How to Streamline Your Vector Construction
Dr. Joan Marcano | Northwest Territory Manager at VectorBuilder
Joan Marcano is the Northwest Territory Manager at VectorBuilder, where he focuses his efforts on establishing relationships with customers to help them achieve their research goals. Prior to joining VectorBuilder, Joan was a Postdoctoral Researcher at National Bioenergy Center of the National Renewable Energy Laboratory, Golden CO. In the laboratory, Joan focused on RNA structural biology and the application of RNA regulatory systems for synthetic biology applications. Joan earned his B.S. in Industrial Biotechnology from the University of Puerto Rico-Mayaguez and his Ph.D. in Biochemistry from the University of Colorado at Boulder.
Every day, more labs realize that vectors are just another reagent in their experiments and have opted to outsource their vector construction allowing their researchers to focus on the main goals for their projects. VectorBuilder can be your research partner by providing you the one-stop solution for all of your vector needs. In today’s presentation, I will go over several of the advantages of outsourcing your vector construction to VectorBuilder. First, I will highlight our unique free Vector Design Studio features that allow you to make plasmid designs in an easy (and fun) way while avoiding common mistakes. Then, I will showcase how our platform will help keep accessible all the information about the plasmids including citations in one place. Next, I will present internal data highlighting our experience of making thousands of vectors and using that knowledge to meet your research needs. Finally, I will touch on our most recent research solutions to support your coronavirus research efforts. Please feel free to ask any questions you have about our services, and we will be looking forward to being your research partner.
Immunoassay Innovations: Tools to Advance Immuno-Oncology Research
Dr. Sigrun Badrnya | Manager Product Development at Thermo Fisher Scientific - Biosciences Division, Protein & Cell Analysis
Sigrun Badrnya is Manager in Product Development at the Thermo Fisher Scientific Center of Excellence for Immunoassays in Vienna, Austria. Sigrun received her PhD in vascular biology from the Medical University of Vienna in Austria. She continued as postdoctoral researcher at the Karolinska Institute in Stockholm, Sweden and within an industry-academia joint project for Randox Laboratories UK, where she focused on the
development of multiplex protein biomarker assays. In her current role she is supervising an R&D team to develop immunoassays across diverse platforms including Invitrogen ELISA, ProcartaPlex and ProQuantum.
The evolving research field of immuno-oncology focuses on an individual’s immune system as innovative treatment approaches to combat cancer. The goal of cancer immunotherapies is to initiate a self-sustaining cycleof cancer immunity, overcome tumor evasion mechanisms and promote conditions that favor immune protection. Immune checkpoint molecules have been identified as critical players in the regulation of NK cell-
and T cell-mediated immune responses. Besides intact transmembrane proteins, soluble isoforms and variants of checkpoint molecules function as immune adjuvants or decoy receptors and may also influence clinical efficacy of checkpoint modulator drugs. The systematic analysis of these soluble biomarkers should help to shed light on the regulation of checkpoint pathways and to monitor response to immunotherapeutic treatment. This
presentation will provide an understanding of immunoassay technologies that enable the simultaneous detection of multiple soluble immune stimulatory and inhibitory factors, and a more comprehensive picture of cancer immunity in a blood sample.
pH-Dependent and Dynamic Interactions of Cystatin C with Heparan Sulfate
Xiaoxiao Zhang | Research Assistant at University at Buffalo, Department of Oral Biology
Xiaoxiao is an enthusiastic PhD candidate with expertise in bone biology, glycobiology and structure biology. She attended Shanghai Jiao Tong University from 2006-2014, where she got her Bachelor’s Degree in Dentistry and Master’s Degree in Oral and Maxillofacial Implantology. She is now a PhD candidate in Department of Oral Biology, University at Buffalo. Her research interests are protein-heparan sulfate interactions in inflammation and bone homeostasis. She has published 4 research manuscripts, of which she was listed first-author in three. She has received numerous awards as a graduate student researcher, including “UB Awards for Excellence in Research, Scholarship and Creativity”and“James English Research Award for Ph.D. Students”. In addition, she has received research grants from Mark Diamond Research Fund and the National Natural Science Foundation of China.
Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. Cst-3 is expressed and secreted by nearly all human cells and ubiquitously present in tissues and body fluids. However, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. In this report we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of
extracellular matrix. By using heparin Sepharose chromatography, cell surface HS-binding assay and surface plasma resonance (SPR), we discovered that Cst-3 is a HS-binding protein only at acidic pH (£6.5). Using an HS oligosaccharides microarray, we found that HS hexasaccharide was sufficient to mediate robust Cst-3–HS interaction. By NMR and site-directed mutagenesis, we identified two HS binding regions in Cst-3: the highly
dynamic N-terminal segment (residue 1-12) and a flexible region located between residue 70-94. Among residues important for HS-interaction, His90 is the most likely mediator for pH-dependent HS-binding. Our data demonstrated that the HS-binding site of Cst-3 consisted of two highly dynamic halves, which is unique among known HS- binding proteins. By immunohistochemistry we provided evidence that Cst-3 indeed interacts with
bone matrix HS under low pH environment, highlighting the physiological relevance of our discovery. In addition, we used Cst-3 to inhibit papain in the presence or absence of HS, and results showed that HS-Cst-3 interactions dampen the inhibitory effect of Cst-3.
