We sincerely thank the Whitehall Foundation for their funding support and startup funds from The Scripps Research Institute for carrying out this work.

1. Preparation of Abdominal Ganglia, Electrophysiological Measurements, and Isolation of Single Identified Neurons from Abdominal Ganglion of Aplysia californica
<ol>
	<li>Maintain Aplysia in the laboratory aquarium with circulating artificial seawater (ASW) at 16 &#176;C under 12:12 light:dark conditions.</li>
	<li>Isolation of abdominal ganglion.
	<ol>
		<li>Anesthetize animals by injecting 380 mM MgCl2 solution for 5-10 min&#160;(equivalent to 30-35% of the animal&#39;s body weight).</li>
		<li>Identify the abdominal ganglion based on its position in the central nervous system24,28.</li>
		<li>Remove the ganglia by surgical operation and immediately store in artificial seawater consisting of 450 mM NaCl, 10 mM KCl, 10 mM CaCl2, 55 mM MgCl2, 2.5 mM NaHCO3, and 10 mM HEPES (pH 7.4).</li>
	</ol>
	</li>
	<li>Protease treatment.
	<ol>
		<li>Incubate abdominal ganglion for 30 min&#160;at 34 &#176;C&#177;0.5 &#160;in a Petri dish with 0.1% protease (dispase) diluted in ASW described above. 
		NOTE: The amount of the protease used needs to be standardized with the age and weight of the animals. In general, older and bigger animals would require more protease.</li>
	</ol>
	</li>
	<li>Removal of ganglia sheath.
	<ol>
		<li>After the enzyme treatment, pin down the ganglia to the Sylgard Silicone base of a cell chamber (&#216;= 1 cm; vol. = 0.3 ml) and perfuse with ASW (flow rate: 150 &#956;l/min) at room temperature (18 &#176;C&#177;2 ). 
		NOTE: In order to increase the visibility of identified neurons, place the ganglion on the top of a polycarbonate glass cylinder (Figure 1) (diameter &#216;= 2 mm modified cell chamber) that can be easily illuminated from the bottom using a binocular stereomicroscope with 20X&#160;and 40X&#160;magnification.</li>
		<li>Carefully remove the sheath from the ganglion by using forceps and microscissors.</li>
	</ol>
	</li>
	<li>Simultaneous electrophysiological recording from two neurons.
	<ol>
		<li>Identify neuron of interest.</li>
		<li>Impale the neuron with a single intracellular sharp microelectrode with resistance 10-15 M&#937; filled with 3 M KCl solution.</li>
		<li>Record neuronal activity (10 kHz band pass) using an intracellular recording system.</li>
		<li>Process the recording data using electrophysiology software such as Axograph. 
		NOTE: One can easily carry out recording from multiple neurons. Neurons L7, L11 or R15 and R2 are easily identified based on their position. Two amplifiers and two micromanipulators are required for simultaneously recording from two L7 and R15 neurons (Figure 2).</li>
	</ol>
	</li>
	<li>Drug application.
	<ol>
		<li>Apply drug of choice into the cell chamber by gentle pipetting or inject directly into neurons.</li>
		<li>Carry out electrophysiological measurements. 
		NOTE: Depending on the experimental objective, one could measure different electrophysiological parameters of neurons such as the changes in membrane potential (MP) or action potential (AP) before, during and after the drug application.</li>
	</ol>
	</li>
	<li>Isolation of single neurons.
	<ol>
		<li>At the conclusion of the electrophysiological experiments, stop the perfusion of ASW and wash the ganglion with 100% ethanol. &#160;Exposure to ethanol will petrify the neurons and, using fine forceps, make it easy to remove individual neurons without damaging other neurons from the ganglion.</li>
		<li>Remove single neurons under a stereomicroscope and transfer to a small Eppendorf tube containing ice cold 500 &#956;l RNA extraction reagent. Isolated single neurons can be stored in RNA extraction reagent at -80&#160;&#176;C for future analysis.</li>
	</ol>
	</li>
</ol>
2. RNA Isolation from Single Neurons and Gene Expression Analysis by Quantitative Real Time PCR (qPCR)
<ol>
	<li>Precautions to minimize RNA degradation: 
	Ribonucleases (RNases) degrade RNA and are very stable and active enzymes. Take the following steps during handling RNAs.
	<ol>
		<li>Clean the working area and instruments with RNase deactivating agents.</li>
		<li>Wear gloves at all times while handling RNA and change gloves often.</li>
