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Carnegie Mellon University

24 ARTICLES PUBLISHED IN JoVE

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Bioengineering

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly
John M. Szymanski 1, Quentin Jallerat 1, Adam W. Feinberg 1,2
1Department of Biomedical Engineering, Carnegie Mellon University, 2Department of Materials Science and Engineering, Carnegie Mellon University

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

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Biology

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
Amanda L. Soohoo 1, Shanna L. Bowersox 1, Manojkumar A. Puthenveedu 1
1Department of Biological Sciences, Carnegie Mellon University

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

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Biology

A Method for Microinjection of Patiria minata Zygotes
Alys M. Cheatle Jarvela 1, Veronica Hinman 1
1Department of Biological Sciences, Carnegie Mellon University

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

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Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Shenglan Li *1,2, Haipeng Xue *1,2, Bo Long 1,2,5, Li Sun 1,2,6, Tai Truong 1,2,4,7, Ying Liu 1,2,3
1Department of Neurosurgery, The University of Texas Health Science Center at Houston, 2Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 3The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 4Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, 5Department of Anesthesiology, Shengjing Hospital, China Medical University, 6Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 7Biology Department, University of West Georgia

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

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Immunology and Infection

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform
Wenjie Xu 1, Norma V. Solis 2, Scott G. Filler 2, Aaron P. Mitchell 1
1Department of Biological Sciences, Carnegie Mellon University, 2Infectious Diseases, Harbor-UCLA Medical Center

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

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Immunology and Infection

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel
Asako Tajima 1, Wen Liu 2, Isha Pradhan 1, Suzanne Bertera 1, Robert A. Lakomy 1, William A. Rudert 1, Massimo Trucco 1,3, Wilson S. Meng 2, Yong Fan 1,3
1Institute of Cellular Therapeutics, Allegheny Health Network, 2Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, 3Department of Biological Sciences, Carnegie Mellon University

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

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Environment

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency
Nicola Beresford 1, Alice Baynes 1, Rakesh Kanda 1, Matthew R. Mills 2, Karla Arias-Salazar 2, Terrence J. Collins 2, Susan Jobling 1
1Institute of Environment Health and Societies, Brunel University London, 2Department of Chemistry, Carnegie Mellon University

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

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Bioengineering

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials
Keith C. Heyde *1,2, Felicia Y. Scott *3, Sung-Ho Paek 3, Ruihua Zhang 3, Warren C. Ruder 3,4
1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

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Neuroscience

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0
Rashed Harun 1,2,3, Christine M. Grassi 2, Miranda J. Munoz 2,4, Amy K. Wagner 1,2,3
1Center for Neuroscience, University of Pittsburgh, 2Department of Physical Medicine & Rehabilitation, University of Pittsburgh, School of Medicine, 3Safar Center for Resuscitation Research, University of Pittsburgh, 4Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

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Bioengineering

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications
Ming-Cheng Chen *1, John R. Lake *1, Keith C. Heyde 2, Warren C. Ruder 1,2
1Department of Bioengineering, University of Pittsburgh, 2Department of Mechanical Engineering, Carnegie Mellon University

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Neuroscience

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping
Bradford Z. Mahon 1,2, Jeffrey A. Mead 3, Benjamin L Chernoff 2, Maxwell H. Sims 5, Frank E. Garcea 4, Emily Prentiss 5, Raouf Belkhir 2, Sam J. Haber 1, Sarah B. Gannon 5, Steve Erickson 5, Kelly A. Wright 5, Michael Z. Schmidt 5, Audrey Paulzak 1, Vanessa C. Milano 1, David A. Paul 1, Kenneth Foxx 1, Madalina Tivarus 7,8, Jacob W. Nadler 6, Jacqueline M Behr 1, Susan O. Smith 1, Yan Michael Li 1, Kevin Walter 1, Webster H. Pilcher 1
1Department of Neurosurgery, University of Rochester Medical Center, 2Department of Psychology, Carnegie Mellon University, 3Public Relations and Communications, University of Rochester Medical Center, 4MOSS Rehabilitation Research Institute, Cognitive Neuroscience, 5University of Rochester Medical Center, 6Department of Anesthesiology and Perioperative Medicine, University of Rochester Medical Center, 7Department of Imaging Sciences, University of Rochester Medical Center, 8Department of Neuroscience, University of Rochester Medical Center

