Targeted Enrichment and Nanopore Sequencing of RNA Transcripts from Breast Cancer Risk Genes

Aberrant alternative splicing of hereditary breast and ovarian cancer genes has been associated with an increased susceptibility for breast and/or ovarian cancer. However, studying the effects of gene variants on splicing patterns proves to be difficult for classifying Variants of Uncertain Significance (VUS) – variants which lack the evidence to be categorised as benign or pathogenic. This is particularly the case for the BRCA1 gene, which is poorly expressed, with a long transcript length. In this study, a novel RNA enrichment technique using the Oxford Nanopore MinION long-read sequencer was developed and tested to alleviate these limitations. The main hypotheses were that direct RNA sequencing can identify full-length splice isoforms of genes of interest, and that these results can be enhanced using a novel gene enrichment technique. Total RNA was extracted from a control lymphoblastoid cell line and reverse-transcribed into cDNA. Second-strand cDNA synthesis was carried out by incorporating a T7-polymerase binding site attached to a gene-specific primer for the genes of interest. In vitro transcription was carried out with T7-polymerase to enrich the RNA isoforms, which were then sequenced with the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, UK). Several bioinformatic pipelines were evaluated, as bespoke pipelines for analysing this novel dataset were unavailable. The final pipeline consisted of alignment to the Human Genome Reference Build 38 with Minimap2, conversion to BAM files, sorting and indexing with Samtools. The output file was visualised on the Integrative Genomics Viewer, and the reads were manually counted and processed in Excel. The results exhibited evidence of transcript enrichment for three genes of interest (BRCA1, RAD51C, CHEK2) and a control gene (GAPDH). Of note, GAPDH presented isoform abundance ratios which were similar in three separate experiments, indicating potentially quantitative and reproducible linear enrichment. Full-length isoforms of GAPDH, RAD51C and BRCA1 were able to be sequenced with the Direct RNA Sequencing Kit. Additionally, novel isoforms which have not been previously described for RAD51C were detected, which include Δ 3,5-6, Δ 3,7 and Δ 4,7. Novel methodologies and proof-of-principle experiments were executed to enrich and analyse specific transcripts using direct RNA nanopore sequencing. The enrichment method should be further validated with other approaches such as RT-qPCR and optimised for use with relevant genes. Provided that a bespoke pipeline for long direct RNA sequencing becomes available, this novel method may provide a powerful new approach for interpreting the clinical significance of variants of uncertain significance which may impact on splicing patterns

Positron emission tomographic imaging of Copper 64- and Gallium 68-labeled chelator conjugates of the somatostatin agonist Tyr3-octreotate

The bifunctional chelator and radiometal have been shown to have a direct effect on the pharmacokinetics of somatostatin receptor (SSTR)-targeted imaging agents. We evaluated three Y3-TATE analogues conjugated to NOTA-based chelators for radiolabeling with 64 Cu and 68 Ga for small-animal positron emission tomographic/computed tomograhic (PET/CT) imaging. Two commercially available NOTA analogues, p-SCN-Bn-NOTA and NODAGA, were evaluated. The p-SCN-Bn-NOTA analogues were conjugated to Y3- TATE through β-Ala and PEG 8 linkages. The NODAGA chelator was directly conjugated to Y3-TATE. The analogues labeled with 64 Cu or 68 Ga were analyzed in vitro for binding affinity and internalization and in vivo by PET/CT imaging, biodistribution, and Cerenkov imaging ( 68 Ga analogues). We evaluated the effects of the radiometals, chelators, and linkers on the performance of the SSTR subtype 2–targeted imaging agents and also compared them to a previously reported agent, 64 Cu-CB-TE2A-Y3-TATE. We found that the method of conjugation, particularly the length of the linkage between the chelator and the peptide, significantly impacted tumor and nontarget tissue uptake and clearance. Among the 64 Cu- and 68 Ga-labeled NOTA analogues, NODAGA-Y3-TATE had the most optimal in vivo behavior and was comparable to 64 Cu-CB-TE2A-Y3-TATE. An advantage of NODAGA-Y3-TATE is that it allows labeling with 64 Cu and 68 Ga, providing a versatile PET probe for imaging SSTr subtype 2-positive tumors

