This work is supported by a National Scientist Development grant from the American Heart Association: 10SDG3500034 and UTMB-NCB start up funds to SSM. The authors acknowledge the Cryo-EM and Scientific Computing facilities at the Sealy Center for Structural Biology at UTMB (<a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a>), as well as Drs. Steve Ludtke and Ed Egelman for help with the 2D and 3D helical reconstruction algorithms.

1. Sample Preparation
<ol>
	<li>Buffer exchange human FVIII-BDD14 and porcine FVIII-BDD15 against HBS-Ca buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH = 7.4) and concentrate to 1.2 mg/ml. Keep the protein solution at -80 &#176;C.</li>
	<li>Prepare lipid nanotubes (LNT) by mixing GalactosylCeramide (GC) and phosphatidylserine (PS) at 1:4 w/w ratio in chloroform. Evaporate the chloroform under argon and solubilize the lipids in HBS buffer to 1 mg/ml. Keep the LNT solution at 4 &#176;C....

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+cDNA+encoding+human+antihaemophilic+factor.">Molecular cloning of a cDNA encoding human antihaemophilic factor.</a> Nature. 312, 342-347 (1984).</li><li>Fay, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factor+VIII+structure+and+function.">Factor VIII structure and function.</a> International journal of hematology. 83, 103-108 (2006).</li><li>Stoilova-McPhie, S., Lynch, G. C., Ludtke, S. J., Pettitt, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+membrane-bound+factor+VIII.">Domain organization of membrane-bound factor VIII.</a> Biopolymers. , (2013).</li><li>Pipe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemophilia:+new+protein+therapeutics.">Hemophilia: new protein therapeutics.</a> Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program. 2010, 203-209 (2010).</li><li>Gatti, L., Mannucci, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+porcine+factor+VIII+in+the+management+of+seventeen+patients+with+factor+VIII+antibodies.">Use of porcine factor VIII in the management of seventeen patients with factor VIII antibodies.</a> Thrombosis and haemostasis. 51, 379-384 (1984).</li><li>Parmenter, C. D., Cane, M. C., Zhang, R., Stoilova-McPhie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+coagulation+Factor+VIII+bound+to+lipid+nanotubes.">Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes.</a> Biochemical and biophysical research communications. 366, 288-293 (2008).</li><li>Parmenter, C. D., Stoilova-McPhie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+recombinant+human+coagulation+factor+VIII+to+lipid+nanotubes.">Binding of recombinant human coagulation factor VIII to lipid nanotubes.</a> FEBS letters. 582, 1657-1660 (2008).</li><li>Wassermann, G. E., Olivera-Severo, D., Uberti, A. F., Carlini, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+urease+activates+blood+platelets+through+a+lipoxygenase-mediated+pathway.">Helicobacter pylori urease activates blood platelets through a lipoxygenase-mediated pathway.</a> Journal of cellular and molecular medicine. 14, 2025-2034 (2010).</li><li>Wilson-Kubalek, E. M., Chappie, J. S., Arthur, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helical+crystallization+of+soluble+and+membrane+binding+proteins.">Helical crystallization of soluble and membrane binding proteins.</a> Methods in enzymology. 481, 45-62 (2010).</li><li>Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+helical+filaments+and+tubes.">Reconstruction of helical filaments and tubes.</a> Methods in enzymology. 482, 167-183 (2010).</li><li>Lusher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+introduction+of+recombinant+factor+VIII--a+clinician's+experience.">Development and introduction of recombinant factor VIII--a clinician's experience.</a> Haemophilia : the official journal of the World Federation of Hemophilia. 18, 483-486 (2012).</li><li>Thim, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+new+recombinant+factor+VIII+(N8).">Purification and characterization of a new recombinant factor VIII (N8).</a> Haemophilia : the official journal of the World Federation of Hemophilia. 16, 349-359 (2010).</li><li>Doering, C. B., Healey, J. F., Parker, E. T., Barrow, R. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+algorithm+for+the+reconstruction+of+helical+filaments+using+single-particle+methods.">A robust algorithm for the reconstruction of helical filaments using single-particle methods.</a> Ultramicroscopy. 85, 225-234 (2000).</li><li>Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iterative+helical+real+space+reconstruction+method:+surmounting+the+problems+posed+by+real+polymers.">The iterative helical real space reconstruction method: surmounting the problems posed by real polymers.</a> Journal of structural biology. 157, 83-94 (2007).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> Journal of structural biology. 152, 36-51 (2005).</li><li>Stoilova-McPhie, S., Villoutreix, B. O., Mertens, K., Kemball-Cook, G., Holzenburg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+structure+of+membrane-bound+coagulation+factor+VIII:+modeling+of+the+factor+VIII+heterodimer+within+a+3-dimensional+density+map+derived+by+electron+crystallography.">3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography.</a> Blood. 99, 1215-1223 (2002).</li><li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+density+maps+with+UCSF+Chimera.">Visualizing density maps with UCSF Chimera.</a> Journal of structural biology. 157, 281-287 (2007).</li></ol>

