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Biochemistry

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Authors: Reka A. Haraszti
Reka A. HarasztiAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4671
, Andrew Coles
Andrew ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
, Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
, Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
,  date: 6/20/2017, 206 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2338.

[Abstract] Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural ...

Root Aliphatic Suberin Analysis Using Non-extraction or Solvent-extraction Methods

Authors: Camille Delude
Camille DeludeAffiliation: Laboratoire de Biogenèse Membranaire, UMR 5200, CNRS – Université de Bordeaux, INRA Bordeaux Aquitaine, Villenave d’Ornon, France
Bio-protocol author page: a4651
, Sollapura J. Vishwanath
Sollapura J. VishwanathAffiliation 1: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Government of Canada, Ottawa, Canada
Affiliation 2: Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Canada
Bio-protocol author page: a1466
, Owen Rowland
Owen RowlandAffiliation: Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Canada
Bio-protocol author page: a1468
 and Frédéric Domergue
Frédéric DomergueAffiliation: Laboratoire de Biogenèse Membranaire, UMR 5200, CNRS – Université de Bordeaux, INRA Bordeaux Aquitaine, Villenave d’Ornon, France
For correspondence: frederic.domergue@u-bordeaux.fr
Bio-protocol author page: a1467
,  date: 6/20/2017, 196 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2331.

[Abstract] Here we describe both non-extraction and solvent-extraction methods for root aliphatic suberin analysis. The non-extraction method is fast as roots are directly depolymerized using acidic transmethylation. However, suberin aliphatic components are isolated together with all the other acyl chains making up the lipids (e.g., membranes) present in roots. ...

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
, Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
, Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
 and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
,  date: 5/5/2017, 511 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

[Abstract] A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein ...

Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells

Authors: Satu Hänninen
Satu HänninenAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
For correspondence: satu.m.hanninen@helsinki.fi
Bio-protocol author page: a4478
, Pentti Somerharju
Pentti SomerharjuAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a4479
 and Martin Hermansson
Martin HermanssonAffiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Bio-protocol author page: a4480
,  date: 5/5/2017, 387 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2268.

[Abstract] Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope ...

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
,  date: 5/5/2017, 349 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

[Abstract] The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps. ...

Liposome Flotation Assays for Phosphoinositide-protein Interaction

Authors: Helene Tronchere
Helene TronchereAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
Bio-protocol author page: a4209
 and Frederic Boal
Frederic BoalAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
For correspondence: frederic.boal@inserm.fr
Bio-protocol author page: a4210
,  date: 3/5/2017, 856 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2169.

[Abstract] Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different ...

Transient Transfection-based Fusion Assay for Viral Proteins

Authors: Melina Vallbracht
Melina VallbrachtAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4197
, Christina Schröter
Christina SchröterAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4205
, Barbara G. Klupp
Barbara G. KluppAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4206
 and Thomas C. Mettenleiter
Thomas C. MettenleiterAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
For correspondence: thomas.mettenleiter@fli.de
Bio-protocol author page: a4207
,  date: 3/5/2017, 799 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2162.

[Abstract] Membrane fusion is vital for entry of enveloped viruses into host cells as well as for direct viral cell-to-cell spread. To understand the fusion mechanism in more detail, we use an infection free system whereby fusion can be induced by a minimal set of the alphaherpesvirus pseudorabies virus (PrV) glycoproteins gB, gH and gL. Here, we describe an ...

Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry

Authors: Jennifer A.E. Payne
Jennifer A.E. PayneAffiliation 1: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Affiliation 2: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
For correspondence: jennifer.payne@monash.edu
Bio-protocol author page: a3952
, Tzong-Hsien Lee
Tzong-Hsien LeeAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3953
, Marilyn A. Anderson
Marilyn A. AndersonAffiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Bio-protocol author page: a3954
 and Marie-Isabel Aguilar
Marie-Isabel AguilarAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3955
,  date: 1/5/2017, 818 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2087.

[Abstract] Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid ...

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
, Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
, Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
,  date: 12/5/2016, 1251 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2037.

[Abstract] Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. ...

Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes

Authors: Michel Lafleur
Michel LafleurAffiliation: Department of chemistry, Université de Montréal, Montréal (Québec), Canada
For correspondence: michel.lafleur@umontreal.ca
Bio-protocol author page: a3731
 and Alexandre Therrien
Alexandre TherrienAffiliation: Department of chemistry, Université de Montréal, Montréal (Québec), Canada
Bio-protocol author page: a3732
,  date: 11/20/2016, 941 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2016.

[Abstract] Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by ...
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Measurement of Liver Triglyceride Content

Author: Hani Jouihan
Hani JouihanAffiliation: Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, Stanford, USA
For correspondence: hanijouihan@hotmail.com
Bio-protocol author page: a52
,  date: 7/5/2012, 15711 views, 11 Q&A
DOI: https://doi.org/10.21769/BioProtoc.223.

[Abstract] This assay is designed to measure relative lipid accumulation of experimental treatments compared to controls. The reagent measures the concentration of glycerol released after lysing the cells and hydrolyzing the triglyceride molecules. The triglyceride concentration can then be determined from the ...

