A Clean One-Pot Synthesis of Structurally Ordered Linear, Monocyclic, and Bicyclic Oligosiloxanes.

A highly selective cross-coupling reaction between Si-OAc (AcO = acetoxy) and Si-OH compounds that generates unsymmetrical and symmetrical oligosiloxanes concurrent with the release of acetic acid has been developed. The high selectivity arises from the reactivity difference that depends on the varying number of acetoxy groups present, thus facilitating a clean one-pot synthesis of oligosiloxanes. For instance, the reactions of di-, tri-, or tetraacetoxysilanes with silanols furnish acetoxy-containing di- and trisiloxanes in high yield. Two equivalents of tetraacetoxysilane can react with various silanediols to form 1,1,1,3,3,3-hexaacetoxytrisiloxanes, which subsequently react with a second molecule of a silanediol to selectively afford 1,1,3,3-tetraacetoxycyclotetrasiloxanes. The cyclotetrasiloxanes further react with a third molecule of silanediol to provide unprecedented bicyclic pentasiloxanes with acetoxy groups at the bridgehead silicon atoms. Applications of the acetoxy-containing products as efficient surface-treatment agents and new building blocks for highly heat-resistant materials are demonstrated.

Polyhedral Particles with Controlled Concavity by Indentation Templating.

Current methods for fabricating microparticles offer limited control over size and shape. Here, we demonstrate a droplet microfluidic method to form polyhedral microparticles with controlled concavity. By manipulating Laplace pressure, buoyancy, and particle rheology, we generate microparticles with diverse shapes and curvatures. Additionally, we demonstrate the particles provide increased capture efficiency when used for particle-templated emulsification. Our approach enables microparticles with enhanced chemical and biological functionality.

Dual-layered hydrogels allow complete genome recovery with nucleic acid cytometry.

Targeting specific cells for sequencing is important for applications in cancer, microbiology, and infectious disease. Nucleic acid cytometry (NAC) is a powerful approach for accomplishing this because it allows specific cells to be isolated based on sequence biomarkers that are otherwise impossible to detect. However, existing methods require specialized microfluidic devices, limiting adoption. Here, a modified workflow is described that uses particle-templated emulsification (PTE) and flow cytometry to conduct the essential steps of cell detection and sorting normally accomplished by microfluidics. Our microfluidic-free workflow allows facile isolation and sequencing of cells, viruses, and nucleic acids and thus provides a powerful enrichment approach for targeted sequencing applications.

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays.

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Cytoneme-mediated signaling essential for tumorigenesis.

Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models and tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. We observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. We also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and we determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. Our results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.

Direct quantification of EGFR variant allele frequency in cell-free DNA using a microfluidic-free digital droplet PCR assay.

Analysis of liquid biopsy samples is a promising diagnostic intervention for noninvasive detection and monitoring of cancer genotypes. However, current methods used to assess mutation status are either costly, in the case of next-generation sequencing-based assays, or lacking in sensitivity, in the case of bulk quantitative PCR measurements. Digital droplet PCR (ddPCR) is at once a sensitive and low-cost method for detecting rare cancer mutations and measuring their variant allele frequency. In this chapter, we describe a method for conducting ddPCR assays without microfluidics in a process called "particle-templated emulsification" (PTE). Using hydrogel particles and a standard benchtop vortexer to rapidly emulsify large volumes, the method forgoes the specialized instrumentation required for conventional ddPCR assays and is capable of high experimental throughput. To assess the quantitative performance of the method, we apply PTE ddPCR to analysis of variant allele frequency in EGFR, a commonly mutated gene in lung adenocarcinomas.

Particle-Templated Emulsification for Microfluidics-Free Digital Biology.

The compartmentalization of reactions in monodispersed droplets is valuable for applications across biology. However, the requirement of microfluidics to partition the sample into monodispersed droplets is a significant barrier that impedes implementation. Here, we introduce particle-templated emulsification, a method to encapsulate samples in monodispersed emulsions without microfluidics. By vortexing a mixture of hydrogel particles and sample solution, we encapsulate the sample in monodispersed emulsions that are useful for most droplet applications. We illustrate the method with ddPCR and single cell culture. The ability to encapsulate samples in monodispersed droplets without microfluidics should facilitate the implementation of compartmentalized reactions in biology.

Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.