Retrograde Signaling by a mtDNA-Encoded Non-Coding RNA Preserves Mitochondrial Bioenergetics
Dr. Anna Blumental-Perry | Assistant Professor at University at Buffalo, Department of Biochemistry | Jacobs School of Medicine & Biomedical Sciences
Dr. Blumental-Perry earned a PhD in Genetics and Molecular Biology from the Hebrew University of Jerusalem in Israel. Dr. Blumental-Perry completed her post-doctoral in Cell Biology and Physiology at the University of Pittsburgh. The focus of her research has been to understand proteostasis imbalance in lung disease, with a focus on endoplasmic reticulum and mitochondria malfunction in smokers. Dr. Blumental-Perry concentrated on alveolar epithelial type II cells responses to cigarette smoke, because those cells are local
progenitors with the ability to repair the damage and replenish the loss of alveolar epithelial type I cells.
Authors: BLUMENTAL-PERRY ANNA1, JOBAVA RAUL2, BEDERMAN ILYA2, PERRY NOA2, PRENDERGAST ERIN2, YE ZHI-WEI3, TOWNSEND DANYELLE3, HATZOGLOU MARIA2 AND YARON PERRY1
1State University of New York at Buffalo, NY/USA,
2Case Western Reserve University, OH/USA,
3Medical University of South Carolina, SC/USA,
Introduction: Alveolar epithelial type II (AETII) cells are important for lung epithelium maintenance and function1.
Material & Methods: The study used MLE12, and isolated AETII cells, and mouse Cigarette Smoke (CS) -exposure model; RNAseq and qPCR to identify CS-exposure altered RNAs; FISH and sub-cellular fractionation to study cellular distribution of RNAs; metabolic labeling and seahorse technology to analyze bioenergetics.
Results: AETII cells from mouse lungs exposed to CS increase the levels of the mitochondria-encoded non-coding RNA, mito-ncR-805, generated by the control region of the mitochondrial genome, and stored in a granular-like form in unstressed cells. CS-exposure causes dispersal of the granular form of mito-ncR-805, and its redistribution from mitochondria to the nucleus both in vitro and in vivo. mito-ncR-805 demonstrated protective effects associated with positive regulation of mitochondrial bioenergetics. mito-ncR-805 do not relate to steady-state transcription or replication of the mitochondrial genome. Nuclear mito-ncR-805 levels were correlated with the expression of a subset of nuclear-encoded genes for mitochondrial proteins.
Conclusion: The study reveals an unrecognized mitochondria stress-associated retrograde signaling2,3, and put forward the idea that mito-ncR-805 represents a novel subtype of small non-coding RNAs that are regulated in a tissue- or cell-type specific manner to protect cells under physiological stress4,5.
1 Hogan, B. Stemming Lung Disease? N Engl J Med 378, 2439-2440, doi:10.1056/NEJMcibr1803540 (2018).
2 Kim, K. H., Son, J. M., Benayoun, B. A. & Lee, B. The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress. Cell Metabolism 28, 516-524, doi:10.1016/j.cmet.2018.06.008 (2018).
3 Cardamone, M. D. et al. Mitochondrial Retrograde Signaling in Mammals Is Mediated by the Transcriptional Cofactor GPS2 via Direct Mitochondria-to-Nucleus Translocation. Mol Cell 69, 757-772 e757, doi:10.1016/j.molcel.2018.01.037 (2018).
4 Guo, X. et al. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature, 579, 427-432, doi:10.1038/s41586-020-2078-2 (2020).
5 Fessler, E. et al. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature, 579, 433-437, doi:10.1038/s41586-020-2076-4 (2020).
Single Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells
Soumyaroop Bhattacharya | Senior Associate at University of Rochester, Department of Pediatrics - Neonatology Division
Soumyaroop Bhattacharya is currently working as a Senior Associate in Pediatrics,
in School of Medicine and Dentistry. He has previously served as a Bioinformatician in the Lung Biology Center at the Harvard Medical School. Mr. Bhattacharya has obtained B.S in Agriculture from Banaras Hindu University in India. He followed it with M.S in Biotechnology, Medical Informatics and M.Ed. in Science Education in the United States.
Research: Soumyaroop Bhattacharya is a Senior Associate in Neonatology Division of Pediatrics Department. His research interests include gene expression analysis of lung development and diseases, involving biomarker discovery and algorithm development for data analysis, and he is currently leading multiple projects on Genome-wide expression profiling studies on lung diseases and development. He is also collaborating with investigators in numerous other bioinformatics studies. Mr. Bhattacharya has also been involved in development and maintenance 'The Lung Transcriptome' (http://lungtranscriptome.bwh.harvard.edu), a web resource for pulmonary genomics, which provides expression microarray databases and analytical tools to the lung research community.
