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20 ARTICLES PUBLISHED IN JoVE

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Biology

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech
Orit Shefi 1, Claire Simonnet 2, Alex Groisman 2, Eduardo R Macagno 1
1Division of Biological Sciences, University of California San Diego - UCSD, 2Department of Physics, University of California San Diego - UCSD

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

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Biology

Measuring the Kinetics of mRNA Transcription in Single Living Cells
Yehuda Brody 1, Yaron Shav-Tal 1
1The Mina and Everard Goodman Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

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Bioengineering

High-throughput Protein Expression Generator Using a Microfluidic Platform
Yair Glick *1, Dorit Avrahami *1, Efrat Michaely 1, Doron Gerber 1
1The Mina & Everard Goodman Faculty of Life Sciences, The Nanotechnology Institute, Bar-Ilan University

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

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Immunology and Infection

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms
Yana Reznick 1, Ehud Banin 2, Anat Lipovsky 3, Rachel Lubart 3, Pazit Polak 4, Zeev Zalevsky 4
1Department of Physics, Bar-Ilan University, 2The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 3Department of Chemistry, Bar-Ilan University, 4Faculty of Engineering, Bar-Ilan University

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

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JoVE Journal

Real-time Live Imaging of T-cell Signaling Complex Formation
Elad Noy *1, Maor H. Pauker *1, Mira Barda-Saad 1
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

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Bioengineering

Designing a Bio-responsive Robot from DNA Origami
Eldad Ben-Ishay 1, Almogit Abu-Horowitz 1, Ido Bachelet 1
1Faculty of Life Sciences and the Institute for Nanotechnology & Advanced Materials, Bar-Ilan University

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

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Engineering

Time Multiplexing Super Resolving Technique for Imaging from a Moving Platform
Asaf Ilovitsh 1, Shlomo Zach 2, Zeev Zalevsky 1
1Faculty of Engineering, Bar-Ilan University, 210 Nachum Street, Kfar Saba, Israel

A method for overcoming the optical diffraction limit is presented. The method includes a two-step process: optical phase retrieval using iterative Gerchberg-Saxton algorithm, and imaging system shifting followed by repetition of the first step. A synthetically increased lens aperture is generated along the direction of movement, yielding higher imaging resolution.

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JoVE Journal

Folding and Characterization of a Bio-responsive Robot from DNA Origami
Yaniv Amir 1, Almogit Abu-Horowitz 1, Ido Bachelet 1
1Faculty of Life Sciences and the Institute for Nanotechnology & Advanced Materials, Bar-Ilan University

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

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Chemistry

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation
Nir Qvit 1, Opher S. Kornfeld 1
1Department of Chemical and Systems Biology, Stanford University School of Medicine

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

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Neuroscience

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster
Shir Zer 1, Julia Ryvkin 1, Harel J. Wilner 1, Hila Zak 1, Anat Shmueli 1, Galit Shohat-Ophir 1
1The Mina & Everard Goodman Faculty of Life Sciences, The Leslie and Susan Gonda Multidisciplinary Brain Research Center, Bar-Ilan University

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

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Engineering

Scanning SQUID Study of Vortex Manipulation by Local Contact
Eylon Persky 1, Anna Kremen 1, Shai Wissberg 1, Yishai Shperber 1, Beena Kalisky 1
1Department of Physics and Institute of Nanotechnology and Advanced Materials, Bar-Ilan University

We present a protocol for manipulation of individual vortices in thin superconducting films, using local mechanical contact. The method does not include applying current, magnetic field or additional fabrication steps.

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Genetics

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq
Ivana Grbesa 1, Miriam Tannenbaum 1, Avital Sarusi-Portuguez 1, Michal Schwartz 1, Ofir Hakim 1
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

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Cancer Research

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates
Orit Ravid-Hermesh 1, Naomi Zurgil 1, Yana Shafran 1, Elena Afrimzon 1, Maria Sobolev 1, Yaron Hakuk 1, Zehavit Bar-On Eizig 1, Mordechai Deutsch 1
1Physics Department, Bar-Ilan University

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

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Bioengineering

Designing Porous Silicon Films as Carriers of Nerve Growth Factor
Michal Rosenberg 1, Neta Zilony 2,3, Orit Shefi 2,3, Ester Segal 1,4
1Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, 2Faculty of Engineering, Bar-Ilan University, 3Bar-Ilan Institute of Nanotechnologies and Advanced Materials, 4Russell Berrie Nanotechnology Institute, Technion - Israel Institute of Technology

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

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Cancer Research

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients
Vikrant Palande 1, Dorith Raviv Shay 1, MILANA Frenkel-Morgenstern 1,2,3
1Cancer Genomics and BioComputing of Complex Diseases laboratory, The Azrieli Faculty of Medicine, Bar-Ilan University, 2The Data Science Institute, Bar-Ilan University, 3The Dangoor Centre For Personalized Medicine, Bar-Ilan University

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

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Behavior

Generating Acute and Chronic Experimental Models of Motor Tic Expression in Rats
Esther Vinner *1, Katya Belelovsky *1, Izhar Bar-Gad 1
1The Leslie & Susan Goldschmied (Gonda) Multidisciplinary Brain Research Center, Bar-Ilan University

We present protocols for generating acute and chronic experimental models of tic expression in freely behaving rats. The models are based on striatal cannula implantation and subsequent GABAA antagonist application. The acute model uses transient injections whereas the chronic model utilizes prolonged infusions via a subcutaneous implanted mini-osmotic pump.

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Bioengineering

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons
Ganit Indech 1,4, Reut Plen 2,4, Dafna Levenberg 2,4, Naor Vardi 1,4, Michal Marcus 2,4, Alejandra Smith 2,4, Shlomo Margel 3,4, Orit Shefi 2,4, Amos Sharoni 1,4
1Department of Physics, Bar-Ilan University, 2Faculty of Engineering, Bar-Ilan University, 3Department of chemistry, Bar-Ilan University, 4The Institutes of Nanotechnology & Advanced Materials, Bar-Ilan University

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

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Immunology and Infection

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions
Shalhevet Azriel *1,2, Hadar Bootz *1,2, Alon Shemesh *1,2, Sivan Amidror 1,2, Nissan Yissachar 1,2
1The Goodman Faculty of Life Sciences, Bar-Ilan University, 2Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

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Biology

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy
Nikola Lukic *1, Trishna Saha *1, Stefanie Lapetina 1, Michal Gendler 1, Gilad Lehmann 1, Anthony J. Koleske 2,3, Hava Gil-Henn 1
1The Azrieli Faculty of Medicine, Bar-Ilan University, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Neuroscience, Yale University

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

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Biochemistry

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology
Yana Lerner *1, Surya Sukumaran *1, Mei-Sze Chua 2, Samuel K. So 2, Nir Qvit 1
1The Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, 2Asian Liver Center, Department of Surgery, Stanford University School of Medicine

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

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