In this work a methodology is presented to differentiate between two membrane-bound organizations of highly homologous proteins: human and porcine FVIII self-assembled on lipid nanotubes in the conditions encountered in the human body.

In the described procedure, human and porcine FVIII are successfully organized helically on lipid nanotubes, which is the most critical step. The next critical step is to preserve the sample in thin amorphous ice by flash freezing&#1...

Blood coagulation Factor VIII (FVIII) is a large glycoprotein of 2,332 amino acids organized in six domains: A1-A2-B-A3-C1-C2&#160;3. Upon Thrombin activation FVIII acts as the cofactor to Factor IXa within the membrane-bound Tenase complex. Binding of activated FVIII (FVIIIa) to FIXa in a membrane-depending manner enhances FIXa proteolytic efficiency more than 105 times, which is critical for efficient blood coagulation4. Despite the important role FVIII plays in coagulation and the Tenase complex formation, the functional membrane-bound FVIII structure is yet to be resolved.
To address this, single lipid bilayer nanotubes (LNT) rich in phosphatidylserine (PS), capable of binding FVIII with high affinity8,9 and resembling the activated platelet surface have been developed10. Consecutive helical organization of FVIII bound to LNT has been proven to be effective for structure determination of FVIII membrane-bound state by Cryo-EM5. Functionalized LNT are an ideal system to study protein-protein and protein-membrane interactions of helically organized membrane-associated proteins by Cryo-EM11,12. Cryo-EM has the advantage over traditional structural methods such&#160;as X-ray crystallography and NMR, as the specimen is preserved at closest to the physiological environment (buffer, membrane, pH), without additives and isotopes. In the case of FVIII, studying the membrane-bound structure with this technique is even more physiologically relevant, as the LNT resemble closely by size, shape and composition the pseudopodia of the activated platelets where the Tenase complexes assemble in vivo.
Defects and deficiency of FVIII cause Hemophilia A, a severe bleeding disorder affecting 1 in 5,000 males of the human population4,6. The most effective therapy for Hemophilia A is life-long administration of recombinant human FVIII (hFVIII). A significant complication of the recombinant FVIII Hemophilia A therapy is the development of inhibitory antibodies to the human form affecting approximately 30% of Hemophilia A patients13. In this case, porcine FVIII (pFVIII) concentrate is used, as porcine FVIII displays low cross-reactivity with inhibitory antibodies against human FVIII and forms functional complexes with human FIXa7. Establishing the membrane-bound organization of both porcine and human FVIII forms is important to understand the structural basis of FVIII cofactor function and implications for blood hemostasis.
In this study, we describe a combination of lipid nanotechnology, Cryo-EM, and structure analysis designed to resolve the membrane-bound organization of two highly homologous FVIII forms. The presented Cryo-EM data and 3D structures for helically organized porcine and human FVIII on negatively charged LNT show the potential of the proposed nanotechnology as basis for structure determination of FVIII and membrane-bound coagulation factors and complexes in a physiological membrane environment.

