Biology subjects

Hill, S. A.

Publications and source records attributed to Hill, S. A..

3 recordsLinked to original sources

bioRxiv2021

A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called endosomal escape problem, i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, "TAT-CaM," evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

cell biology↗

bioRxiv2021

Green Fluorescent Carbon Dots as Targeting Probes for LED-Dependent Bacterial Killing

The emergence of antimicrobial resistance represents a significant health and economic challenge worldwide. The slow pace of antibacterial discovery necessitates strategies for optimal use of existing agents, including effective diagnostics able to drive informed prescribing; and development of alternative therapeutic strategies that go beyond traditional small-molecule approaches. Thus, the development of novel probes able to target bacteria for detection and killing, and that can pave the way to effective theranostic strategies, is of great importance. Here we demonstrate that metal-free green-emitting fluorescent carbon dots (FCDs) synthesized from glucosamine HCl and m-phenylenediamine, and featuring 2,5-deoxyfructosazine on a robust amorphous core, can label both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacterial pathogens within 10 minutes of exposure. Moreover, effective killing of Gram-positive and -negative bacteria can be induced by combining FCD treatment with irradiation by LED light in the visible range. Cell-based, electron microscopy and Tandem Mass Tag (TMT) proteomic experiments indicate that FCD administration in combination with LED exposure gives rise to local heating, ROS production, and membrane- and DNA-damage, suggesting multiple routes to FCD-mediated bacterial killing. Our data identify FCDs as materials that combine facile synthesis from low-cost precursors with labelling and light-dependent killing of clinically important bacterial species, and that thus warrant further exploration as the potential bases for novel theranostics.

microbiology↗

bioRxiv2019

Sonic hedgehog signaling in astrocytes mediates cell-type-specific synaptic organization

Astrocytes have emerged as integral partners with neurons in regulating synapse formation and function, but the mechanisms that mediate these interactions are not well understood. Here, we show that Sonic hedgehog (Shh) signaling between neurons and astrocytes is required for establishing structural organization and remodeling of cortical synapses in a cell type-specific manner. In the postnatal cortex, Shh signaling is active in a subpopulation of mature astrocytes localized primarily in deep cortical layers. Selective disruption of Shh signaling in astrocytes produces a dramatic increase in synapse number specifically on layer V apical dendrites that emerges during adolescence and persists into adulthood. Dynamic turnover of dendritic spines is impaired in mutant mice and is accompanied by an increase in neuronal excitability and a reduction of the glial-specific, inward-rectifying K+ channel Kir4.1. These data identify a critical role for Shh signaling in astrocyte-mediated modulation of neuronal activity required for sculpting synapses.

neuroscience↗