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

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Medicine

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion
Aleksandra Klimas 1, Octavian Bucur 2, Brigdet Njeri 1, Yongxin Zhao 1
1Department of Biological Sciences, Carnegie Mellon University, 2QPathology

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

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Cancer Research

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane
Moe Ishihara *1, Junhui Hu *1, Xiaoyu Zhang 2, YongHyeon Choi 3, Anthony Wong 4, Celine Cano-Ruiz 5, Rongwei Zhao 6, Ping Tan 7, Jonathan L. Tso 1, Lily Wu 1,8
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Bioengineering, Hanyang University, 4Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 6School of Life Sciences, Beijing Normal University, 7Department of Urology, West China Hospital, Sichuan University, 8Department of Urology, David Geffen School of Medicine, University of California, Los Angeles

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

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Immunology and Infection

Clarifying and Imaging Candida albicans Biofilms
Frederick Lanni 1, Katherine Lagree 1, Manning Y. Huang 1, Lan Yan 2, Carol A. Woolford 1, Aaron P. Mitchell 1
1Department of Biological Sciences, Carnegie Mellon University, 2Department of Pharmacology, Second Military Medical University

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Bioengineering

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures
Sriram Kumar 1, Ying Liu 1, Rebecca E. Taylor 1,2,3
1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Electrical and Computer Engineering, Carnegie Mellon University

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

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Biology

Chemical Dimerization-Induced Protein Condensates on Telomeres
Rongwei Zhao 1, David M. Chenoweth 2, Huaiying Zhang 1
1Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University, 2Department of Chemistry, School of Arts and Sciences, University of Pennsylvania

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.  The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

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Bioengineering

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep
Rei Ukita 1, John W. Stokes 1, W. Kelly Wu 1, Jennifer Talackine 1, Nancy Cardwell 1, Yatrik Patel 1, Clayne Benson 5, Caitlin T. Demarest 1, Erika B. Rosenzweig 3, Keith Cook 2, Emily J. Tsai 4, Matthew Bacchetta 1,6
1Department of Thoracic Surgery, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Pediatrics, Columbia University Medical Center, Columbia University Irving Medical Center, 4Department of Medicine, Columbia University Medical Center, Columbia University Irving Medical Center, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

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Genetics

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform
Chengming Xu *1, Riqing Wei *2, Hui Lin 1, Leiyu Deng 1, Li Wang 3, Deyang Li 4, Honghui Den 5, Wensong Qin 1, Ping Wen 1, Ying Liu 1, Yingsong Wu 2, Qiang Ma 2, Jinliang Duan 1
1Centre for Women, Children, and Reproduction, Guangxi Key Laboratory of Metabolic Diseases Research, 2Department of Biopharmaceutics, School of Laboratory Medicine and Biotechnology, Southern Medical University, 3Department of Obstetrics and Gynecology, Chinese PLA General Hospital, 4Clinical Laboratory, Northern Theater Air Force Hospital, 5Guangzhou Darui Reproduction Technology Co., Ltd.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

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Developmental Biology

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte
Laura Adams *1, Ying Liu *1, Barbara Durrant 2, Elena Ruggeri 2, Carly Young 2, Rebecca Krisher 3, Irina Polejaeva 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2Reproductive Sciences, Beckman Center for Conservation Research, San Diego Zoo Wildlife Alliance, 3Genus PLC

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P < 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

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Engineering

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces
Steven V. Iasella 1, Sourav Barman 1, Clara Ciutara 1, Boxun Huang 1, Michael L. Davidson 2, Joseph A. Zasadzinski 1
1Department of Chemical Engineering and Materials Science, University of Minnesota, 2Department of Chemical Engineering, Carnegie Mellon University

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

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Bioengineering

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System
Utku M. Sönmez 1, Nolan Frey 2, Jonathan S. Minden 2, Philip R. LeDuc 1
1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biological Sciences, Carnegie Mellon University

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

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Biology

Universal Molecular Retention with 11-Fold Expansion Microscopy
Brendan R. Gallagher 1, Aleksandra Klimas 1, Zhangyu Cheng 1, Yongxin Zhao 1
1Department of Biological Sciences, Carnegie Mellon University

Presented here is a new version of expansion microscopy (ExM), Magnify, that is modified for up to 11-fold expansion, conserving a comprehensive array of biomolecule classes, and is compatible with a broad range of tissue types. It enables the interrogation of the nanoscale configuration of biomolecules using conventional diffraction-limited microscopes.

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