique literary artifact, digital poetry can be regarded as an ouroboric recursive practice that builds on the extant traditions of early experimentations with print, film, and video poetry as well as net art. Understanding how digital poetry operates as an art world allows us to legitimize and recognize the importance of digital culture and its impact on contemporary art and culture

Derivation of EDK and iPDK cells.

<p><b>A.</b> Summary of the differentiation protocol used for the derivation of EDK and iPDK cells from hESCs and hiPSCs. <b>B.</b> Cells differentiated from hESC (EDK) and hiPSC (iPDK) demonstrate typical fibroblast morphology. Bars, 100 µm.</p