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  • Accelerating clinical and translational research for biomarker discovery through advanced, standardized cell isolation methodologies

    Contains 1 Component(s) Recorded On: 02/05/2018

    ​Specimens are not meant to live in a freezer. Their sole purpose in life is to produce data. Biorepositories are critical to accelerating clinical and translational research technologies and discoveries. Human subject research depends on the availability of standardized biorepository methods for collection, storage, processing, and distribution of biological specimens alongside associated patient metadata.

    Specimens are not meant to live in a freezer. Their sole purpose in life is to produce data. Biorepositories are critical to accelerating clinical and translational research technologies and discoveries. Human subject research depends on the availability of standardized biorepository methods for collection, storage, processing, and distribution of biological specimens alongside associated patient metadata. Stanford Medicine’s growth across the Bay Area has created an opportunity for us to connect participants to bench-side research in ways never before possible. Our biobank has an emphasis on more advanced sample processing geared at downstream, functional analysis using viable cell suspensions. Work is often performed in tandem with specialized assay groups, such as the Human Immune Monitoring Center (directed by Dr. Holden Maecker) to leverage cutting-edge technologies such as CyTOF, single-cell RNA sequencing, flow cytometry, and immunoassay. Many of these assays require specimen types from humans that have been processed using very specific techniques and methodologies to prevent the introduction of artifacts. In particular, the standardization of the procedures for cell isolation is critical to the success of the translational research; by increasing throughput and minimizing ‘hands-on’ time, applications for biomarker discovery have a chance to be accelerated and reproduced.

    At Stanford Medicine, the lab of Dr. Irving Weissman has coordinated the build out of a unique biorepository dedicated to collection and advanced, standardized processing of tissue and tumors into viable single-cell suspensions. Over the course of the last two years, tumors with match normal tissue have been collected alongside archives of clinical, pathology and surgical notes. In November 2016, Sydney Gordon, a graduate student in Weismann’s lab, discovered the novel increased expression of PD-1 on tumor macrophages in colon cancer of mouse models. Sydney was hoping to translate her findings into humans and did so by taking advantage of Stanford’s biorepository. Within a matter of two months, Sydney was able to repeat her discovery on human samples that had been preserved for functional analysis. She validated her findings of increased PD-1 expression on human colon cancer tumors, opening the door to multiple discussions around new drug therapeutics in cancer immunotherapy. Key to her success was the standardization and care taken to procure and process the solid tissues into single-cell suspensions with minimal impact to the cell surface antigens and cryopreserve the cells viably. Automating parts of this pipeline presents an opportunity to greatly improve the throughput, while also standardizing the methodology away from technician variability.

    Rohit Gupta

    Stanford University

    I began my research at Stanford in 2003 and now oversee harmonization and alignment efforts for biobanking infrastructure, alongside directing the largest research focused, ambulatory care unit at Stanford, the Clinical and Translational Research Unit (CTRU). Both efforts are part of Spectrum’s vision to provide innovative and compliant resources for research services to the research community both locally and globally. Notably, I’ve led and established the biorepository and associated data infrastructure for multiple large-scale biobanks, including those associated with Spectrum, Parker Institute for Cancer Immunotherapy, Stem Cell Institute, Institute for Immunology/Transplantation/Infection, Blood and Bone Marrow Transplant Biobank, Google Baseline, and many more.

  • Leveraging label-free dynamic mass redistribution technology to study G protein-coupled receptor ligand pharmacodynamics

    Contains 1 Component(s) Recorded On: 02/05/2018

    ​Label-free dynamic mass redistribution (DMR) technology represents a powerful approach to studying G protein-coupled receptor (GPCR) signaling in cultured cells.

    Label-free dynamic mass redistribution (DMR) technology represents a powerful approach to studying G protein-coupled receptor (GPCR) signaling in cultured cells. Recently, our laboratory has leveraged DMR to study multiple facets of human adrenergic receptor biology, including:
    1) Deconvoluting the α1D-adrenergic receptor (ADRA1D) PDZ-protein macromolecular complex. Tandem-affinity purification/mass spectrometry identified novel PDZ-protein interactors syntrophin and scribble for the ADRA1D in human cells. DMR assays subsequently revealed syntrophin and scribble differentially enhance agonist efficacy. 2) Investigating the importance of PDZ-ligands for GPCR agonist pharmacodynamics. DMR screens were used to assess the importance of PDZ-protein interactions for agonist pharmacodynamics of 24 human GPCRs containing PDZ-ligands in their distal C-termini. DMR agonist concentration response-curves were generated for full length and PDZ-ligand truncated GPCRs expressed in human cells. 3) Structure-function analysis of GPCR structural domains. SNAP-technology revealed the ADRA1D undergoes constitutive N-terminal domain proteolytic cleavage in human cells. DMR assays indicate this N-terminal cleavage event enhances ADRA1D signaling properties. 4) Identification and pharmacological characterization of endogenous adrenergic receptors in human cancer cell lines. DMR assays examining subtype-selective adrenergic receptor drugs revealed previously undetectable adrenergic receptors in SW480 human colon carcinoma cells. Schild plot analysis with adrenergic receptor subtype-selective antagonists permitted pharmacological characterization of functional adrenergic receptors expressed in SW480 cells. DMR data facilitated subsequent examination of adrenergic receptor stimulation on SW480 cell fate. To conclude, label-free DMR technology is a diverse, powerful tool that can be used to study both transfected and endogenous GPCRs in cultured cells; to deconvolute functional modules of GPCR macromolecular complexes; to address the importance of specific structural domains for GPCR function; and can be combined with traditional analytical methods to facilitate pharmacological characterization of ligand-receptor interactions.  

    Chris Hague

    University of Washington - Pharmacology

    Dr. Hague's research focuses on the molecular pharmacology of G protein-coupled receptors (GPCRs). The Hague lab currently focuses on proteomic identification of GPCR macromolecular complexes in human cells, and molecular deconvolution of functional roles of de novo interacting proteins. The long-term goal of the Hague lab is to identify novel GPCR protein-protein interaction interfaces that have the potential to be targeted by novel small molecules to modulate GPCR function. Dr. Hague received his B.Sc from McMaster University (Hamilton, ON, Canada) in Pharmacology, his Ph.D. from Creighton University (Omaha, NE) in Pharmacology, and performed his post-doctoral fellowship in the Department of Pharmacology at Emory University (Atlanta, GA). Dr. Hague has been a faculty member in the Department of Pharmacology at the University of Washington since 2005.

  • Primary Cell 3D Pancreatic Cancer Organoid Models for Phenotypic High-throughput Therapeutic Screening

    Contains 1 Component(s) Recorded On: 02/05/2018

    ​Pancreatic cancer remains a leading cause of cancer-associated death, with a median survival of ~ 6 months and 5-year survival rate less than 8%. The tumor microenvironment promotes tumor initiation and progression, and is associated to cancer metastasis and drug resistance.

    Pancreatic cancer remains a leading cause of cancer-associated death, with a median survival of ~ 6 months and 5-year survival rate less than 8%. The tumor microenvironment promotes tumor initiation and progression, and is associated to cancer metastasis and drug resistance. Traditional high throughput screening (HTS) assays for drug discovery use lab adapted 2D monolayer cancer cell models, which inadequately recapitulate the physiologic context of cancer. Primary cell 3D cell culture models have recently received renewed recognition not only due to their ability to better mimic the complexity of in vivo tumors but, are now cost effective and efficient. Here we describe phenotypically relevant 3D cell culture in ultra-low-attachment high density 384 and 1536 well plates using a magnetic force-based bioprinting technology. We have validated HTS amenable 2D and 3D spheroid/organoid-based cytotoxicity assays using 4 pancreatic cancer-associated cell lines against 5 known anti-cancer agents, and thereby screened ~3,400 drugs from Approved Drug and National Cancer Institute (NCI) collections. Assay quality was notable with Z’ averaging >0.8 across all assays and cell lines. As anticipated, results from the 3D screen were significantly different from the parallel screen performed on 2D cell monolayers. Collectively, these data indicate that a complex 3D cell culture can be adapted for quantitative HTS and may improve the disease relevance of assays used for therapeutic screening. Further analysis provides a basis for expedited translation into clinical study due to their well-known pharmacology in humans.

    Shurong Hou

    The Scripps Research Institute - FL

    Shurong Hou is a postdoctoral fellow in The Scripps Research Institute Molecular Screening Center, who has dedicated herself to early drug discovery. She is interested in assay development of biochemical and cell-based assays for high throughput screening, especially developing physiologically relevant 3D tumor models for cancer drug discovery.

  • Mining novel CRISPR systems for new genome engineering tools

    Contains 1 Component(s) Recorded On: 02/05/2018

    ​CRISPR systems exist broadly throughout prokaryotic life and constitute an incredible diversity of adaptive immunity mechanisms. Here we present a framework to computationally mine and experimentally characterize novel CRISPR systems for useful bioengineering tools. ​

    CRISPR systems exist broadly throughout prokaryotic life and constitute an incredible diversity of adaptive immunity mechanisms. Here we present a framework to computationally mine and experimentally characterize novel CRISPR systems for useful bioengineering tools.

    Patrick Hsu

    Salk Institute for Biological Studies

    More information coming!

  • Cloud-based qPCR analysis software for rapid-throughput screening of antisense oligonucleotides

    Contains 1 Component(s) Recorded On: 02/05/2018

    Advances in automation, miniaturization and microfluidics have enabled researchers to develop high-throughput qPCR assays that generate thousands of data points per day creating an increased burden on downstream data management, computation and analysis. Here we present a software tool to complement these high-throughput methods by simplifying the computation and visualization of screening results for standard-curve experiments.

    One distinct advantage of the antisense oligonucleotide (ASO) platform has over other therapeutic approaches is the ability to rapidly screen for safe and effective ASO compounds against new molecular targets.  While RNA screening assays will often utilize sequencing technology like RNA-Seq, today, the gold standard for rapid and cost effective gene expression quantification remains quantitative polymerase chain reactions (qPCR).  Advances in automation, miniaturization and microfluidics have enabled researchers to develop high-throughput qPCR assays that generate thousands of data points per day creating an increased burden on downstream data management, computation and analysis.  Here we present a software tool to complement these high-throughput methods by simplifying the computation and visualization of screening results for standard-curve experiments.  Our qPCR pipeline uses modern cloud technology provided by Amazon Web Services to permit users to dynamically generate workflows for a vast array of plate-based qPCR assays.  Our visualization tools make use of the HELM notation (Hierarchical Editing Language for Macromolecules) to both visualize the genomic targets as well as the distinct chemistries employed in SAR screens.

    Donald Milton

    Ionis Pharmaceuticals

    I have over 20 years of biotech software experience in both academic and industry settings.  I began my career at Molecular Simulations Inc (now Biovia) developing bioinformatics tools before the first draft of the Human Genome. After a short diversion in the aviation industry, I returned to bioinformatics as a developer at the Protein Data Bank where I created and managed some notable projects including the ImmersivePDB; highlighted in the book “Advances in Computers” by Marvin Zelkowitz.   I joined the Research Bioinformatics Department at Genentech (gRED) in 2010 and managed multiple software projects for core labs including research pathology and next-generation sequencing.   In 2013 I accepted a position at Ionis Pharmaceuticals where I currently lead software development for the Drug Discovery department.  My current focus is in-silico oligonucleotide drug design and software to support high-throughput in-vitro screening. 

  • A High Throughput Imaging Assay for the Quantification of Gene Expression Dynamics at the Single Cell Level

    Contains 1 Component(s) Recorded On: 02/05/2018

    We will describe the design and implementation of a high-throughput imaging assay consisting of panels of cell lines stably expressing a variety of endogenous genes tagged with MS2-stem loops, automated live-cell confocal microscopy for the long-term visualization of the expression dynamics of these genes at the single allele level, automated image processing for cell and transcription site tracking in time-lapse series, and the generation of gene expression trajectories for hundreds of cells per sample.

    The establishment and maintenance of gene expression programs is essential for cellular differentiation and organism development. For this reason, gene expression is tightly regulated at the level of mRNA transcription, splicing, and translation. Recently, a combination of genetically encoded fluorescent reporters capable of binding and visualizing mRNA transcripts in living cells, such as MS2 stem loops and MS2-GFP, and of image processing techniques to detect, track and measure these transcripts has enabled the characterization of the dynamic regulation of these processes in live cells. We will describe the design and implementation of a high-throughput imaging assay consisting of panels of cell lines stably expressing a variety of endogenous genes tagged with MS2-stem loops, automated live-cell confocal microscopy for the long-term visualization of the expression dynamics of these genes at the single allele level, automated image processing for cell and transcription site tracking in time-lapse series, and the generation of gene expression trajectories for hundreds of cells per sample. Furthermore, we will show practical implementations of this imaging-based assay to measure the transcriptional kinetics of several independently MS2-repeats-tagged genes, and to quantify changes in transcriptional on/off cycles for a glucocorticoid receptor (GR) regulated locus. Overall, the development of this approach opens the possibility of screening focused chemical or oligo siRNA libraries to identify and characterize novel molecular mechanisms regulating gene expression dynamics.

    Gianluca Pegoraro

    National Cancer Institute/NIH

    Gianluca Pegoraro uses high-throughput imaging to identify and characterize molecular mechanisms regulating basic cellular processes such as nuclear architecture, gene expression and the DNA damage response. 

