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  • CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

    Contains 1 Component(s) Recorded On: 02/07/2018

    The effects of synthetic compounds on signaling pathways are often evaluated using overexpressed genetic reporters. It is now possible with CRISPR/Cas9 to better preserve native biology by appending reporters directly onto the endogenous genes. For this purpose, we introduced the HiBiT peptide (11 amino acids) as a small reporter tag capable of producing bright and quantitative luminescence through complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT).

    The effects of synthetic compounds on signaling pathways are often evaluated using overexpressed genetic reporters.  It is now possible with CRISPR/Cas9 to better preserve native biology by appending reporters directly onto the endogenous genes.  For this purpose, we introduced the HiBiT peptide (11 amino acids) as a small reporter tag capable of producing bright and quantitative luminescence through complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT).  The small size of HiBiT minimally alters protein structure, while the luminescent assay provides sensitive analysis at very low expression levels.  Using CRISPR/Cas9, we demonstrated that HiBiT can be rapidly and efficiently integrated into the genome to serve as a quantitative tag for endogenous proteins.  Without requiring clonal isolation of the edited cells, we were able to determine changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli.  In combination with fluorescent antibodies, we further used energy transfer from HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation.  These assay methods allowed dynamics in protein abundance and covalent modifications to be assessed within 24-48 hours of introducing synthetic oligonucleotides together with Cas9 into the cells, thus circumventing the prerequisite for molecular cloning.

    Keith Wood

    PROMEGA CORPORATION

    Keith Wood is Head of Research, Advanced Technologies and Senior Research Fellow at Promega Corporation. Widely regarded for his work in bioluminescence, he currently leads a cross‐disciplinary team focused on long‐range innovation in biochemical and cellular research.  His current research centers on the development of bioanalytical capabilities, including novel bioluminescent chemistries, intracellular detection technologies, and efficient isolation methods for protein and drug complexes.  He has authored over 64 journal articles and book chapters and is an inventor for 164 issued and 124 pending U.S. and foreign patents.  Keith received his Ph.D. in Biochemistry at University of California‐San Diego, where he also performed post‐doctoral research before joining Promega in 1990.

  • Raman spectroscopy of isogenic breast cancer cells derived from organ-specific metastases reveals distinct biochemical signatures

    Contains 1 Component(s) Recorded On: 02/07/2018

    Objective characterization of the biomolecular divergences of metastatic lesions, which distinguish them from the primary tumor, remains challenging but is crucial for better understanding of organ-specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cells directly from organ explants that show phenotypic differences from the primary tumor cell line.

    Objective characterization of the biomolecular divergences of metastatic lesions, which distinguish them from the primary tumor, remains challenging but is crucial for better understanding of organ-specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cells directly from organ explants that show phenotypic differences from the primary tumor cell line. Leveraging label-free Raman spectroscopic measurements on these isogenic metastatic breast cancer cells from the brain, spine, lung and liver, we designed decision algorithms to enable accurate differentiation without requiring staining or human interpretation. The Raman spectroscopy-based decision models show significant diagnostic power in resolving these isogenic cell lines by analyzing the nucleic acid, protein, lipid and metabolite content. The latter differences were validated through metabolomic analyses that revealed tissue of origin distinctions between the cell lines. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, and open the door for use of Raman spectroscopy to define organ-specific smart chemotherapeutic approaches.

    Chi Zhang

    Johns Hopkins University

    Mr. Chi Zhang is a Ph.D candidate in Mechanical Engineering, Johns Hopkins University. His current research efforts in Dr. Ishan Barman’s lab are directed towards application of Raman spectroscopy and multivariate data analysis to develop novel quantitative approaches for addressing unmet needs in the biochemical study of cancers. He recent mainly focuses on breast cancer cell biochemical and biomechanical variances diagnosis during metastasis by using Raman spectroscopy and machine learning techniques. He also works on kidney stone identification and otitis media effusion analysis. He has published five papers in journals such as Analytical chemistry and Accounts of Chemical Research. He has been awarded the Whiting School Doctoral Fellowship and Mechanical Engineering Departmental Fellowship by Johns Hopkins University.

  • The Ig Nobel Prizes: The Fine Line Between Sound and Silly Science

    Contains 1 Component(s) Recorded On: 02/07/2018

    The Ig Nobel Prizes are renowned as a spoof alternative to the Nobel Prizes. The annual Ig Nobel awards ceremony is a celebration of curious, imaginative studies that make people laugh. Yet while studies of cats behaving like liquids or frogs levitating inside of a magnet might have you chortling, its founder Marc Abrahams has an equally important purpose in mind for the Prizes: to get you to think.

