O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (

BackgroundPraziquantel (PZQ) is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism) screening with adult worms in vitro and/or animal models of disease—tools that limit automation and throughput with modern microtiter plate-formatted compound libraries.MethodsA partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to ‘reposition’ (re-profile) drugs as anti-schistosomals with potential savings in development timelines and costs.FindingsMultiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of ‘hit’ drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource.ConclusionsTo accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that interfaces schistosomula with microtiter plate-formatted compound libraries. The workflow has identified various compounds and drugs as hits in vitro and leads, with the prescribed oral efficacy, in vivo. Efforts to improve throughput, automation, and rigor of the screening workflow are ongoing.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

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Poster #3732 * expected fold changes measured by orthogonal methods No CNV called in 24 normal samples (in duplicates) CNV specificity = 100% *excluding known polymorphism sites For Research Use Only. Not for use in diagnostic procedures.

Recent studies have highlighted the importance of gene fusions and splice variants in solid tumor profiling1. Next-generation sequencing can be an effective means of detecting these alterations in FFPE samples using RNA rather than DNA, as a single chimeric RNA transcript could result from numerous alterations in DNA2. To that end, Illumina developed TruSight® Tumor 1703, a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes. Along with a DNA workflow, the assay includes a RNA workflow for the identification of splice variants and gene fusions. Following sequencing on the NextSeq® or HiSeq® instruments, TruSight® Tumor 170 offers an analytical pipeline which initiates variant calling. These algorithms were first optimized against the simulated read data from >350 fusions and splice variants reported in the RNA content of the gene panel. A hybrid approach of read alignment and assembly was used to enhance the fusion calling sensitivity. Deliberate filters were designed to reduce false positive calling from sequence homologs, polymerase read-through, or FFPE artifacts. For splice variant calling, a panel of FFPE non-cancerous samples were used to capture false positive mutation calls. With endogenous RNA splicing in cellular physiology, exon-boundary probes were added in the hybrid capture to enhance enrichment efficiency. To the best of our knowledge, there is not yet a standard definition for the limit of detection (LoD) in detecting gene fusions and splice variants from NGS data. We propose to define the LoD of a fusion calling and splice variant NGS panel as the lowest molecule count of a chimeric transcript that could be reliably detected with a sufficient number of supporting sequencing reads. To determine the LoD of TruSight® Tumor 170 using this definition, we mixed cell lines expressing a panel of known fusions and splice variants to measure the copy number of each chimeric transcript. Using these samples we examined the ability of the assay to confidently detect the alterations using 40 ng of RNA input. To demonstrate the analytical sensitivity and specificity of this NGS based assay, we compiled a panel of 49 mixed samples and validated the molecule count to be near the LoD of 5 copies per ng RNA input by PCR. The sensitivity was >98% for fusions and 100% for splice variants. For understanding the limit of blank (LoB) of the assay, another panel of 40 samples not harboring fusions and splice variants was also assessed by TruSight® Tumor 170. These samples demonstrated a ~97% specificity for fusion calling and >95% specificity for splice variant calling. These results indicate that the TruSight® Tumor 170 panel analysis can identify lowly expressed fusions and splice variants from a small amount of compromised RNA from solid tumor samples at high analytical sensitivity and specificity. 1 Klijn et al. (2015) 2 Maher et al. (2009) 3 For Research Use Only. Citation Format: Tingting Du, June Snedecor, Jennifer S. LoCoco, Xiao Chen, Laurel Ball, Allan Castaneda, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Manh Do, Sarah Dumm, Yonmee Han, Mike Havern, Chia-Ling Hsieh, Tingting Jiang, Suzanne Johansen, Scott Lang, Rachel Liang, Jaime McLean, Yousef Nassiri, Austin Purdy, Jason Rostron, Jennifer Silhavy, Natasha Talago, Li Teng, Kevin Wu, Clare Zlatkov, Chen Zhao, Ali Kuraishy, Karen Gutekunst, Sohela De Rozieres, Matthew Friedenberg, Anne C. Jager, Han-Yu Chuang. Analytical performance of TruSight® Tumor 170 in the detection of gene fusions and splice variants using RNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2017-565

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