		<li>Use only RNase free plastics to handle RNAs.</li>
	</ol>
	</li>
	<li>Isolating Total RNA from Single neurons:
	<ol>
		<li>Standard Trizol protocol for RNA isolation can be used to isolate RNAs from single neurons. Briefly add 20% v/v of chloroform to Trizol, mix well by brief vortexing.</li>
		<li>Place the tubes on rotator for 10 min&#160;at 4 &#176;C. Spin the Trizol-Chloroform mix at 12,000 x g for 15 min&#160;at 4 &#176;C.</li>
		<li>Collect the aqueous phase and transfer to a fresh tube.</li>
		<li>Add sodium acetate solution (pH 5.5) to a final concentration of 0.3 M, 100 ng/ml of the coprecipitant GlycoBlue, and 2.5 volumes of 100% ethanol to precipitate RNA.</li>
		<li>Mix the samples well and incubate at -80 &#176;C overnight.</li>
		<li>Spin the samples at 12,000 x g for 20 min&#160;at 4 &#176;C and a small blue pellet will be visible at the bottom of the tube.</li>
		<li>Remove the supernatant and wash the pellet with 850 &#956;l of 75% ice-cold ethanol at 12,000 x g for 10 min&#160;at 4 &#176;C.</li>
		<li>Remove the supernatant carefully and air-dry the RNA pellet for 7-10 min, maximum. 
		NOTE: Make sure that RNA is not left for long to dry as over-drying the RNA pellet makes solubilization very difficult.</li>
		<li>Once dried, resuspend the RNA pellet in 10 &#956;l of RNAse free water and determine RNA concentration. 
		NOTE: Determine RNA concentration using a spectrophotometer. When not using right away, store RNA at -80 &#176;C.</li>
	</ol>
	</li>
	<li>aRNA synthesis from single neurons:
	<ol>
		<li>Amplify the RNA (one or two rounds depending on experimental objective) using commercially available linear RNA amplification system. 
		NOTE: The total RNA from single neurons ranged from ~10-60 ng. Expected yield of first round of amplification is ~100-300 ng and the second round of amplification is ~0.8-1.5 &#956;g of RNA.</li>
	</ol>
	</li>
	<li>Reverse transcription of RNA:
	<ol>
		<li>To generate cDNA from aRNA, use 1.0 &#956;g of aRNA in 20 &#956;l of a reverse transcription reaction. Several kits are commercially available for this purpose.
		 NOTE: cDNA was synthesized according to the manufacturer&#39;s protocol using the following settings on thermal cycler. 
		5 min&#160;at 25 &#176;C 
		30 min&#160;at 42 &#176;C 
		5 min&#160;at 85 &#176;C 
		Hold at 4 &#176;C</li>
		<li>After the reverse transcription step, store the cDNA at -20 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>Primer design and standardization: 
	Several excellent software programs are available, free of charge, for designing primers for real time amplifications.
	<ol>
		<li>Select 18-24 mer oligonucleotides that produce 70-110 bp amplicons and synthesize the primers using commercial sources. 
		NOTE: Two to three primer pairs should be designed for each gene of interest. Each primer set needs to standardized by qPCR. The forward and reverse primer pairs that were used for gene CREB1 (Figure 4) are below:
		 Primer set 1 
		Forward: 5&#39;-TGATTCTGACGCAAAGAAAAGA-3&#39; 
		Reverse: 5&#39;-ACCGTGAGCAGTCAGTTGTAGA-3&#39; 
		Primer set 2 
		Forward: 5&#39;-AGGGAATGTCGATGAAAGAAGA-3&#39; 
		Reverse: 5&#39;-GACACACAGGAAGTATGCCAAA-3&#39;</li>
		<li>To select the best primer to be used for the downstream analyses, monitor Ct value and the shape of the amplification curve in qPCR. 
		NOTE: Primers that give Ct (Cycle threshold) values between 10-35 in the 40 round amplification and smooth curves during the exponential phase of the PCR followed by a smooth plateau are optimal for gene expression analyses.</li>
	</ol>
	</li>
	<li>Quantitative Real Time PCR (qPCR): 
	Note: Use appropriate tubes or plates that are compatible with the qPCR machine.
	<ol>
		<li>Dilute cDNA to 5x&#160;with nuclease free water.</li>
		<li>To 2 &#956;l of diluted cDNA, add 8 &#956;l of a qPCR master mix containing 2 &#956;l of H2O, 5 &#956;l of 2x&#160;SYBR Green master mix, and 1.0 &#956;l of 10 &#956;M (each) forward and reverse primer.</li>
		<li>Close the tubes and seal the plate after pipetting cDNA and master mix. Mix the contents by gentle tapping and spinning down at 1,500 rpm for 30 sec.</li>
		<li>Proceed to setting up the qPCR machine.</li>
	</ol>
	</li>
</ol>

