King Abdullah University of Science and Technology (KAUST)

10 ARTICLES PUBLISHED IN JoVE

Chemistry

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

Satoshi Habuchi¹, Takuya Yamamoto^2,3, Yasuyuki Tezuka³

¹Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), ²Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, ³Department of Organic and Polymeric Materials, Tokyo Institute of Technology

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

Biology

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Francesco Castagnetti^1,2, Elisabetta Fiacco³, Carol Imbriano⁴, Lucia Latella^1,2

¹Department of Medicine, Institute of Translational Pharmacology, Italian National Research Council, ²Epigenetics and Regenerative Medicine, IRCCS Fondazione Santa Lucia, ³Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), ⁴Department of Life Sciences, University of Modena and Reggio Emilia

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Chemistry

Imine Metathesis by Silica-Supported Catalysts Using the Methodology of Surface Organometallic Chemistry

Maha A. Aljuhani¹, Jérémie D.A. Pelletier¹, Jean-Marie Basset¹

¹KAUST Catalysis Center, Division of Physical Sciences and Engineering, King Abdullah University of Science and Technology (KAUST)

A new group IV metal catalyst for imine metathesis is prepared by grafting amine metal complex onto dehydroxylated silica. Surface metal fragments are characterized using FT-IR, elemental microanalysis, and solid-state NMR spectroscopy. Further dynamic nuclear polarization surface enhanced NMR spectroscopy experiments complement the determination of the coordination sphere.

Genetics

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Mohamed A. Sobhy^*1, Amer Bralić^*1, Vlad-Stefan Raducanu¹, Muhammad Tehseen¹, Yujing Ouyang¹, Masateru Takahashi¹, Fahad Rashid¹, Manal S. Zaher¹, Samir M. Hamdan¹

¹Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST)

Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Engineering

Rendering SiO₂/Si Surfaces Omniphobic by Carving Gas-Entrapping Microtextures Comprising Reentrant and Doubly Reentrant Cavities or Pillars

Sankara Arunachalam¹, Eddy M. Domingues¹, Ratul Das¹, Jamilya Nauruzbayeva¹, Ulrich Buttner², Ahad Syed², Himanshu Mishra¹

¹Water Desalination and Reuse Center (WDRC), Biological and Environmental Science and Engineering (BESE) Division, King Abdullah University of Science and Technology (KAUST), ²Core Labs, King Abdullah University of Science and Technology (KAUST)

This work presents microfabrication protocols for achieving cavities and pillars with reentrant and doubly reentrant profiles on SiO₂/Si wafers using photolithography and dry etching. Resulting microtextured surfaces demonstrate remarkable liquid repellence, characterized by robust long-term entrapment of air under wetting liquids, despite the intrinsic wettability of silica.

Engineering

Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO₂/Si/SiO₂ Wafers for Green Desalination

Ratul Das¹, Sankara Arunachalam¹, Zain Ahmad¹, Edelberto Manalastas¹, Ahad Syed², Ulrich Buttner², Himanshu Mishra¹

Presented here is a stepwise protocol for realizing gas-entrapping membranes (GEMs) from SiO₂/Si wafers using integrated circuit microfabrication technology. When silica-GEMs are immersed in water, the intrusion of water is prevented, despite the water-loving composition of silica.

Biology

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Pedro M. Cardoso¹, Ahmed A. Alsaggaf¹, Helena M. Villela¹, Raquel S. Peixoto¹

¹Red Sea Research Center (RSRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST)

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Genetics

Chromatin Immunoprecipitation in the Cnidarian Model System Exaiptasia diaphana

Mascha Fiona Dix^*1, Peng Liu^*2, Guoxin Cui¹, Francesco Della Valle², Valerio Orlando², Manuel Aranda¹

¹Red Sea Research Center (RSRC), Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), ²KAUST Environmental Epigenetics Program (KEEP), Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST)

A chromatin immunoprecipitation (X-ChIP) assay against the histone mark H3K4me3 for the model organism Exaiptasia diaphana is presented. The specificity and effectiveness of the assay are confirmed by quantitative PCR and next-generation sequencing. This protocol enables the increased investigation of protein-DNA interactions in the sea anemone E. diaphana.

Immunology and Infection

Gene Editing of Primary Rhesus Macaque B Cells

Harald Hartweger¹, Rajeev Gautam², Yoshiaki Nishimura², Fabian Schmidt^3,5, Kai-Hui Yao¹, Amelia Escolano^1,6, Mila Jankovic¹, Malcolm A. Martin², Michel C. Nussenzweig^1,4

¹Laboratory of Molecular Immunology, The Rockefeller University, ²Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ³Laboratory of Retrovirology, The Rockefeller University, ⁴Howard Hughes Medical Institute, The Rockefeller University, ⁵Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), ⁶Vaccine and Immunotherapy Center, Wistar Institute

We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies.

Biology

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

Atheer Althobaiti¹, Kareem Hamdan¹, Mohamed A. Sobhy¹, Renad Rawas¹, Masateru Takahashi¹, Olga Artyukh¹, Muhammad Tehseen¹

¹Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST)

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.