Coherent mixing of mechanical excitations in nano-optomechanical structures

The combination of the large per-photon optical force and small motional mass achievable in nanocavity optomechanical systems results in strong dynamical back-action between mechanical motion and the cavity light field. In this Article, we study the optical control of mechanical motion within two different nanocavity structures, a zipper nanobeam photonic crystal cavity and a double-microdisk whispering-gallery resonator. The strong optical gradient force within these cavities is shown to introduce significant optical rigidity into the structure, with the dressed mechanical states renormalized into optically bright and optically dark modes of motion. With the addition of internal mechanical coupling between mechanical modes, a form of optically controlled mechanical transparency is demonstrated in analogy to electromagnetically induced transparency of three-level atomic media. Based upon these measurements, a proposal for coherently transferring radio-frequency/microwave signals between the optical field and a long-lived dark mechanical state is described

THE MYTH OF SELF RELIANCE: Economic Lives Inside a Liberian Refugee Camp. Naohiko Omata. New York: Berghahn Books, 2017

THE MYTH OF SELF RELIANCE: Economic Lives Inside a Liberian Refugee Camp. Naohiko Omata. New York: Berghahn Books, 2017. 194 pages, ISBN 9781785335648 (hardback)

Assessing Attitudes Towards Tobacco Advertising in Winooski, VT

Introduction. Smoking rates are 15% in Vermont and higher in low-income populations. Winooski, Vermont is vulnerable to high tobacco use rates given that 23.6% of Winooski residents live below the poverty line. Tobacco advertising, which has been shown to have a direct, dose-dependent association with youth tobacco use, is highly prevalent in stores in Winooski.https://scholarworks.uvm.edu/comphp_gallery/1091/thumbnail.jp

Autophagy Is Required for Glucose Homeostasis and Lung Tumor Maintenance

Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance, and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy related 7 (Atg7), throughout adult mice. Here, we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2 to 3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non–small cell lung cancer (NSCLC) initiation by activation of oncogenic KrasG12D and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with preexisting NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This antitumor activity occurred before destruction of normal tissues, suggesting that acute autophagy inhibition may be therapeutically beneficial in cancer. Significance: We systemically ablated cellular self-cannibalization by autophagy in adult mice and determined that it is dispensable for short-term survival, but required to prevent fatal hypoglycemia and cachexia during fasting, delineating a new role for autophagy in metabolism. Importantly, acute, systemic autophagy ablation was selectively destructive to established tumors compared with normal tissues, thereby providing the preclinical evidence that strategies to inhibit autophagy may be therapeutically advantageous for RAS-driven cancers.Val Skinner FoundationNational Institutes of Health (U.S.) (RC1 CA147961)Rutgers Cancer Institute of New JerseyRutgers Cancer Institute of New Jersey (P30 CA072720)National Institutes of Health (U.S.) (R01 CA163591)National Institutes of Health (U.S.) (R37 CA53370)National Institutes of Health (U.S.) (R01 CA130893

Unifying the Framework of Multi-Layer Network and Visual Analytics

International audienceThe notion of multi-layer networks introduces a general framework and common vocabulary for existing ideas in complex network theory. In doing so, it is possible to understand and compare these dierent ideas in a new and more fruitful manner. However, to make this operationalizable to the visualization and visual analytics community, we need more clarity. For example: What is a layer? What are the semantics of interlayer edges, and specifically, identity links between layers? Can dierent multilayered networks be expressed or implemented in the same way? And vice versa, can one multilayered network be expressed or implemented in dierent ways

Measuring the Invisible Higgs Width at the 7 and 8 TeV LHC

The LHC is well on track toward the discovery or exclusion of a light Standard Model (SM)-like Higgs boson. Such a Higgs has a very small SM width and can easily have large branching fractions to physics beyond the SM, making Higgs decays an excellent opportunity to observe new physics. Decays into collider-invisible particles are particularly interesting as they are theoretically well motivated and relatively clean experimentally. In this work we estimate the potential of the 7 and 8 TeV LHC to observe an invisible Higgs branching fraction. We analyze three channels that can be used to directly study the invisible Higgs branching ratio at the 7 TeV LHC: an invisible Higgs produced in association with (i) a hard jet; (ii) a leptonic Z; and (iii) forward tagging jets. We find that the last channel, where the Higgs is produced via weak boson fusion, is the most sensitive, allowing branching fractions as small as 40% to be probed at 20 inverse fb for masses in the range between 120 and 170 GeV, including in particular the interesting region around 125 GeV. We provide an estimate of the 8 TeV LHC sensitivity to an invisibly-decaying Higgs produced via weak boson fusion and find that the reach is comparable to but not better than the reach at the 7 TeV LHC. We further estimate the discovery potential at the 8 TeV LHC for cases where the Higgs has substantial branching fractions to both visible and invisible final states.Comment: 23 pages, 7 figures. v2: version published in JHEP. 8 TeV analysis adde

A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed