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Collaborative Phenotyping at King's College London: HipSci and the Stem Cell HotelContains 1 Component(s) Recorded On: 02/05/2018
This presentation will review in particular the characterisation of a large panel of human induced pluripotent stem cells, focusing on the integration of high content imaging data with genomics.
We work in the framework of the Human Induced Pluripotent Stem Cells Initiative (HipSci) project, funded by the Wellcome Trust and MRC (www.hipsci.org). Here, we will present in particular the characterisation of a large panel of human induced pluripotent stem cells, focusing on the integration of high content imaging data with genomics. Imaging over 100 human iPS cell lines from healthy donors we have observed evidence for inter-individual variability in cell behaviour. Cells were plated on different concentrations of fibronectin and phenotypic features describing cell morphology, proliferation and adhesion were obtained by high content imaging as in our previously reported method. Furthermore, we have used dimensionality reduction approaches to understand how different extrinsic (fibronectin concentration), intrinsic (cell line or donor) and technical factors affected variation. We have identified with our platform specific RNAs associated with intrinsic or extrinsic factors and single nucleotide variants that account for outlier cell behaviour. We will also mention significant progress in the integration of dynamic imaging data with other datasets. By leveraging the expertise derived on this project, we now provide to internal and external scientists a dedicated laboratory space for collaborative cell phenotyping to study how intrinsic and extrinsic signals impact on human cells to develop assays for disease modeling and drug discovery and to identify new disease mechanisms.
King's College London
Davide Danovi holds an MD from University of Milan and a PhD in Molecular Oncology from the European Institute of Oncology where he demonstrated the causative role of the HdmX protein in human cancer. He completed his postdoctoral training working with Prof. Austin Smith and Dr. Steve Pollard at the University of Cambridge and at University College London where he developed a screening platform to isolate compounds active on human neural stem cells from normal or brain tumour samples. Prior to his current role, he worked as principal scientist at a novel biotechnology company founded to isolate drugs for regenerative medicine using innovative stem cell technologies.
Identification of new negative regulators of ciliogenesis in breast cancer cells through high-throughput siRNA screeningContains 1 Component(s) Recorded On: 02/05/2018
Three-dimensional spheroid assays are considered valid models to recapitulate features of tumors and, combined with new technologies of automated imaging and analysis, will contribute to a better understanding of ciliogenesis and breast cancer and to an important step in anticancer drug research.
Breast cancer is a major cause of death in women in the world. The basal subtypes, also recognized as triple negative breast cancers (TBNC), are the most aggressive type and account for the highest mortality rate in patients. Currently, there are no FDA approved targeted therapies for TNBC, and innovative approaches are necessary to develop new therapeutic options. The primary cilium is a membrane-bound, cell surface projection assembled from centrosomes and singularly expressed in the majority of cells in the human body, serving as a cellular 'antenna' in the recognition and transduction of extra-cellular stimuli, such as growth factors. This organelle forms during cellular quiescence and disassembles when cells enter the cell cycle and proliferate. Interestingly, primary cilia are frequently lost in malignant tumors, such as breast tumors. Thus primary cilia may play a repressive role in regulating cell proliferation and could lower breast cancer development. In order to identify negative regulators of ciliogenesis that could represent target for new drugs, we performed a high content screen using an arrayed library containing pooled siRNAs targeting 23,000 human genes in triplicate on Hs578T cells, a basal B breast cancer cell line which forms cilia at low frequency. Detecting cilia by automated immunofluorescence staining and imaging, we identified 350 candidate genes (~1-2%) that increased the number of ciliated cells. Candidate genes were retested in secondary screens in additional cell lines to distinguish the genes involved in cilia formation common to all cell lines and the ones specific to the (sub)types of (breast) cancer. There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers. In order to test the candidate genes from the 2D cell culture experiments in a tertiary screen to see their effect on tumor growth, migration and invasion, we grew Hs578T cells in ultra-low attachment (ULA) 96-well roundbottomed plates, where tumor cell suspensions formed a three-dimensional structure within 24 h. Three-dimensional spheroid assays are considered valid models to recapitulate features of tumors and, combined with new technologies of automated imaging and analysis, will contribute to a better understanding of ciliogenesis and breast cancer and to an important step in anticancer drug research.
NYU Pelmutter Cancer Institute
Since my Pharmacy studies, I wanted to work in basic research. I did an internship in the Neuropharmacology Center of the Pharmacy University of Milan where I learned basic proteomic research (Mallei A, et al., 2014). During my Master’s degree, I was in charge of the validation of a small scale siRNA screen on ciliogenesis. During my Ph.D., I focused on the characterization of two new Nephronophthisis candidate genes (Failler M et al., 2014). I used high resolution imaging (SIM and STED microscopy) and participated in setting up this imaging platform at our institution (Alby C et al., 2015). I now wish to continue understanding the role of ciliary dysfunction in cancer. Under the supervision of my mentor, I performed high-throughput siRNA screen in a breast cancer cell lines and identified candidate genes that allow cilia growth in these cells.
Integrating high resolution mass spectrometry with cheminformatics for standardized, routine non-targeted metabolomicsContains 1 Component(s) Recorded On: 02/05/2018
Over the past 20 years, metabolomics has evolved into using either multi-targeted assays, usually with nominal mass resolution spectrometers, or non-targeted approaches with high resolution mass spectrometry. We will here show that how to merge targeted approaches with high quality non-targeted discovery metabolomics.
Over the past 20 years, metabolomics has evolved into using either multi-targeted assays, usually with nominal mass resolution spectrometers, or non-targeted approaches with high resolution mass spectrometry. We will here show that how to merge targeted approaches with high quality non-targeted discovery metabolomics. We will highlight the importance of advanced, open access data processing, the proper use of quality controls and internal standards, and full reporting of raw data as well as result data. At the NIH West Coast Metabolomics Center, we use 17 mass spectrometers in the central facility for providing data, informatics services and collaborative research for over 400 projects and more than 25,000 samples per year. These services include commercial assays for plasma analytics, the p180 kit, in addition to steroid, bile acid and oxylipin assays for more than 100 target compounds. Most projects, however, use our three integrated non-targeted metabolomics assays: primary metabolism for up to 200 identified compounds per study using GC-TOF MS, complex lipids for more than 600 identified lipids per study using high resolution liquid chromatography / tandem mass spectrometry and more than 150 identified compounds per study for biogenic amines using hydrophilic interaction chromatography/ high resolution mass spectrometry.
We use standardized data processing in free-access MS-DIAL 2.0 software that is far superior standard solutions with respect to data deconvolution, compound identification and false positive/false negative peak detection. This software is now integrated with MS-FINDER 2.0 software for predicting and annotating spectra of biomarkers with unknown chemical structures. Both programs work excellently for high resolution GC-MS and LC-MS data. In addition, we harness the power of legacy data from more than 2,000 projects we have acquired since 2004 that is available to the biomedical and biological research community at large, the BinVestigate interface to our BinBase metabolome database. We showcase how the integrated use of these resources identified novel epimetabolites in cancer metabolism, both on a prospective cohort scale (in lung cancer) and as new epitranscriptome metabolites from modified RNA molecules (in a range of cancers except for liver cancer).
UC Davis, NIH West Coast Metabolomics Center
Prof. Oliver Fiehn has pioneered developments and applications in metabolomics with over 220 publications to date. He aims at understanding metabolism on a comprehensive level. In order to leverage data from these diverse sets of biological systems, his research laboratory focuses on standardizing metabolomic reports and establishing metabolomic databases, for example the MassBank of North America that hosts over 200,000 public metabolite mass spectra and BinBase, a resource of over 100,000 samples covering more than 2,000 studies. He develops and implements new approaches and technologies in analytical chemistry for covering the metabolome, from increasing peak capacity by ion mobility to compound identifications through cheminformatics workflows and software. He collaborates with a range of investigators in human diseases through statistics, text mining and pathway-based mapping. He studies fundamental biochemical questions from metabolite damage repair to the new concept of epimetabolites.
Supervising the Unsupervised: Maximizing Biological Impact in Cellular ImagingContains 1 Component(s) Recorded On: 02/05/2018
Avoiding “black box” algorithms, instead favouring those which could be interrogated by biological and data scientists alike, led to faster and more relevant analysis cycles, and helped cement a “marriage” between statistical significance and biological relevance. Here, we discuss the analytical methodologies invoked to achieve this.