Authors: Soumyaroop Bhattacharya1, Jacquelyn Lillis2, Cameron Baker2, Minzhe Guo3, Soula Donolopoulus4, Jason R. Myers2, Gautam Bandopadhyay1, Heidie L. Huyck1, Ravi S. Misra1, Denise Al-Alam4, Yan Xu3, Jeffrey A. Whitsett3, Gloria S. Pryhuber1, Thomas J. Mariani1
1Department of Pediatrics, and 2Genomic Research Center, University of Rochester, 3Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and 4Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA
Rationale: While studies of animal models have defined molecular mechanisms controlling cell diversity during lung morphogenesis, data from late stage human lung development represents a significant knowledge gap. The Lung Molecular Atlas Program (LungMAP) has worked to fill this gap by creating a structural, cellular and molecular atlas for human and mouse lung.
Methods: We used single cell RNA sequencing (RNAseq) to generate transcriptional profiles of 5500 cells obtained from the newborn human lung and available through the LungMAP Human Tissue Center Biorepository. Previously frozen single cell isolates were captured, and library preparation was completed on the Chromium 10X system. Reads generated on a HISeq2500 were aligned to GRCh38. Canonical correlation analysis, clustering, cluster marker gene identification and tSNE representation were performed in Seurat. Marker genes for individual clusters were assessed for cell type and pathway annotations using Toppgene functional analysis tool (ToppFun). To compare human cells with neonatal mouse lung cells, we obtained RNA expression data from 32,000 mouse cells at postnatal day (PN) 1, 3, 7 and 10 generated by the LungMAP Cincinnati Research Center.
Results: Transcriptional analysis of newborn human lung cells identified clusters of closely related cells, consistent with distinct populations of endothelial, epithelial, fibroblasts, pericytes and smooth muscle cells (Figure 1). Epithelial cells were under-represented, but expressed known markers (SFTPC, HOPX, NKX2-1). Multiple, distinct populations of immune cells, including macrophages and lymphocytes (both B and T cells), were identified in the newborn human lung. Signature genes from each of these populations were identified and used to predict regulatory pathways active in each cell population. Neonatal mouse and newborn human lung cells were highly coherent, facilitating the identification of distinct cell populations, including AT1, AT2 and ciliated epithelial cells. Maturation states of human and mouse cells were compared. Human cell populations identified in the newborn lung were similar to those in early postnatal (PN1/3) or alveolar stages (PN7/10) of mouse lung development.
Conclusion: We generated a comprehensive molecular map of the cellular landscape of neonatal human lung using single-cell RNAseq. Ongoing analysis and validation will further refine the cellular landscape to provide a comprehensive characterization of the developing human lung.
The Most Important Mouse in the World - Your Guide to the C57BL/6 Mouse
Dr. Philip Dubé | Field Applications Scientist at Taconic Biosciences
Dr. Philip Dubé is a Field Applications Scientist at Taconic Biosciences. He earned a PhD in Physiology and an Honors B.Sc. in Pharmacology from the University of Toronto and completed postdoctoral fellowships at Vanderbilt University and Children’s Hospital Los Angeles. Dr. Dubé has over sixteen years of experience in the use of rodent models, with extensive knowledge of the application and execution of successful research studies.
The C57BL/6 mouse is the most widely used strain in mouse research, but did you know there is no standard B6 strain? Learning the characteristics and potential of each B6 substrain will inform more effective study design in metabolism, obesity, diabetes, immunology, behavior, and oncology.Understanding the C57BL/6 Mouse In this exclusive webinar, we’ll cover the origins of the C57BL/6 inbred mouse, the applications of its various substrains, and the implications of this variability for study design and the generation of genetically modified mice.
Characteristics and applications of C57BL/6 mice
Using the C57BL/6 as a genetic engineering background
Variations between B6 substrains — and how they affect your results
How to Turbo Charge your Research with CRISPR
Alex Pomerantz | Sr. Account Specialist at Synthego
Alex is the Senior Account Specialist for Synthego supporting the Midwest of the US and Central & East of Canada. He received his Bachelor of Science in Cell and Molecular Biology with a minor in Chemistry from The State University of California, Sacramento. Upon completion of his Bachelor’s in 2011, he worked in a number of lab technician positions, one being at Genentech, performing diagnostic testing in support of patient
monitoring, and drug discovery. He then moved from the lab to sales to support researchers with general lab supplies. In August 2018, Alex joined Synthego to support researchers utilizing the advances in genome engineering enabled by the power of CRISPR.
Join us to learn more about the easiest way to start using CRISPR in your research. See how your lab can use genome engineering to drive your research forward and accelerate discoveries. This webinar will review the process of how to optimize CRISPR genome engineering to create knockout cell lines as well as point mutations, gene tags, and other knockins at your target gene of interest. With an optimized CRISPR strategy you can edit genes not only in immortalized cancer cells but also in physiologically relevant induced pluripotent stem cells and primary cells.