This project was funded in part by the National Institutes of Health Project number 5P20GM103499-19 through the Student Initiated Research Project Program. This work was also partially supported by Clemson&#39;s Creative Inquiry Program. We also acknowledge Nicholas L&#39;Amoreaux and Aon Ali who initially worked on creating this protocol, publishing their first paper cited here14.

1. Spherical polymersome formation using a solvent injection method
<ol>
	<li>Dissolution of polyesters in organic solvent 
	NOTE: Only one polyester should be dissolved in its respective organic solvent at a time to form polymersomes.
	<ol>
		<li>Dissolve polyesters PEG-PLA or PEG-b-poly(lactic-co-glycolic acid) (PEG-PLGA) in dimethyl sulfoxide (DMSO) at a concentration of 1.5% weight. Specifically, dissolve 0.015 g of selected polyester in 1 mL of DMSO (15 mg/mL). Full dissolution of the polymer may require periods of up to 15 min of vortexing.</li>
	</ol>
	</li>
	<li>While the polyester is dissolving in organic solvent, set up the solvent injection apparatus according to Figure 1.
	<ol>
		<li>Place a stir plate directly below the vertical syringe pump. Place a 5 mL glass vial with 1 mL of Type II deionized water and a miniature stir bar on the stir plate.</li>
		<li>Adjust the syringe pump&#39;s height to allow for the tip of the needle to be fully immersed in Type II deionized water.</li>
		<li>Set the infusion rate of the syringe pump to 5 &#181;L/min. 
		NOTE: If a small volume syringe pump is used, the adapter with the syringe can be set up on a ring stand. If a large volume syringe pump is used, the pump can be placed vertically on a lab jack to adjust the height.</li>
	</ol>
	</li>
	<li>Perform the solvent injection by drawing the organic solvent and polyester solution (step 1.1.1) into a 27 G needle with a &#189;&#34; needle length.
	<ol>
		<li>Place the needle into the syringe pump and make sure it is entirely secure. Adjust the pusher block to touch the syringe plunger&#39;s end.</li>
		<li>Start the stir plate so that the water is spinning at 100 rpm, and then start the syringe pump.</li>
	</ol>
	</li>
	<li>Once the syringe pump has fully infused the organic solvent and polymer into the stirring water, remove from the stir bar and cap the glass vial.</li>
	<li>Characterize the polymersomes via dynamic light scattering (DLS).
	<ol>
		<li>Take 1 mL of water, now with a small percentage of organic solvent and polymer, and place in a 1 mL cuvette.</li>
		<li>Using the settings from Table 1, perform DLS by placing a 1 mL cuvette into the system and set up the run. Read and collect the polymersome intensity-weighted diameter and polydispersity index (PDI). 
		NOTE: A plastic cuvette works fine in this case, as the amount of organic solvent is very low. However, a glass cuvette will work as well if any concerns exist.</li>
	</ol>
	</li>
	<li>Confirm spherical polymersome formation using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).
	<ol>
		<li>Optimize TEM and SEM protocols based on the equipment available. Representative results were obtained at 120 kV in the TEM and 5.0 kV in the SEM.</li>
		<li>If polymersomes are not visible using EM, apply uranyl acetate as a background stain. 
		&#8203;NOTE: Details on TEM and SEM imaging for the shape modulation of polyester-based polymersomes can be found in reference14. Information on electron microscopy techniques for soft nanoparticles is detailed in reference16.</li>
	</ol>
	</li>
</ol>
2. Dialysis to remove organic solvent
<ol>
	<li>Wash a 300 kDa dialysis membrane according to protocols provided by the manufacturer.</li>
	<li>Add 1 mL of polymersome solution into the reservoir of the dialysis device.</li>
	<li>Place the dialysis device in a 250 mL beaker with 150 mL of Type II deionized water on a stir plate. Set the stir plate to a speed that allows for gentle movement of the dialysis device and leave to stir overnight. 
	NOTE: If a vortex is formed during dialysis, the speed needs to be decreased.</li>
	<li>After the dialysis is completed, extract the 1 mL polymersome solution from the dialysis device. Characterize the polymersome solution, following step 1.5. 
	NOTE: Collection of this information is relevant to determine the success of the shape modulation protocol, as one should be able to identify an increase in PDI if the polymersome has been elongated.</li>
</ol>
3. Dialysis against salt gradients
<ol>
	<li>Create 150 mL of desired salt buffer, with either 50 mM, 100 mM, or 200 mM concentration of sodium chloride based on the final desired polymersome properties. In general, increased salt concentration leads to increased polymersome elongation.</li>
	<li>Take the polymersome solution that was dialyzed and characterized and re-place into the dialysis device. Place the loaded dialysis device into 150 mL of the desired salt solution and allow to gently stir for 18 h. 
	&#8203;NOTE: Shape-modulated polymersomes can be stored and maintain their shape in an isotonic solution for periods of up to 7 days.</li>
</ol>
4. Shape modulated polymersome characterization
<ol>
	<li>After the shape modulation, perform polymersome characterization via DLS, TEM, and SEM. If polymersomes are not visible using EM, apply uranyl acetate as a background stain.</li>
	<li>Perform DLS measurements as described in step 1.5, paying particular attention to PDI measurements compared to spherical polymersomes, as a change in PDI suggests effective shape change in polymersomes.</li>
	<li>Ensure the use of appropriate controls for imaging, especially non-shape modulated polymersomes, to ensure the method&#39;s success.</li>
</ol>

