Kodak Moments

Poem reminiscing about a childhood interaction with her father. Despite being past her bedtime, she waits until her father gets home to hear about his day. The speaker likens the memory to a photograph captured in time

In Vivo Imaging of Transport and Biocompatibility of Single Silver Nanoparticles in Early Development of Zebrafish Embryos

Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo, and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5- 46 nm) transport in and out of embryos through chorion pore canals (CPCs), and exhibit Brownian diffusion (not active transport), with ∼26 times lower diffusion coefficient (3×10-9 cm2/s) inside the chorionic space than that in egg water (7.7×10-8 cm2/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer (nm) spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities

Silver Nanoparticles Induce Developmental Stage-Specific Embryonic Phenotypes in Zebrafish

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0–0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c ≪ 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c \u3c 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop to a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develops abnormality. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development

Uncovering Shakespeare\u27s Sisters in Special Collections and College Archives, Musselman Library

Foreword by Professor Suzanne J. Flynn I have taught the first-year seminar, Shakespeare’s Sisters, several times, and over the years I have brought the seminar’s students to the Folger Shakespeare Library in Washington, D.C. There, the wonderful librarians have treated the students to a special exhibit of early women’s manuscripts and first editions, beginning with letters written by Elizabeth I and proceeding through important works by seventeen and eighteenth-century women authors such as Aemelia Lanyer, Anne Finch, Aphra Behn, and Mary Wollstonecraft. This year I worked with Carolyn Sautter, the Director of Special Collections and College Archives, to give my 2018 seminar students the opportunity to produce a sequel to the Folger exhibit of early modern women writers. Special Collections houses an impressive array of first editions from the nineteenth and twentieth centuries, many of them acquired from Thomas Y. Cooper, the former editor of the Hanover Evening Sun newspaper, who donated over 1600 items to Musselman Library in 1965. Working with Kerri Odess-Harnish, we chose first editions of eight significant works of literature written by American and British women from the mid-nineteenth through the mid-twentieth centuries. The students worked in pairs, researching a single book and producing a report that outlines important biographical facts about the author, the book’s publication and reception history, and finally the significance of the book in the years since its publication. We hope that our project will draw attention to the wealth of literary treasures housed in Special Collections at Musselman Library, but especially to these works by eight of “Shakespeare’s Sisters.

Single Nanoparticle Plasmonic Spectroscopy for Study of the Efflux Function of Multidrug ABC Membrance Transporters of Single Live Cells

ATP-binding cassette (ABC) membrane transporters exist in all living organisms and play key roles in a wide range of cellular and physiological functions. The ABC transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, leading to multidrug resistance. Despite extensive study, their efflux molecular mechanisms remain elusive. In this study, we synthesized and characterized purified silver nanoparticles (Ag NPs) (97 ± 13 nm in diameter), and used them as photostable optical imaging probes to study efflux kinetics of ABC membrane transporters (BmrA) of single live cells (B. subtilis). The NPs with concentrations up to 3.7 pM were stable (non-aggregated) in a PBS buffer and biocompatible with the cells. We found a high dependence of accumulation of the intracellular NPs in single live cells (WT, Ct-BmrAEGFP, ΔBmrA) upon the cellular expression level of BmrA and NP concentration (0.93, 1.85 and 3.7 pM), showing the highest accumulation of intracellular NPs in ΔBmrA (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in ΔBmrA increases nearly proportionally with the NP concentration, while those in WT and Ct-BmrA-EGFP do not. This result suggests that the NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane and they are extruded out of cells by BmrA transporters, similar to conventional pump substrates (antibiotics). This study shows that such large substrates (84-100 nm NPs) can enter into the live cells and be extruded out of the cells by BmrA, and the NPs can serve as nm-sized optical imaging probes to study the size-dependent efflux kinetics of membrane transporters in single live cells in real time

Phase II Trial of IL-12 Plasmid Transfection and PD-1 Blockade in Immunologically Quiescent Melanoma.

PurposeTumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; "tavo") electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL).Patients and methodsTavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.v.) was administered every 3 weeks. The primary endpoint was objective response rate (ORR) by RECIST, secondary endpoints included duration of response, overall survival and progression-free survival. Toxicity was evaluated by the CTCAE v4. Extensive correlative analysis was done.ResultsThe combination of tavo and pembrolizumab was well tolerated with adverse events similar to those previously reported with pembrolizumab alone. Patients had a 41% ORR (n = 22, RECIST 1.1) with 36% complete responses. Correlative analysis showed that the combination enhanced immune infiltration and sustained the IL-12/IFNγ feed-forward cycle, driving intratumoral cross-presenting dendritic cell subsets with increased TILs, emerging T cell receptor clones and, ultimately, systemic cellular immune responses.ConclusionsThe combination of tavo and pembrolizumab was associated with a higher than expected response rate in this poorly immunogenic population. No new or unexpected toxicities were observed. Correlative analysis showed T cell infiltration with enhanced immunity paralleling the clinical activity in low cpCTL tumors

Study of Charge-Dependent Transport and Toxicity of Peptide-Functionalized Silver Nanoparticles Using Zebrafish Embryos and Single Nanoparticle Plasmonic Spectroscopy

Nanomaterials possess unusually high surface area-to-volume ratios and surface-determined physicochemical properties. It is essential to understand their surface-dependent toxicity in order to rationally design biocompatible nanomaterials for a wide variety of applications. In this study, we have functionalized the surfaces of silver nanoparticles (Ag NPs, 11.7 ±+2.7 nm in diameter) with three biocompatible peptides (CALNNK, CALNNS, CALNNE) to prepare positively (Ag-CALNNK NPs+ζ), negatively (Ag-CALNNS NPs−2ζ), and more negatively charged NPs (Ag-CALNNE NPs−4ζ), respectively. Each peptide differs in a single amino acid at its C-terminus, which minimizes the effects of peptide sequences and serves as a model molecule to create positive, neutral, and negative charges on the surface of the NPs at pH 4-10. We have studied their charge-dependent transport into early developing (cleavage-stage) zebrafish embryos and their effects on embryonic development using dark-field optical microscopy and spectroscopy (DFOMS). We found that all three Ag-peptide NPs passively diffused into the embryos via their chorionic pore canals, and stayed inside the embryos throughout their entire development (120 h), showing charge-independent diffusion modes and charge-dependent diffusion coefficients. Notably, the NPs create chargedependent toxic effects on embryonic development, showing that the Ag-CALNNK NPs+ζ (positively charged) are the most biocompatible while the Ag-CALNNE NPs−4ζ (more negatively charged) are the most toxic. By comparing with our previous studies of the same sized citrated Ag and Au NPs, the Ag-peptide NPs are much more biocompatible than the citrated Ag NPs, and nearly as biocompatible as the Au NPs, showing the dependence of nanotoxicity upon the surface charges, surface functional groups, and chemical compositions of the NPs. This study also demonstrates powerful applications of single NP plasmonic spectroscopy for quantitative analysis of single NPs in vivo and in tissues, and reveals the possibility of rational design of biocompatible NPs

In Vivo Quantitative Study of Sized-Dependent Transport and Toxicity of Single Silver Nanoparticles Using Zebrafish Embryos

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes and high surface area-to-volume ratios) and promise a wide variety of applications, ranging from the design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advances in nanotechnology. In this study, we have synthesized and characterized purified and stable (nonaggregation) silver nanoparticles (Ag NPs, 41.6 ± 9.1 nm in average diameter) and utilized early developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe the diffusion and toxicity of Ag NPs. We found that single Ag NPs (30-72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose-and size-dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤0.02 nM), 75-91% of embryos developed into normal zebrafish. At the higher concentrations of NPs (≥0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02-0.2 nM), embryos developed into various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6 ± 3.5 nm), we found striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6 ± 9.1 nm) are more toxic than the smaller Ag NPs (11.6 ± 3.5 nm)

Rats selectively bred for low aerobic capacity have reduced hepatic mitochondrial oxidative capacity and susceptibility to hepatic steatosis and injury

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65411/1/jphysiol.2009.169060.pd

Mapping Copy Number Variation by Population Scale Genome Sequencing

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.Organismic and Evolutionary Biolog