Preparation of Outer Membrane Vesicle from Escherichia coli

Authors: Oh Youn Kim
Oh Youn KimAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1053
, Bok Sil Hong
Bok Sil HongAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1054
, Kyong-Su Park
Kyong-Su ParkAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1055
, Yae Jin Yoon
Yae Jin YoonAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1056
, Seng Jin Choi
Seng Jin ChoiAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1057
, Won Hee Lee
Won Hee LeeAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1058
, Tae-Young Roh
Tae-Young RohAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1059
, Yoon-Keun Kim
Yoon-Keun KimAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1060
 and Yong Song Gho
Yong Song GhoAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
For correspondence: ysgho@postech.ac.kr
Bio-protocol author page: a1061
,  date: 12/5/2013, 5878 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.995.

[Abstract] Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various ...

Protocol for Macrophage Depletion from Mice

Authors: Zuoxiang Xiao
Zuoxiang XiaoAffiliation: Cancer Inflammation Program, NCI-Frederick, NIH, Frederick, USA
For correspondence: xiaoz2@mail.nih.gov
Bio-protocol author page: a1391
 and Qun Jiang
Qun JiangAffiliation: Cancer Inflammation Program, NCI-Frederick, NIH, Frederick, USA
Bio-protocol author page: a1392
,  date: 6/5/2014, 5803 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1144.

[Abstract] Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have ...

Lipid Extraction from Mouse Feces

Authors: Daniel Kraus
Daniel KrausAffiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, USA
Present address: Medizinische Klinik und Poliklinik I, Universitätsklinikum Würzburg, Würzburg, Germany
For correspondence: daniel.kraus@uni-wuerzburg.de
Bio-protocol author page: a1930
, Qin Yang
Qin YangAffiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, USA
Present address: Department of Medicine, Physiology and Biophysics, University of California, Irvine, USA
Bio-protocol author page: a1931
 and Barbara B. Kahn
Barbara B. KahnAffiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, USA
Bio-protocol author page: a1932
,  date: 1/5/2015, 5564 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1375.

[Abstract] The analysis of feces composition is important for the study of energy metabolism, which comprises various measurements of energy intake, energy expenditure, and energy wasting. The current protocol describes how to measure energy-dense lipids in mouse feces using a modification of the method proposed ...

Quantification of Bacterial Fatty Acids by Extraction and Methylation

Authors: Mark Politz
Mark PolitzAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
Bio-protocol author page: a938
, Rebecca Lennen
Rebecca LennenAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
Bio-protocol author page: a939
 and Brian Pfleger
Brian PflegerAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
For correspondence: pfleger@engr.wisc.edu
Bio-protocol author page: a557
,  date: 11/5/2013, 5537 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.950.

[Abstract] This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty acids, both free and bound, from a bacterial culture, ...

In vitro Lipid Binding Assay

Authors: Yan Zhang
Yan ZhangAffiliation: National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing, China
Bio-protocol author page: a588
 and Xiaochen Wang
Xiaochen WangAffiliation: National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing, China
For correspondence: wangxiaochen@nibs.ac.cn
Bio-protocol author page: a317
,  date: 5/5/2013, 5353 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.694.

[Abstract] This is a protocol to examine in vitro protein-lipid binding using membrane strips coated with various lipids. It has been successfully used to study in vitro interaction between lipids and C. elegans proteins....

Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants

Authors: Liwei Chen
Liwei ChenAffiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1609
 and Wei Ning Chen
Wei Ning ChenAffiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore
For correspondence: wnchen@ntu.edu.sg
Bio-protocol author page: a1610
,  date: 9/5/2014, 5078 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1219.

[Abstract] Metabolite and fatty acid analysis play important roles in evaluating the metabolic state of microorganisms. To examine the growth state and metabolism response of cells to environmental stress or genetic modification, intracellular and extracellular metabolites including fatty acids are usually analyzed ...

Wax Analysis of Stem and Rosette Leaves in Arabidopsis thaliana

Authors: Tegan M Haslam
Tegan M HaslamAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
For correspondence: tegan.haslam@botany.ubc.ca
Bio-protocol author page: a612
 and Ljerka Kunst
Ljerka KunstAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
For correspondence: ljerka.kunst@ubc.ca
Bio-protocol author page: a611
,  date: 6/5/2013, 4812 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.782.

[Abstract] The primary aerial surfaces of all land plants are coated by a lipidic cuticle, which restricts non-stomatal water loss and protects the plant from pathogens, herbivores, and ultraviolet radiation. The cuticle is made up of two components: cutin, a polymer of hydroxy- and epoxy- long-chain fatty acid ...

In vitro Lipid Transfer Assay

Authors: Yan Zhang
Yan ZhangAffiliation: National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing, China
Bio-protocol author page: a588
 and Xiaochen Wang
Xiaochen WangAffiliation: National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing, China
For correspondence: wangxiaochen@nibs.ac.cn
Bio-protocol author page: a317
,  date: 5/5/2013, 4523 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.691.

[Abstract] This is a protocol to detect lipid transfer activity of NRF-5, a member of the LPS binding/lipid transfer protein family. The lipid transfer activity is examined by using isotope-labeled cholesterol and liposomes, and tested in two directions (Figure 1): from proteins to liposomes and from liposomes ...

Glycophosphosphingolipid (GSPL) Purification Protocol

Author: Tripti De
Tripti DeAffiliation: Division of Infectious Disease and Immunology, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
For correspondence: triptide@iicb.res.in
Bio-protocol author page: a175
,  date: 12/5/2012, 4493 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.299.

[Abstract] Glycosylated ceramide phosphorylinositol are present in many species of fungi and mushrooms and bacteria and parasitic organisms like leishmania. These are usually membrane raft associated and are not easily extracted by conventional methodologies. This extraction method gives higher yield of the glycolipid. ...
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