The authors would like to sincerely thank the Department of Cell Biology, Central South University, Changsha, China, for providing the instrumentation facilities.

NOTE: Before preparing the extract, the obtained bark material was washed twice using deionized water and dried in a shaded place. The shade-dried barks were stored in an airtight container.
1. Preparation of Eucommia ulmoides bark extract
<ol>
	<li>Chop the bark collected from the Eucommia ulmoides tree into small pieces using scissors.</li>
	<li>Wash the chopped bark materials twice with double distilled water.</li>
	<li>Dry the bark pieces under 37 &#176;C for 24 h at shaded conditions.
	<ol>
		<li>Adjust the duration of the drying process based on the quantity of bark utilized for the study. Ensure the barks are completely dried before chopping the barks into small pieces. Avoid direct sunlight.</li>
	</ol>
	</li>
	<li>Transfer 20 g of shade-dried barks into a conical flask containing 220 mL of sterile double distilled water and heat at 130 &#176;C for 20 min.
	<ol>
		<li>The color of the reaction solution changes to light yellow. The changes in color of the solution occur after 10 min. Allow the solution to heat for another 10 min. Adjust the volume of double distilled water based on the sample quantity.</li>
	</ol>
	</li>
	<li>Store the crude extract containing polyisoprene at 4 &#176;C for further use. The formation of a thread-like structure indicates the presence of polyisoprene in the extracts.</li>
</ol>
2. Biosynthesis of E. ulmoides bark-mediated ZnO nanoparticles
<ol>
	<li>Add 1 M Zinc nitrate dihydrate Zn (NO3)2 to 50 mL of deionized water in a 500 mL conical flask. Stir continuously using magnetic stirring (60 RPM). The Zn (NO3)2 takes 30 min to dissolve completely.</li>
	<li>Add 15 mL of E. ulmoides bark extract dropwise to 20 mL of 1 M Zinc nitrate dihydrate (Zn (NO3)2) solution.</li>
	<li>Place the covered reaction mixture onto a magnetic stirrer, switch on the stirrer, and spin at (60 RPM) for 3 h.</li>
	<li>Add 1 N sodium hydroxide NaOH (3 mL) solution dropwise to the reaction mixture to adjust the pH to 9. Add NaOH until the solution mixture turns milky white and the pH is not more than 9. The solution becomes a milky white color when ZnO nanoparticles are formed.
	<ol>
		<li>The preparation of 1 M of Zinc nitrate dihydrate Zn (NO3)2 solution volume may vary based on the experimental needs. Make sure to use the freshly prepared bark extracts. If the pH exceeds 10, it will result in nanoparticle aggregation.</li>
	</ol>
	</li>
	<li>Transfer the synthesized Eu-ZnO-NPs to a 50 mL centrifuge tube and centrifuge it at 100 x g for 5 min at 4 &#176;C. 
	NOTE: The impurities can be avoided by immediate washing.</li>
	<li>Collect the washed Eu-ZnO-NPs in a glass plate and dry them at 45-50 &#176;C for 1 h in a hot air oven. 
	NOTE: If the Eu-ZnO-NPs are kept for more than 30 min can affect the physicochemical nature of the nanoparticles</li>
</ol>
3. Size confirmation using TEM
<ol>
	<li>Prepare 1 mg/mL of nanoparticles in DDH2O and load 5 &#181;L of Eu-ZnO-NP sample in the copper grid and wait until it completely dries. Vortex the sample prior to loading on the copper grid.</li>