The exciting challenge of imaging data is the sheer number of options to recognize and retrieve meaningful content; while some turn to the ever-growing algorithmic tool-shed of machine learning, others utilize a priori knowledge of the biology at hand to arrive at the answer. With a balance between these two paramount, we implemented a hybrid workflow to re-analyse compound data in a phenotypic COPD screen. Allowing biological subject matter expertise to guide data-driven decisions, and vice-versa, we used a combination of knowledge-based, supervised, and unsupervised methods to de-convolute patient-derived macrophages into patient-specific subpopulations. At this level of granularity, we could discern previously masked effects of compounds on healthy and diseased cells, both in their physical properties and population makeup. These differences proved to be key when understanding the underlying phenotypic changes. Avoiding “black box” algorithms, instead favouring those which could be interrogated by biological and data scientists alike, led to faster and more relevant analysis cycles, and helped cement a “marriage” between statistical significance and biological relevance. Here, we discuss the analytical methodologies invoked to achieve this.
Degree in mathematics and computational biology from Cambridge University. Started at GSK October 2016.
Developing and Implementing a Scientific Data Strategy for PharmaContains 1 Component(s) Recorded On: 02/05/2018
As the use of predictive modeling, analytics and machine learning increases to address the challenges of declining R&D productivity and increasing pressures for demonstrating product value, a cohesive scientific data strategy and scalable approaches are required to handle the ever increasing variety of data types, data sources, data models and analytics patterns.
The discovery research paradigm requires integration of a broad range of human biology data and knowledge in order to generate and explore diverse hypotheses. Scientists often spend a significant amount of their time and resources in analytics and informatics projects trying to find, access, understand, curate and integrate data. While scientific information is generally managed effectively for its primary use, it often lacks the accessibility and context that facilitates secondary use and cross-functional integration on-demand. As a result, much of the research informatics efforts across the pharmaceutical industry are focused on creating single point solutions to these challenges within a particular problem space or functional area. As the use of predictive modeling, analytics and machine learning increases to address the challenges of declining R&D productivity and increasing pressures for demonstrating product value, a cohesive scientific data strategy and scalable approaches are required to handle the ever increasing variety of data types, data sources, data models and analytics patterns. It also calls for a reevaluation of data access rules, accountability, and data stewardship culture to realize business strategic goals while managing risk.
Nicole is currently a director in Merck's Scientific Information Management organization. She is an epidemiologist by training and began her career in academia conducting large-scale observational research studies before joining Merck. She now leads the Scientific Data Development team at Merck, responsible for defining and executing a data strategy to improve the utility of Merck’s scientific information across the company’s drug development pipeline through data-centric, analytics-focused solutions.
Novel Graphene Field Effect Biosensing Technology for Binding KineticsContains 1 Component(s) Recorded On: 02/05/2018
We introduce a breakthrough electrical label-free biosensor that provides a new approach to measuring binding kinetics. This approach uses a label-free technique called Field Effect Biosensing (FEB) to measure biomolecular interactions.
We introduce a breakthrough electrical label-free biosensor that provides a new approach to measuring binding kinetics. This approach uses a label-free technique called Field Effect Biosensing (FEB) to measure biomolecular interactions. Field effect biosensors use a semiconducting material to monitor changes in binding potential of biomolecules such as proteins, nuceotides, peptides, and small molecules conjugated to the semiconductor surface. Practical use of this technology for biology requires use of a biocompatible semiconductor such as graphene. Graphene is a 2-dimensional sheet of sp2 hybridized carbon that is well known for its excellent electrical conductivity, high surface area, and unique biocompatibility. Basic electronic devices using graphene were first demonstrated in 2004; this work won the Nobel prize in 2010. In nanotechnology labs, graphene biosensors have pushed existing limits of detection for label free sensors and have shown the ability to measure a large range of biochemical interactions from detecting DNA SNPs to small molecules binding to GPCRs.
We will present our architecture and implementation of graphene based FEB biosensors for label free kinetics. In our architecture, FEB measures the current through a graphene biosensor with targets conjugated to the surface and used as a functional active-biology gate dielectric. Any interaction or binding that occurs with the target causes a change in conductance that is monitored in real-time. We will also present data from our recently published research demonstrating sensitivity into the pM range to inflammation markers (IL-6) and Zika viral antigen (ZIKV NS1). High precision measurements of protein kinetics captured using this technology, commercially available as the Agile R100, are comparable to both ELISA and standard label free biomolecule characterization tools. Specifically, we show an improvement in signal-to-noise and in lower limit of detection. These results demonstrate that graphene-based platforms are highly attractive biological sensors for next generation kinetics characterization.
Late Night with LRIG
Accelerating clinical and translational research for biomarker discovery through advanced, standardized cell isolation methodologiesContains 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.
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 pharmacodynamicsContains 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.
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 ScreeningContains 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.
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 toolsContains 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.
Salk Institute for Biological Studies
More information coming!