    The Ig Nobel Prizes are renowned as a spoof alternative to the Nobel Prizes. The annual Ig Nobel awards ceremony is a celebration of curious, imaginative studies that make people laugh. Yet while studies of cats behaving like liquids or frogs levitating inside of a magnet might have you chortling, its founder Marc Abrahams has an equally important purpose in mind for the Prizes: to get you to think. Once something becomes generally understood and accepted, then it comes to be seen as serious and important. Almost everybody either forgets or doesn’t become aware that this thing started out as something that everyone else regarded as nuts! Any scientific discovery seems like such an easy thing after it has been discovered, and it almost never was. Your understanding of a study might change – drastically – if you spend time looking at its details!

    Marc Abrahams

    Co-founder/Editer, Annals of Improbable Research

    Marc Abrahams writes about research that makes people LAUGH, then THINK. Marc founded Ig Nobel Prize Ceremony in 1991, and serves as its Master of Ceremonies. He co-founded and edits the magazine Annals of Improbable Research (AIR), hosts the Improbable Research weekly podcast (distributed by CBS), and wrote This is Improbable, The Ig Nobel Prizes, and other books. He edits and writes much of the web site and blog www.improbable.com, and the monthly newsletter mini-AIR.

  • A 3D High-Content Screening assay as in vitro model to study polycystic kidney disease

    Contains 1 Component(s) Recorded On: 02/06/2018

    ​Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either the Pkd1 or Pkd2 gene. The most important characteristic of this disease is the formation of cysts in the kidney, which reduces renal function and will lead to end stage renal disease.

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either the Pkd1 or Pkd2 gene. The most important characteristic of this disease is the formation of cysts in the kidney, which reduces renal function and will lead to end stage renal disease.  Although it is known that ADPKD is caused by mutations in the Pkd1 or Pkd2 gene, it is not yet understood why this mutation leads to cyst formation.  Since cysts cannot form in conventional in vitro 2D cell culture, current research on ADPKD relies heavily on the use of animal models. The lack of proper in vitro models makes the study of this disease all the more challenging.  To address this, we developed a 3D high-content in vitro screening assay usable for mechanistic studies as well as target discovery in ADPKD. This culture system uses kidney collecting duct Pkd1 KO cells, which spontaneously form small cysts when cultured in our 3D hydrogel.  In the presence of the test compounds, cAMP inducer Forskolin is added to stimulate the cyst swelling. To examine the effect of the compounds on the swelling, cysts are fixed, stained and imaged. The 3D image stacks are analyzed with our OminerTM image analysis software, capable of measuring many phenotypic characteristics, including cyst size, nucleus shape and thickness of cyst wall. This also enables us to identify compounds that are effective and do not influence cell viability, and discard compounds which have undesired therapeutic profiles. These methods are optimized for the use of lab automation, capable of testing large compound libraries in a single experiment. To follow up on previously presented work (Booij et al, SLAS Discovery, 2017) , we screened a collection of 2320 natural products and bioactive compounds. Multiple hit compounds were identified and validated in vitro. Based on the phenotypic profile, we then selected  several of these hit compounds for in vivo validation in mouse models. One of these compounds proved effective in reducing cyst progression and collagen deposition in a dose-dependent manner.

    In these experiments, we show that this 3D in vitro screening model can be used to select compounds that have the desired phenotypic profile, which was validated in vivo. These results prove the applicability and reliability of this model in Drug Discovery for ADPKD.

    Hester Bange

    Leiden University

    Hester graduated in 2016 as MSc in Bio-Pharmaceutical Sciences from Leiden University (8.5/10 average). Following very successful successful Master internship at the division of Toxicology at the Leiden Academic Centre for Drug Research, Hester started her PhD at Leiden University spin-off company OcellO BV. in September 2016 on a collaborative project with Leiden University and the Leiden University Medical Centre. Het PhD research is titled "3D Models for Cystopathies - the missing link in translational Medicine", and focuses on the development on high content 3D in vitro screening models for diseases such as polycystic kidney disease and cystic fibrosis. 

  • New approaches for single cell genome sequencing and mutation analysis

    Contains 1 Component(s) Recorded On: 02/06/2018

    This presentation reviews two new methods for single-cell genome analysis, one that requires no microfluidics or specialized equipment for direct single-cell genome amplification and another that leverages culture-based amplification rather than biochemical amplification to enable studies of de novo mutations in single cells.​

    Microfluidics and whole-genome amplification are enabling single-cell genomic analyses.  At the same time, these technologies limit single-cell genomic studies by imposing cost and complexity (microfluidics) and degrading data quality (whole-genome amplification). Here I will present two new methods for single-cell genome analysis, one that requires no microfluidics or specialized equipment for direct single-cell genome amplification and another that leverages culture-based amplification rather than biochemical amplification to enable studies of de novo mutations in single cells.