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A., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+target+geometry+in+phagocytosis.">Role of target geometry in phagocytosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (13), 4930-4934 (2006).</li><li>Banerjee, A., Qi, J., Gogoi, R., Wong, J., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nanoparticle+size,+shape+and+surface+chemistry+in+oral+drug+delivery.">Role of nanoparticle size, shape and surface chemistry in oral drug delivery.</a> Journal of Controlled Release. 238, 176-185 (2016).</li><li>Kolhar, P., 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=Using+shape+effects+to+target+antibody-coated+nanoparticles+to+lung+and+brain+endothelium.">Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium.</a> Proceedings of the National Academy of Sciences. 110 (26), 10753-10758 (2013).</li><li>Meng, F., Zhong, Z., Feijen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+polymersomes+for+programmed+drug+delivery.">Stimuli-responsive polymersomes for programmed drug delivery.</a> Biomacromolecules. 10 (2), 197-209 (2009).</li><li>Iqbal, S., Blenner, M., Alexander-Bryant, A., Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes+for+therapeutic+delivery+of+protein+and+nucleic+acid+macromolecules:+from+design+to+therapeutic+applications.">Polymersomes for therapeutic delivery of protein and nucleic acid macromolecules: from design to therapeutic applications.</a> Biomacromolecules. 21 (4), 1327-1350 (2020).</li><li>Discher, D. 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The authors have nothing to disclose.

Self-assembled systems are notoriously uncontrollable. Their final properties, including size, shape, and structure, are driven by the chosen amphiphile&#39;s hydrophobic properties and the solvent environment selected. Amphiphilic block copolymers tend towards spherical shapes, which minimizes Gibb&#39;s free energy and leads to the thermodynamic equilibrium23, thus forming polymersomes. Because of their equilibrium nature, polymersomes are significantly more challenging to elongate or alter in s...

Shape modulation is a relatively new and efficient way to improve nanoparticle-mediated drug delivery. Not only does the change in morphology increase the surface area of particles, which in turn allows for a greater carrying capacity, but it also has implications across the board to improve stability, circulation time, bioavailability, molecular targeting, and controlled release1. Polymersomes, the nanoparticle of focus in this method, tend to thermodynamically self-assemble into a spherical shape, which has proven to be impractical in cellular uptake and is more easily detected in the immune system as a foreign body. Being able to elongate the structure into a prolate or a rod will allow the drug carrier to evade macrophages by mimicking native cells and more successfully deliver to their desired target2,3,4,5,6,7. The significant benefits of polymersomes, including membrane-bound protection of payloads, stimuli-responsiveness of the membrane, and dual encapsulation of hydrophilic and hydrophobic drugs8,9,10, that make them strong candidates for drug delivery are maintained during shape modulation.
There are many different methods in modulating polymersomes&#39; shapes, and each comes with its respective advantages and disadvantages. However, most of these methods fall into two categories: solvent-driven and salt-driven osmotic pressure change11. Both approaches aim to overcome the bending energy present after polymersomes are formed in a spherical equilibrium shape. By introducing an osmotic pressure gradient, polymersomes can be forced to bend into elongated structures despite strong bending energies11,12.
The solvent-based method explores shape change inspired by the work of Kim and van Hest13. They plasticized polymersomes in an organic solvent and water mixture to trap the organic solvents in the vesicle membrane and drive water out of the vesicle core. Eventually, the particle&#39;s internal volume is so low that it elongates. While this method has shown promise, it lacks practicality. This method requires different solvents for each individual polymeric backbone involved in the modulation. Therefore, it is not widely applicable to promote shape change. Conversely, the salt-based method is uniform and utilizes one universal driver that can introduce osmotic pressure to many block copolymer-based polymersomes.
This project utilizes the salt-based method introduced by L&#39;Amoreaux et al14. This protocol involves two rounds of dialysis. One aims at purifying and solidifying poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) polymersomes by removing organic solvent that may have gotten trapped in the bilayer during production, and one that promotes the shape change. The second dialysis step introduces a 50 mM NaCl solution that creates an osmotic pressure gradient to drive the shape change. This method is supported by Salva et al., who note that hypertonic stress in a solution will cause the vesicle to shrink15. This method builds on a previously published method14 looking at two different polyester-based polymersomes and various salt gradients from 50-200 mM NaCl. Polyesters are used due to their biocompatibility and biodegradation. The salt gradient has varying effects on the shape depending on the hydrophobicity of the block copolymer backbone. It can be used to create prolates, rods, and stomatocytes. This salt-driven method was chosen because of the ease of replication and experimental versatility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15*45 vials screw thread w/cap attached</td>
			<td>Fisherbrand</td>
			<td>9609104000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Chemical</td>
			<td>D128-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>BDH</td>
			<td>BDH1115-1LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoremp stirrers, hotplates, and stirring hotplates</td>
			<td>Fisher scientific</td>
			<td>CIC00008110V19</td>
			<td></td>
		</tr>
		<tr>
			<td>LEGATO 130 SYRINGE PUMP</td>
			<td>kd Scientific</td>
			<td>788130</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG(1000)-b-PLA(5000), Diblock Polymer</td>
			<td>Polysciences Inc</td>
			<td>24381-1</td>
			<td>note the molecular weights when replicating</td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) (2000) Methyl ether-block-poly(lactide-co-glycolide) (4500)</td>
			<td>Sigma aldrich</td>
			<td>764825-1G</td>
			<td>note the molecular weights when replicating</td>
		</tr>
		<tr>
			<td>Single-Use Syringe/BD PrecisionGlide Needle combination, sterile, BD medical</td>
			<td>BD medical</td>
			<td>BD305620</td>
			<td>Tuberculin</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modulating shape of polyester based polymersomes using osmotic pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Table 2 presents expected results when following the protocol step 1. Note that DMSO is used as a solvent for both PEG-PLA and PEG-PLGA in polymersomes formation. Deviation from this solvent is possible, as other water-miscible solvents will dissolve the copolymers but is expected to change results. It is expected that PDI will be less than 0.2, indicating the formation of monodisperse polymersomes17. Note that increasing hydrophobicity leads to increased deviation in both polymer...