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			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
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			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
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			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
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			<td style="width:447px;">cooled to -175 &#176;C</td>
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			<td style="width:447px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
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			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
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			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
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			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
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			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
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			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
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			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
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			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
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			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
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			<td height="31" style="height:31px;">EM software Spider</td>
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			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
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			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
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			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
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			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

Recombinant human and porcine FVIII were successfully organized helically on negatively charged single bilayer LNT, resembling the activated platelet surface. The helical organization of the human and porcine FVIII-LNT was consistent through the collected digital micrographs (Figure&#160;2). &#160;The control LNT and the human and porcine FVIII-LNT helical tubes were selected and segmented with the e2helixboxer.py GUI and initial data sets created with the e2workflow.py GUI, Single parti...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

helical-organization-blood-coagulation-factor-viii-on-lipid

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JoVE Journal

Bioengineering

12.2K ビュー.  University of Texas Medical Branch. ヒトおよびブタ：我々は、2つの高度に相同なFVIII形態の膜結合構造を解決するために適用クライオ電子顕微鏡法、脂質ナノテクノロジー、および構造解析の組み合わせを提示する。螺旋状に負に荷電した脂質ナノチューブ（LNT）上の二つの機能の​​組換えFVIIIフォームを整理するために私たちの研究室で開発された方法論が記載されている。 

ビデオ: Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is...

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Here, we describe in detail the operation procedures and precautions to be taken during herbs-partitioned moxibustion on the navel in the treatment of primary dysmenorrhea in rats with cold coagulation and blood stasis, which can significantly improve the writhing reaction of dysmenorrhea rats and alleviate...

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Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based...

Improved Sensitivity for Tissue Factor Activity Assay in Extracellular Vesicles

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reconstitution of membrane-tethered minimal actin cortices on supported lipid bilayers

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence...

pulling membrane nanotubes from giant unilamellar vesicles

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in...

procoagulant platelet characterization by measuring phosphatidylserine exposure and microvesicle release from human purified platelets

Author Spotlight: Studying Platelet Coagulation Responses in Diverse Pathological Conditions

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

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detection of rhesus factor in human blood samples using magnetic levitation

This video demonstrates the detection of the Rhesus (Rh) factor in blood samples using a magnetic levitation instrument. The magnetic field aligns and levitates beads and blood cells based on density. Treating the samples with anti-Rh antibodies results in intermediate-density bead-cell complexes&#39; formation, allowing for the differentiation of Rh-positive and Rh-negative red blood...

Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar...

detection and quantification of tunneling nanotubes using 3d volume view images

Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images

Tunneling nanotubes (TNTs) are primarily open-ended F-actin membrane nanotubes that connect neighboring cells, facilitating intercellular communication. The notable characteristic that distinguishes TNTs from other cell protrusions is the hovering nature of the nanotubes between cells. Here, we characterize TNTs by constructing a 3D volume view of confocal z-stack images.

To prepare the experimental tube containing only beads, add 174 microliters of HBSS with calcium and magnesium, 1 microliter of IgG control beads, 1 microliter of heavy beads, with a 1.2 gram per milliliter density, and 24 microliters of 500 millimolar...

in vitro reconstitution of self-organizing protein patterns on supported lipid bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

growth factor-coated bead placement on dorsal forebrain explants

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex.   These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants.  Methods are given for using both Affi-Gel beads and heparin acryllic beads.

determination of the procoagulant activity of extracellular vesicle (ev) using ev-activated clotting time (ev-act)

Author Spotlight: Deciphering Coagulation Disorders in Traumatic Brain Injury Patients 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent...

Assessing Blood Hypercoagulation of EV-Rich Plasma

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transport of surface-modified carbon nanotubes through a soil column

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

self-assembling morphologies obtained from helical polycarbodiimide copolymers and their triazole derivatives

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of...

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&amp;#946; on the Epithelial-to-Mesenchymal Transition

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...