	<li>Load the copper grid containing Eu-ZnO-NPs on the TEM sample holder and acquire images at 50x and 100x magnifications. 
	NOTE: Grids must be picked up using tweezers during this step.</li>
</ol>
4. Cytotoxicity assessment
<ol>
	<li>Seed 1 &#215; 104 HUVECs in each well of a 96 well plate and place it in a 5% CO2 37 &#176;C incubator.</li>
	<li>Add 10 &#181;L of various concentrations 0, 10, 20, 30, 40, and 50 &#181;g/mL of Eu-ZnO-NPs into the 90% confluent cells and incubate it for 24 h.</li>
	<li>After incubation, remove the old medium without disturbing the cells, add 10 &#181;L of CCK-8 solution to each well containing 90 &#181;L of fresh DMEM medium, and incubate in a 5% CO2, 37 &#176;C incubator.</li>
	<li>Measure the absorbance of cells treated with CCK8 solution at 450 nm using a spectrophotometer. 
	NOTE: The absorbance was measured immediately within 15 min to avoid the changes in absorbance.</li>
</ol>
5. Preparation of HUVEC cells for scratch assay
<ol>
	<li>Seed an appropriate (1 &#215; 105) quantity of HUVECs in 12 well culture plates containing Dulbecco&#39;s modified Eagle medium with 10% fetal bovine serum (FBS) and 1% pen-strep and incubate it in a 5% CO2 37 &#176;C incubator.
	<ol>
		<li>Before performing the scratch assay, check the confluency using the inverted microscope. 
		NOTE: For performing the scratch assay 6 well plates or 12 plates will be utilized based on the requirements, and the cell density will vary for different culture plates. The usage of 70%-80% confluent cells is ideal and recommended for scratch assay. The volume of DMEM medium may vary based on the culture plates utilized in the study. For instance, 6 well plates require 1-1.5 mL of culture medium, and 12 well plates require 0.5-1.0 mL of growth medium.</li>
	</ol>
	</li>
	<li>Gently make a scratch using a sterile 200 &#181;L pipette tip in the representative wound with a wound width of 200 &#181;m.</li>
	<li>Ensure that the tip used to make scratches on the cell monolayer makes contact with the surface of the cells. 
	NOTE: Every time a scratch is made, use the sterile tip.</li>
	<li>Remove the complete medium and wash the HUVEC monolayer using 1 mL of 1x PBS to remove the detached cells. 
	NOTE: Ensure the detached monolayer cells are completely removed from the respective wells. Confirm that no damage has occurred in the wound-created area.</li>
	<li>To evaluate the wound healing potential of synthesized Eu-ZnO-NPs, add 0 (Control), 10, and 20 &#181;g/mL concentrations of Eu-ZnO nanoparticles combined with a complete medium with 10% FBS into the wells. Maintain the experimental plates at 37 &#176;C in a 5% CO2 incubator. 
	NOTE: Do not disturb the cell monolayer while adding the fresh, complete medium. Ensure that the monolayer cells remain undisturbed during the incubation.</li>
	<li>Acquire microphotograph at 0 h and 24&#160;h using an inverted microscope. Use the annotation tool to measure the wound closure in different time intervals.
	<ol>
		<li>Calculate the percentage of wound closure using the following formula: Wound closure (%) = [(cell migration (in &#181;m) at 0 h - cell migration (in &#181;m) at 24 h)/ cell migration (in &#181;m) at 0 h] x 100.</li>
	</ol>
	</li>
</ol>