    Paul Blainey

    MIT Department of Biological Engineering and Broad Institute of MIT and Harvard

    Dr. Blainey trained in mathematics, chemistry, biophysics, microfluidics, and genomics before joining the Broad Institute and the Department of Biological Engineering at MIT as a faculty member in 2012. Dr. Blainey’s laboratory integrates microfluidic, molecular, and imaging tools to address new challenges in single-cell analysis, genomic screening, and therapeutics development.

  • Development Of 3-Dimensional Human Cortical Spheroid Platforms With High Homogeneity And Functionality For High Throughput And High Content Screening

    Contains 1 Component(s) Recorded On: 02/06/2018

    Here we describe the development of a highly homogenous human induced Pluripotent Stem ell (hiPSC)-derived cortical spheroid screening platform in 384 well format, composed of cortical neurons and astrocytes. Immunofluorescence analysis indicated that these derived neurons and astrocytes display key markers of cellular identity as well as maturity, such as synaptic proteins and glutamate transporters.

    The human cerebral cortex is organized in a complex 3-dimensional (3D) structure comprising different neural cell types. The coordinated work of these different cell types is key for brain function and homeostasis. Recently, much work has been focused on obtaining 3D brain organoids in an attempt to better recapitulate the brain development/function in vitro. However, current protocols may lead to variable organoid size and function, making the use of these powerful tools impractical in a drug screening scenario. Here we describe the development of a highly homogenous human induced Pluripotent Stem ell (hiPSC)-derived cortical spheroid screening platform in 384 well format, composed of cortical neurons and astrocytes. Immunofluorescence analysis indicated that these derived neurons and astrocytes display key markers of cellular identity as well as maturity, such as synaptic proteins and glutamate transporters. Viability assays carried out with compounds with known mechanism of action indicated scaleability and feasibility of the assays, with results comparable to a standard 2D model employing the same culture composition. Kinetic, high trhoughput calcium flux analysis performed in a in a Fluorometric Imaging Plate Reader (FLIPR) highlighted that the spheroids present quantifiable, robust and uniform spontaneous calcium oscillations. The calcium signal was modified with excitatory and inhibitory modulators coherently and in a highly reproducible fashion, confirming the presence of a functionally integrated glutamatergic/GABAergic circuit. High speed confocal imaging confirmed homogenous calcium oscillations at the cellular level, whereas multielectrode array (MEA) analysis demonstrated robust synchronous neurophysiological activity at the network level. Additionally, these cortical organoids are amenable to immunostaining in suspension, enabling scalable high content image-based assays focused on key protein markers. Altogether, the developed 3D cortical spheroid platform can be easily implemented as a reliable high throughput screening platform to investigate complex cortical phenotypes in vitro, as a reliable high-throughput screening platform for toxicology studies, disease modeling and drug testing.

    Cassiano Carromeu

    StemoniX

    Experienced Neuroscientist with expertise in the use of human induced pluripotent stem cells (hiPSCs) for safety pharmacology, toxicology, drug screening and for modeling of neurodevelopmental disorders.

  • DNA-encoded library screening on a GPCR: identification of agonists and antagonist to protease-activated receptor 2 (PAR2) with novel and diverse mechanisms of action.

    Contains 1 Component(s) Recorded On: 02/06/2018

    Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling.

    DNA-encoded library screening on a GPCR: identification of agonists and antagonist to protease-activated receptor 2 (PAR2) with novel and diverse mechanisms of action. Niek Dekker1 AstraZeneca, Innovative Medicines Biotech Unit, Gothenburg, Mölndal SE-431 83, Sweden Protease-activated receptor-2 (PAR2) is irreversibly activated by proteolytic cleavage of the N-terminus which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain eliciting a cellular cascade in response to inflammatory signals and other stimuli. PAR2 is implicated in a wide range of inflammatory and other diseases including cancer. Activation of PAR2 on sensory neurons leads to hyperphophorylation of TRP channels resulting in pain and hyperalgesia. The discovery of small molecule antagonists to PAR2 has proven challenging. DNA-encoded library (DEL) screening on purified PAR2 delivered both antagonists and agonists, exemplified by AZ3451 (SLIGRL PAR2  IP-one IC50 = 23 nM) and AZ8838 (SLIGRL PAR2 IP-one IC50 = 1500 nM), and agonist AZ2429 (EC50 of 53 nM in IP-one). Crystal structures of antagonist bound to the GPCR revealed that AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling.  AZ3451 and AZ8838 were tested in a rat model of PAR2-induced oedema using 2fLIGRL-NH2 (350 µg/paw in 100 µL and trypsin (20 µg/ paw in 100 µL). At a 10 mg/kg dose, both compounds exhibited reduction of paw swelling in both in vivo models. These results confirm that at least two allosteric sites exist on the PAR2 receptor and can be blocked resulting in reversal of in vitro and in vivo PAR2 mediated signaling. DEL screening on purified PAR2 combined with crystallography provided a basis for the development of selective PAR2 antagonists for a range of therapeutic indications.