  • High throughput 2D and 3D cell and whole-organism screenings in nanoliter format on Droplet-Microarray platform

    Contains 1 Component(s) Recorded On: 02/05/2018

    Here I will present our latest developments and results on compound screenings on patient-derived leukemia cells, tumor spheroids and embryonic bodies. Droplet-Microarray is universal platform that is compatible with various biological assays including compound screenings and transfection-based assays on different cell types (adherent and suspension cells, stem cells, and primary cells), 3D spheroids, hydrogels and embryos. We believe that this technology will open a new opportunities for high-throughput screenings that were not affordable or possible with other technologies till now.

    Small molecule high-throughput screenings are essential for the fields of drug discovery and toxicology. Hundreds of millions of compounds are screened every year. In these screenings, compounds are tested against molecules (biochemical screens), cells, 3D cellular systems and even whole organisms. Routine screenings in academia and pharma industry are performed in microtiter plates. The main drawbacks of using microplates for large experiments are, first, relatively high volumes and therefore high reagent and cell consumption, and, second, requirement of pipetting robotics. Due to these reasons not every biological laboratory can afford high throughout experiments. Another essential drawback is incompatibility with large screenings of rare but physiologically relevant cells such as patient-derived primary and stem cells due to restricted amount of cell material. We have developed a technology that allows for screenings of cells in 2D and 3D environment and of whole-organism in miniaturized array format. Droplet-Microarray technology is based on patterns of hydrophilic spots separated from each other by superhydrophobic, water repellent, regions. The difference in wettability of spots and borders generates the effect of discontinuous dewetting and enables spontaneous, without pipetting, formation of arrays of separated droplets of nanoliter to microliter volumes trapping live cells and even small animals. In the past years we developed all necessary protocols for culturing cells in 2D and 3D environment, parallel addition of compounds and reagents to individual droplets and performing various phenotypic assays with read-out based on microscopy. Here I will present our latest developments and results on compound screenings on patient-derived leukemia cells, tumor spheroids and embryonic bodies. Droplet-Microarray is universal platform that is compatible with various biological assays including compound screenings and transfection-based assays on different cell types (adherent and suspension cells, stem cells, and primary cells), 3D spheroids, hydrogels and embryos. We believe that this technology will open a new opportunities for high-throughput screenings that were not affordable or possible with other technologies till now.

    Anna Popova

    Institute of Toxicology and Genetics (ITG) , Karlsruhe Institute of Technology

    Dr. Popova graduated from the department of Cell Biology and Immunology of the Faculty of Biology, Lomonosov Moscow State University in Russia. She performed her M.Sc. on “Investigation of polymorphism of latent membrane protein 1 of Epstein-Barr virus” in Institute for Carcinogenesis, Blokhin Cancer Research Center, Moscow. While working on her Master’s Degree she performed part of the project in Institute for Virus Research, Kyoto University, Kyoto, Japan. After graduation Dr. Popova worked as junior scientist in Engelhardt Institute of Molecular Biology, Moscow, Russia on “Mechanisms of termination of protein translation”. During this project she worked as a guest researcher in Institute for Medical Physics and Biophysics, University Hospital Charite, Berlin, Germany. Dr. Popova obtained her Ph.D. in Department of Dermatology and Allergology, University Medical Centre Mannheim, University of Heidelberg, Germany. Since January 2014 Dr. Popova is working as postdoctoral fellow in Institute of Toxicology and Genetics (ITG), Karlsruhe, Germany.

  • Answers to questions not yet asked: Informatics strategy applied to scientific questions

    Contains 1 Component(s) Recorded On: 02/05/2018

    This presentation will describe how we used different technologies to address the specific needs in various scientific domains in a sustainable way. Topics such as software architecture, data integration, visualization will be considered.

    The amount of scientific data generated in current life science research is too large to be analyzed without informatic tools. While automation and new statistical algorithms provide useful tools for the analysis of large amounts of data, one also needs to ask the appropriate scientific questions to pursue discovery. This presentation will describe how we used different technologies to address the specific needs in various scientific domains in a sustainable way. Topics such as software architecture, data integration, visualization will be considered.

    Yohann Potier

    Novartis Institute for Biomedical Research

    Yohann is a Senior Principal Analyst at the Novartis Institute for Biomedical Research. He studied biotechnology and informatics in France followed by a Ph.D. in computational chemistry at the University of Zurich. Yohann joined PerkinElmer Informatics (formerly CambridgeSoft) as a business analyst where he worked with various life sciences customers to deliver informatics solutions. He has been working for the past 3 years at Novartis in the Scientific Information Systems team. During this time, Yohann worked with scientists and engineers from Novartis providing Informatics tools to perform cutting edge biology research in a collaborative open science environment.