Bioactive ZnO Nanoparticles from Eucommia ulmoides for Wound Healing Enhancement

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L. <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+medicinal+plants+in+the+process+of+wound+healing:+a+literature+review.">Use of medicinal plants in the process of wound healing: a literature review.</a> Pharmaceuticals. 17 (3), 303 (2024).</li><li>Budovsky, A., Yarmolinsky, L., Ben-Shabat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+medicinal+plants+on+wound+healing.">Effect of medicinal plants on wound healing.</a> Wound Repair Regen. 23 (2), 171-183 (2015).</li><li>Yazarlu, O., 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=Perspective+on+the+application+of+medicinal+plants+and+natural+products+in+wound+healing:+A+mechanistic+review.">Perspective on the application of medicinal plants and natural products in wound healing: A mechanistic review.</a> Pharmacol Res. 174, 105841 (2021).</li><li>Zhu, M. Q., Sun, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eucommia+ulmoides+Oliver:+a+potential+feedstock+for+bioactive+products.">Eucommia ulmoides Oliver: a potential feedstock for bioactive products.</a> J Agric Food Chem. 66 (22), 5433-5438 (2018).</li><li>Peng, M., Zhou, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+and+potential+application+of+extracts+and+compounds+from+different+medicinal+parts+(bark,+leaf,+staminate+flower,+and+seed)+of+Eucommia+ulmoides:+A+review.">Biological properties and potential application of extracts and compounds from different medicinal parts (bark, leaf, staminate flower, and seed) of Eucommia ulmoides: A review.</a> Heliyon. 10 (6), e27870 (2024).</li><li>Xing, Y. Y., 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=Inhibition+of+rheumatoid+arthritis+using+bark,+leaf,+and+male+flower+extracts+of+Eucommia+ulmoides.">Inhibition of rheumatoid arthritis using bark, leaf, and male flower extracts of Eucommia ulmoides.</a> Evid Based Complement Alternat Med. 2020, 3260278 (2020).</li><li>Guo, M., 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=Quantitative+detection+of+natural+rubber+content+in+Eucommia+ulmoides+by+portable+pyrolysis-membrane+inlet+mass+spectrometry.">Quantitative detection of natural rubber content in Eucommia ulmoides by portable pyrolysis-membrane inlet mass spectrometry.</a> Molecules. 28 (8), 3330 (2023).</li><li>Yusof, H. M., Rahman, N. A., Mohamad, R., Zaidan, U. H., Samsudin, A. 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The authors have nothing to disclose

The E. ulmoides bark, seed, and leaves are believed to exhibit numerous health benefits. Our results have shown that the synthesis of EU-ZnO nanoparticles was achieved using a simple and cost-effective approach. The aqueous extract was utilized to synthesize the nanoparticles. Heating the bark material at high temperatures may cause degradation of some phytoconstituents and lower the extraction efficiency. The phytoconstituents present in the aqueous herbal extract can reduce the metal ions to form nanoparticles...