    co-authors: Dean G. Brown1, Giles A. Brown2, Robert K.Y. Cheng2, Matt Clark3,Miles S. Congreve2, Robert Cooke2, John Cuozzo3, Andrew S. Doré2, Christoph Dumelin3, Karl Edman1, Rink-Jan Lohman4, Yuhong Jiang4, David P. Fairlie4, Cedric Fiez-Vandal2, Stefan Geschwinder1, Christoph Grebner1, Marie-Aude Guie3, Nils-Olov Hermansson1, Ali Jazayeri2, Patrik Johansson1, Anthony Keefe3, Rudi Prihandoko2, Mathieu Rappas2, Oliver Schlenker2, Eric Sigel3, Arjan Snijder1, Holly Souter3, Linda Sundström1, Benjamin Tehan2, Barry Teobald2, Peter Thornton1Dawn Troast3, Giselle Wiggin2, Ying Zhang3, Andrei Zhukov2 and Fiona H. Marshall2

    1AstraZeneca, Innovative Medicines Biotech Unit, Gothenburg, Mölndal SE-431 83, Sweden
    2Heptares Therapeutics Ltd, Biopark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX, UK
    3X-Chem Inc., 100 Beaver St. Waltham MA 02453
    4Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia

    Niek Dekker

    AstraZeneca

    Strong delivery- and collaboration-focus with experience working in matrix organizations. Science-driven with excellent background in lead-discovery technologies. Worked in large number of early discovery projects (small molecule and biologics), both supporting projects and leading capability projects. Strong team and people skills from a number of years of line management responsibility. Experienced working with external academic and biotech partners on new technologies and with contract research organizations (outsourcing). Portfolio management skills. Excellent leadership skills from working in academia and pharma industries, training and from a range of different roles.
     
    Professional career
    2012-present   Principal scientist in Reagents & Assay Development, Discovery Sciences;
    2008-2012       Delivery Leader CNSP iMed, Cell, Protein & Structural Sciences, Discovery Enabling Capabilities and Sciences, Mölndal, AstraZeneca;
    2004-2008        Associate Director Protein Engineering Section, Structural Chemistry Laboratories, Mölndal, AstraZeneca;
    2000-2004        Team Leader Protein Engineering, Structural Chemistry Laboratories-Mölndal, AstraZeneca, Sweden.;
    1994-2000        Assistant Professor Utrecht University, the Netherlands.

  • Highthroughput Binder Confirmation (HTBC): A new non-combinatorial synthesis platform created to enhance and accelerate hit ID.

    Contains 1 Component(s) Recorded On: 02/06/2018

    Encoded Library Technology (ELT) is a hit identification platform that uses ultra-large collections of chemically diverse DNA-encoded small molecule libraries selected for affinity against a therapeutically relevant target. Recent advances in ELT libraries, library pooling strategies, selections and DNA sequencing have vastly increased the number of actionable chemotypes produced for a given selection campaign.

    Highthroughput Binder Confirmation (HTBC): A new non-combinatorial synthesis platform created to enhance and accelerate hit ID.
    Joseph Franklin, Xiaopeng Bai, Lijun Fan, Kenneth Lind, Heather O’Keefe, Eric Shi, Jennifer Summerfield, Jerry Yap & Jeffrey Messer; NCE Molecular Discovery - GSK

    Encoded Library Technology (ELT) is a hit identification platform that uses ultra-large collections of chemically diverse DNA-encoded small molecule libraries selected for affinity against a therapeutically relevant target. Recent advances in ELT libraries, library pooling strategies, selections and DNA sequencing have vastly increased the number of actionable chemotypes produced for a given selection campaign. In practice, only a small fraction of these chemotypes are synthesized as discrete molecules without the encoding DNA using traditional organic synthesis. To address this bottleneck we developed an automated microscale parallel synthesis platform that uses double stranded DNA with a cleavable linker as a chemical handle. This High Throughput Binder Confirmation (HTBC) platform uses the original DNA-Encoded Library (DEL) chemistry and will recapitulate the products, side-products and intermediates produced in the original library synthesis. The resulting compounds are cleaved from the DNA support and are screened as small molecule mixtures by Affinity Selection Mass Spectrometry. The platform is capable of assessing target engagement for hundreds of compounds per month and is used at GSK to prioritize synthesis decisions for more traditional scale organic synthesis.