Watch this Scientific Journal Video about Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure at JoVE.com

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...

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3.1K Views.  Clemson University. Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Video: Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Ammonia Synthesis at Low Pressure

Ammonia can be synthesized at low pressure by the use of an ammonia selective absorbent. The process can be driven with wind energy, available locally in areas requiring ammonia for synthetic fertilizer. Such wind energy is often called &#34;stranded,&#34; because it is only available far from population centers where it can be directly used.
In the proposed low pressure process, nitrogen is made from air using pressure swing absorption, and hydrogen is produced by electrolysis of water. While these gases can react at approximately 400 &#176;C in the presence of a promoted conventional catalyst, the conversion is often limited by the reverse reaction, which makes this reaction only feasible at high pressures. This limitation can be removed by absorption on an ammine-like calcium or magnesium chloride. Such alkaline metal halides can effectively remove ammonia, thus suppressing the equilibrium constraints of the reaction. In the proposed absorption-enhanced ammonia synthesis process, the rate of reaction may then be controlled not by the chemical kinetics nor the absorption rates, but by the rate of the recycle of unreacted gases. The results compare favorably with ammonia made from a conventional small scale Haber-Bosch process.

Ammonia can be synthesized at low pressure by using a conventional catalyst and an ammonia selective absorbent.

Ammonia can be synthesized at low pressure by the use of an ammonia selective absorbent. The process can be driven with wind energy, available locally in ...

Pattern-based Search of Epigenomic Data Using GeNemo

Compared with the robust text-based search tools for genomic or RNA sequencing data, current methodologies for pattern-based searches of epigenomic and other functional genomic data are very limited. GeNemo is the first online search tool that accomplishes this goal. Users input their functional genomic data in the Browser Extensible Data (BED), Peaks, and bigWig formats, and may search for data in any of the three formats. Users may specify which types of datasets to search against, choosing from a variety of online datasets, with the Encyclopedia of DNA Elements (ENCODE) representing different epigenomic marks, transcriptional factor binding sites, and chromatin hypersensitivities or accessibilities in specific cell types, and developmental stages or species (mouse or human). GeNemo returns a list of genomic regions with matching patterns to the input data, which may be viewed in the browser as well as downloaded in the BED file format. The upgraded GeNemo has improved graphical display, has more robust interface, and is no longer prone to errors due to changes in the University of California, Santa Cruz (UCSC) genome browser. Troubleshooting steps for common problems are discussed. As the amount of functional genomic data is expanding exponentially, there is a critical need to develop and refine new bioinformatic tools such as GeNemo for data analyses and interpretation.

Unlike DNA sequence data, epigenomic data are not readily subjected to text-based searches. Presented here are the procedures to use an upgraded version of GeNemo, a web-based bioinformatics tool, to conduct pattern-based searches for similarities in epigenomic data comparing available online databases including Encyclopedia of DNA Elements with user&#39;s data.

Compared with the robust text-based search tools for genomic or RNA sequencing data, current methodologies for pattern-based searches of epigenomic and ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.