  • Something old, something new: Improving genome editing efficiency over CRISPR with a new generation of TALE nucleases

    Contains 1 Component(s) Recorded On: 02/05/2018

    Taking advantage of the ability to design TALENs to target any sequence and the observation that the success of SNP editing is highly influenced by the proximity of the cut to the desired edit site, we also demonstrate that TALENs can facilitate superior HDR editing efficiency compared to Cas9 by being able to position TALENs at the SNP site regardless of the sequence. This is especially relevant in editing genomic regions with a low abundance of PAMs.

    Genome editing has become easier with the advent of CRISPR-Cas9. However, the CRISPR system has the drawback of requiring a sequence motif (PAM) in order to bind and cleave genomic DNA. Attempts to overcome this limitation have been made by developing a suite of orthogonal Cas9s through directed engineering or through isolation of Cas9 variants with novel PAMs. A more universal approach can be achieved by using Transcription Activator-Like Effector Nucleases (TALENs). In the shadows of the CRISPR revolution, TALENs have been engineered to remove their binding site requirement for a 5’ T, thereby removing any specific sequence requirement. In parallel, improved editing has been achieved through delivery of TALEN mRNA via electroporation and we have developed a high throughput assembly method using pre-made RVD libraries which allows rapid production of TALEN mRNA in a day. We demonstrate that when using TALEN mRNA we can achieve high cleavage efficiency in a variety of cells. Taking advantage of the ability to design TALENs to target any sequence and the observation that the success of SNP editing is highly influenced by the proximity of the cut to the desired edit site, we also demonstrate that TALENs can facilitate superior HDR editing efficiency compared to Cas9 by being able to position TALENs at the SNP site regardless of the sequence. This is especially relevant in editing genomic regions with a low abundance of PAMs.

    Jason Potter

    Thermo Fisher Scientific

    Genome Engineering, Protein Engineering, general molecular biology

  • Development of a 3D-High Throughput Assay to Identify Compounds that Block the Growth of Patient Derived Glioma Stem Cells

    Contains 1 Component(s) Recorded On: 02/05/2018

    Glioblastoma (GBM) is the most aggressive primary brain cancer with a recurrence rate of nearly 100% and a 5-year survival rate less than 5%. Recent studies have shown that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be a major contributor to chemotherapy resistance and responsible for relapse disease. Consequently, identifying compounds that modulate GSC proliferation may dramatically improve treatment response.

    Glioblastoma (GBM) is the most aggressive primary brain cancer with a recurrence rate of nearly 100% and a 5-year survival rate less than 5%. Recent studies have shown that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be a major contributor to chemotherapy resistance and responsible for relapse disease. Consequently, identifying compounds that modulate GSC proliferation may dramatically improve treatment response. While high throughput screening (HTS) assays for drug discovery have traditionally used 2D cancer cell models, these monolayer cultures are not representative of tumor complexity. To increase translational relevance three-dimensional (3D) cell culture models have recently received more recognition. Furthermore, patient derived GSCs can be grown as neurospheres and in vivo can functionally recapitulate the heterogeneity of the original tumor. Using patient derived GSC enriched cultures we have developed a 1536-well spheroid-based cytotoxicity assay. In a pilot screening we have tested ~3,400 drugs comprising most Food and Drug Administration (FDA) approved Drugs. This automation-friendly assay yielded an average S/B of 181.3 ± 1.81 and Z’ of 0.77 ± 0.02 demonstrating a robust assay. Importantly, several compounds were identified as potential anti-GBM drugs from this pilot screen, demonstrating the applicability of this assay for large scale HTS. These studies may provide a basis for expedited drug repositioning into a GBM clinical study due to their well characterized pharmacology and safety profile in humans. 

    Victor Quereda

    The Scripps Research Institute

    I am a Senior Research Associate in Dr. Derek Duckett’s laboratory (The Scripps Research Institute, Florida). My studies focus mainly on the biology of cancer and drug discovery fields. As part of a team and using a multidisciplinary approach we are developing novel small molecule inhibitors to block the growth of Glioma Stem Cells, a major contributor to chemotherapy resistance and responsible for relapse disease in Glioblastoma. In collaboration with the Lead Identification Division at Scripps Florida we have generated a 3D-high throughput method to identify novel inhibitors of Glioma Stem Cell growth. This technique together with a battery of other research methods will be used to identify and validate compounds which could be use alone or in combination to maximize their killing effect in Glioblastoma.