    Joe Franklin

    GlaxoSmithKline

    DNA Encoded Library on-DNA chemistry 

  • Combining CRISPR/Cas9 screening with custom engineered reporter cell lines to identify genes required for tubulin formation

    Contains 1 Component(s) Recorded On: 02/06/2018

    This case-study describes an effective methodology to combine multi-pronged gene-editing with phenotypic screening to enrich our knowledge of gene and molecular interactions in complex biological systems. Further, with an expanded array of reporter cell lines at the researcher’s disposal, this type of strategy can be adjusted to dissect many other relevant pathways and phenotypes.

    Phenotypic high-throughput / high content screens have become popular tools for elucidating molecular and genetic pathways in biological systems.  Phenomics, or high-dimensional biology, incorporates screening methods that can enable many parameters to be tested in concert under similar or identical conditions, providing a potential wealth of information about a specific biological process.  Here we describe the use of a gene-edited reporter cell line, U2OS LMNB1-TUBA1B-ACTB (Sigma-Aldrich CLL1218), to phenotypically detect genes responsible for tubulin formation.  CLL1218 was transduced with CAS9 Blasticidin Lentiviral Particles (Sigma-Aldrich LVCAS9BST) and selected. Following selection, the pool was cloned, and derived clones were then screened for CRISPR/Cas9 activity using a known active gRNA.  Preferred clones were expanded and banked to be used in a semi-automated high-throughput CRISPR library screens to identify modulators of tubulin expression, formation, and distribution.  Proof-of-concept was demonstrated using a set of CRISPR guides specific for vimentin.  Creation of a vimentin knock-out in the CLL1218-Cas9 reporter line alters cell morphology that can be visually detected  on a variety of imagining platforms, including high-content instruments.  This case-study describes an effective methodology to combine multi-pronged gene-editing with phenotypic screening to enrich our knowledge of gene and molecular interactions in complex biological systems. Further, with an expanded array of reporter cell lines at the researcher’s disposal, this type of strategy can be adjusted to dissect many other relevant pathways and phenotypes.

    Mark Gerber

    MilliporeSigma

    Mark joined Sigma-Aldrich in 2006, and has worked in the areas of biotherapeutic production, stem cell applications and gene regulation. In 2014, Mark was recruited to lead the Cell Design Studio team in the engineering of custom cell lines utilizing ZFN, CRISPR and shRNA technologies. Mark obtained his Ph.D. in Biochemistry and Molecular Biology from Saint Louis University School of Medicine where he used RNAi in Drosophila models to elucidate developmental and biochemical roles for RNA polymerase II-associated transcription factors. Following graduate school, Mark served as a Postdoctoral Fellow at Washington University in St. Louis where he investigated signalling pathways involved in the development of human meningioma. 

  • Microfluidics and Commercial Success? Experience and examples of the last 16 years.

    Contains 1 Component(s) Recorded On: 02/06/2018

    The presenter will try to help the audience to understand the factors that have shaped successes and failures to ensure that future ventures try to avoid the pitfalls of the past.

    The presenter has built a group of companies over the last 16 years which exploit Microfluidics in a wide range of ways. Using Microfluidics to make products such as:
    - Pioneering the field of flow chemistry
    - microfluidic components
    - microfluidic 3d printing
    - particle engineering
    - Single cell Biology
    During this time, the Dolomite part of the group was also a leading consulting team in microfluidics, and attacked challenges in very varied application areas from the oil industry, through food, pharmaceuticals and diagnostics to name a few. As a result of this history the presenter has extremely diverse knowledge and experience of the successes and failures of exploitation of microfluidics, as well as understanding why these outcomes were arrived at. This coupled with the presenter having been an Editorial board member of the RSC Journal "Lab on a Chip" since 2012 gives yet another insight from the academic perspective. The presenter will try to help the audience to understand the factors that have shaped successes and failures to ensure that future ventures try to avoid the pitfalls of the past.

    Mark Gilligan

    Blacktrace Holdings Ltd

    https://www.linkedin.com/in/ma...