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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 ...

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. ...

Procedure for Rhamnolipids Quantification Using Methylene-Blue

Authors: Tsiry Rasamiravaka
Tsiry RasamiravakaAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
For correspondence: travaka@yahoo.fr
Bio-protocol author page: a3049
, Olivier M. Vandeputte
Olivier M. VandeputteAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
Bio-protocol author page: a3050
 and Mondher El Jaziri
Mondher El JaziriAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
Bio-protocol author page: a3051
,  date: 4/5/2016, 1854 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1783.

[Abstract] Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g. bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric acid and orcinol based quantification (Helbert ...

[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae

Authors: Sarah C. Plecha
Sarah C. PlechaAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
Bio-protocol author page: a2783
 and Jeffrey H. Withey
Jeffrey H. WitheyAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
For correspondence: jwithey@wayne.edu
Bio-protocol author page: a2784
,  date: 12/20/2015, 1232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1682.

[Abstract] The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how ...

Extraction and Purification of Mycobacterial Mycolic Acids

Authors: Christian M. Dupont
Christian M. DupontAffiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université Montpellier, Montpellier, France
Bio-protocol author page: a1728
 and Laurent Kremer
Laurent KremerAffiliation 1: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université Montpellier, Montpellier, France
Affiliation 2: Inserm, Place Eugène Bataillon, Montpellier, France
For correspondence: lremer@univ-montp2.fr
Bio-protocol author page: a1729
,  date: 10/20/2014, 3789 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1265.

[Abstract] Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a critical ...

Dye Release Experiments with Dextran Loaded Vesicles

Authors: Marc-Antoine Sani
Marc-Antoine SaniAffiliation: School of Chemistry, The University of Melbourne, Melbourne, Australia
For correspondence: msani@unimelb.edu.au
Bio-protocol author page: a1492
, Neil M. O’Brien-Simpson
Neil M. O’Brien-SimpsonAffiliation: Melbourne Dental School, The University of Melbourne, Melbourne, Australia
Bio-protocol author page: a1488
 and Frances Separovic
Frances SeparovicAffiliation: School of Chemistry, The University of Melbourne, Melbourne, Australia
Bio-protocol author page: a1493
,  date: 7/20/2014, 2726 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1190.

[Abstract] Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked ...

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, while the base derivatization protocol (Lennen ...
1 

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, ...

Extraction and Purification of Mycobacterial Mycolic Acids

Authors: Christian M. Dupont
Christian M. DupontAffiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université Montpellier, Montpellier, France
Bio-protocol author page: a1728
 and Laurent Kremer
Laurent KremerAffiliation 1: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université Montpellier, Montpellier, France
Affiliation 2: Inserm, Place Eugène Bataillon, Montpellier, France
For correspondence: lremer@univ-montp2.fr
Bio-protocol author page: a1729
,  date: 10/20/2014, 3789 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1265.

[Abstract] Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that ...

Dye Release Experiments with Dextran Loaded Vesicles

Authors: Marc-Antoine Sani
Marc-Antoine SaniAffiliation: School of Chemistry, The University of Melbourne, Melbourne, Australia
For correspondence: msani@unimelb.edu.au
Bio-protocol author page: a1492
, Neil M. O’Brien-Simpson
Neil M. O’Brien-SimpsonAffiliation: Melbourne Dental School, The University of Melbourne, Melbourne, Australia
Bio-protocol author page: a1488
 and Frances Separovic
Frances SeparovicAffiliation: School of Chemistry, The University of Melbourne, Melbourne, Australia
Bio-protocol author page: a1493
,  date: 7/20/2014, 2726 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1190.

[Abstract] Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted ...

Procedure for Rhamnolipids Quantification Using Methylene-Blue

Authors: Tsiry Rasamiravaka
Tsiry RasamiravakaAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
For correspondence: travaka@yahoo.fr
Bio-protocol author page: a3049
, Olivier M. Vandeputte
Olivier M. VandeputteAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
Bio-protocol author page: a3050
 and Mondher El Jaziri
Mondher El JaziriAffiliation: Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Gosselies, Belgium
Bio-protocol author page: a3051
,  date: 4/5/2016, 1854 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1783.

[Abstract] Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g. bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric ...

[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae

Authors: Sarah C. Plecha
Sarah C. PlechaAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
Bio-protocol author page: a2783
 and Jeffrey H. Withey
Jeffrey H. WitheyAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
For correspondence: jwithey@wayne.edu
Bio-protocol author page: a2784
,  date: 12/20/2015, 1232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1682.

[Abstract] The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able ...

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. ...

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 ...
1 
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