Authors do not have any competing financial interests.

The neuron R15 is involved in regulating cardiovascular, digestive, respiratory, and reproductive systems30. A regularly rhythmic bursting activity of the AP is a feature of R15. As shown in the results section, paired recording of R15 and L7 show that the ganglia preparation has preserved activity of R15 neurons. R15 and L7 neurons responded appropriately to Ach. This ganglia preparation could be maintained up to 8-10 hr&#160;and electrophysiological activity could be continuously monitored. Thus, one could s...

The human brain is extraordinarily complex with almost 100 billion neurons and trillions of synaptic connections. There are almost an equal number of nonneuronal cells that interact with neurons and regulate their function in the brain. Neurons are organized into circuits that regulate specific behaviors. Despite the advances in our understanding of brain functions and neural circuits, little is known about the identity of circuitry components that control a specific behavior. Knowledge of the identities of various components of a circuitry will greatly facilitate our understanding of both cellular and molecular basis of behavior and aid in developing novel therapeutic strategies for neuropsychiatric disorders.
The marine snail Aplysia californica has been a workhorse for determining neuronal circuits underlying specific behaviors1-14. The Aplysia nervous system contains approximately 20,000 neurons that are organized into 9 different ganglia. The neurons of Aplysia are large and can be easily identified based on their size, electrical properties, and position in the ganglia. Aplysia has a rich repertoire of behaviors that can be studied. One of the well-studied behaviors is the gill-withdrawal reflex (GWR). The central components of this reflex are situated in abdominal ganglia. Components of the GWR circuitry have been mapped and contributions of various components determined. Importantly, GWR circuitry undergoes associative and nonassociative learning5,6,15-19. Decades of study on this reflex have also identified several signaling pathways that have a key role in learning and memory20-24.
Several different preparations of Aplysia were used to study cellular and molecular basis of memory storage. These include the intact animal2,3, semi-intact preparation1,7,13,14,16 and reconstitution of major components of neural circuitry25-29. A reduced preparation for exploring Aplysia ganglia for the electrophysiological and molecular analyses of identified neuronal circuits is described here. The following four identified neurons were studied. R15, a bursting neuron, L7 and L11, two different motor neurons and R2, a cholinergic neuron were studied. R2 is the largest neuron described in the invertebrate nervous system. Briefly, this methodology involves protease treatment of ganglia, electrophysiological measurements before and after pharmacological treatments, and isolation of single neurons for quantitative analysis of gene expression. This methodology enables us to combine molecular analyses with simultaneous recording from multiple neurons. This methodology was successfully used to study responses of R15 and L7 neurons to acetylcholine (Ach) by paired intracellular recordings. Following electrophysiological measurements R15 and L7 and other identified neurons such as L11 and R2 were isolated for quantitative polymerase chain reaction (qPCR) analysis of expression of CREB1, a transcription factor important for memory storage.

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			<td height="101" style="height:101px;width:120px;">Aplysia</td>
			<td style="width:123px;">National Aplysia Resource Facility, University of Miami</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">NaCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 3014-1KG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">KCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">P 9333-500G</td>
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		<tr height="27">
			<td height="27" style="height:27px;width:120px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">C5080- 500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:129px;">BP 214-501</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">NaHCO4</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 6297-250G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">HEPES</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">H 3375-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Protease</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:129px;">17105-042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Trizol</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">15596-026</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Chloroform</td>
			<td style="width:123px;">MP Biomedicals</td>
			<td style="width:129px;">2194002</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:120px;">100% Ethanol</td>
			<td style="width:123px;">ACROS</td>
			<td style="width:129px;">64-17-5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:120px;">GlycoBlue</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9515</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">3 M NaOAc, pH 5.5</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9740</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Nuclease free water</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9737</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">MessageAmp II aRNA Amplification Kit</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM1751</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">qScript cDNA SuperMix</td>
			<td style="width:123px;">Quanta Biosciences</td>
			<td style="width:129px;">95048-100</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Power SYBR Green PCR Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">4367659</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">11252-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">15000-08</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Stainless Steel Minutien Pins&#160;</td>
			<td rowspan="2" style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">26002-10 or</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">26002-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Veriti Thermal Cycler</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">Veriti Thermal Cycler</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">5430R Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:129px;">5430R Centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">7900HT Fast Real-Time PCR</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">7900HT Fast Real-Time PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:120px;">Amplifier</td>
			<td>BRAMP-01R</td>
			<td style="width:129px;">NPI Electronics</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Digidata Converter</td>
			<td style="width:123px;">Instrutech ITC-18</td>
			<td style="width:129px;">HEKA ELEKTRONIK</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Micro Manipulator</td>
			<td style="width:123px;">Patch Star</td>
			<td style="width:129px;">Scientifica</td>
		</tr>
	</tbody>
</table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

The weights of animals that were used in this study ranged from 100-200 g. Following the described protocols, we conducted electrophysiological measurements and molecular analysis of neurons of abdominal ganglia isolated from animals ranging from 2-5 g&#160;to 200-300 g.
Standardization of protease treatment is important for successful electrophysiological measurements of neurons in the ganglia. Initially, multiple protease (Dispase) concentrations and durations were used and bursting action p...

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

aplysia-ganglia-preparation-for-electrophysiological-molecular

Research

JoVE Journal

Bioengineering

9.0K Views.  The Scripps Research Institute, Florida. Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Video: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

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 ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...