Medicinal plants and plant-derived compounds have been shown to exhibit numerous health benefits1. The World Health Organization (WHO) reported that 80% of the global population depends upon traditional medicinal plants for primary health care. China is well-recognized and popular for its Traditional Chinese Medicine (TCM) practices. Chinese medicinal herbs have been reported to treat various diseases and utilized for their biological potential. Medicinal plants serve as reservoirs for bioactive compounds and multiple therapeutic roles. Medicinal plants have also been utilized to treat the wounds. There are several types of approaches applied to treat chronic wounds2. A recent investigation revealed that medicinal plants were involved in the wound healing process by providing favorable conditions for healing, free from infections, and fastening tissue regeneration3. Meanwhile, the antibacterial and antifungal properties of bioactive compounds present in medicinal plants can help to cure wounds and fasten wound healing efficiency4.
Metal-based nanomaterials are gaining attention due to their biocompatible and biodegradable properties. Eucommia ulmoides, commonly called the Chinese rubber tree, is a native species of China. The leaves and bark of the tree are used in medicinal practices. Most importantly, the plant species was cultivated in the central and western provinces of China5. Peng et al.6reported that leaves, barks, and staminate flowers were edible with therapeutic potential. In addition, E. ulmoides serve as the best source of lignans, phenylpropanoids, iridoids, flavonoids, amino acids, and trace elements. Furthermore, the bark has been utilized for various biomedical applications such as controlling blood pressure, lowering fat, and promoting antiosteroporosis and hypoglycemic activity7. Hence, undoubtedly, it has been proven that E. ulmoides bark extract has a long history in traditional Chinese medicine. Earlier reports suggested that natural polymer polyisoprene is rich in the barks of Eucommia ulmoides8. Based on the information above, the present work aims to fabricate nanomaterials using Eucommia ulmoides bark extracts. The combination of zinc with bark extract is an attractive choice for preparing nanomaterials. Overall, the ultimate aim of the present investigation was to fabricate a novel hybrid nanomaterial for wound healing applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 well plate</td>
			<td>NEST</td>
			<td>703011</td>
			<td>Used for cell culture and assay</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SCILOGEX</td>
			<td>SC1406</td>
			<td>Used to separate the nanoparticles from the colloidal mixture</td>
		</tr>
		<tr>
			<td>Centrifuge Tube &#8211; 15 mL</td>
			<td>BIOFIL</td>
			<td>CFT-312150</td>
			<td>To centrifuge the synthesized solution&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge tube &#8211; 5 mL</td>
			<td>Biosharp&#160;</td>
			<td>BS-50-CM-S</td>
			<td>To store the nanoparticles</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo scientific</td>
			<td>3010</td>
			<td>To culture the HUVEC cells</td>
		</tr>
		<tr>
			<td>Denoised water</td>
			<td>Millipore</td>
			<td>Not applicable&#160;</td>
			<td>For preparation of the extract&#160;</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Cytiva</td>
			<td>SH30243.01</td>
			<td>Used for cell culture work</td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>Thermo scientific</td>
			<td>42600060</td>
			<td>To hold the filter paper during the filtration</td>
		</tr>
		<tr>
			<td>Glass beakers</td>
			<td>Borosilicate&#160;</td>
			<td>1102-50</td>
			<td>Used to prepare the aqueous extract</td>
		</tr>
		<tr>
			<td>Hot air oven</td>
			<td>Genetimes</td>
			<td>Not Applicable&#160;</td>
			<td>Used to dry the nanoparticles and collect in the powder form</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>KYLIN-BELL</td>
			<td>GL-5250-A</td>
			<td>Used for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse</td>
			<td>Ts2</td>
			<td>Used to take microphotographs&#160;</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>NEST</td>
			<td>753001</td>
			<td>Used to collect the nanoparticles&#160;</td>
		</tr>
		<tr>
			<td>Pipette 1 mL</td>
			<td>Lab Science&#160;</td>
			<td>YEA17AD0055580</td>
			<td>To take/add the specific volume of solution/extract</td>
		</tr>
		<tr>
			<td>Pipette tips 1 mL</td>
			<td>SAINING&#160;</td>
			<td>3014200-T</td>
			<td>To take/add the specific volume of solution/extract</td>
		</tr>
		<tr>
			<td>PTFE Magnetic Mixer Stir Bars</td>
			<td>LAN RAN</td>
			<td>Not applicable&#160;</td>
			<td>Used for nanomaterial synthesis process</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma Alrich</td>
			<td>71690</td>
			<td>Used to adjust pH during the synthesis</td>
		</tr>
		<tr>
			<td>Stainless Scissor</td>
			<td>Deli</td>
			<td>6034</td>
			<td>Used for chopping the bark materials</td>
		</tr>
		<tr>
			<td>T25 tissue culture flask</td>
			<td>NEST</td>
			<td>707001</td>
			<td>Used to maintain the cells&#160;</td>
		</tr>
		<tr>
			<td>Weighing Balance&#160;</td>
			<td>Radwag&#160;</td>
			<td>AS220R2</td>
			<td>Used to weigh the chemicals&#160;</td>
		</tr>
		<tr>
			<td>Whatman filter paper No.1</td>
			<td>Newstar</td>
			<td>GB/T1914-2017</td>
			<td>Used to filter the extract for synthesis</td>
		</tr>
		<tr>
			<td>Zinc nitrate&#160;</td>
			<td>Sigma Alrich</td>
			<td>13778-30-8</td>
			<td>Used as precursor for the nanoparticle&#8217;s synthesis</td>
		</tr>
	</tbody>
</table>

formulation of zinc-based nanomaterials using the eucommia ulmoides bark extract and their wound healing potential

The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here aims to explore the preparation of zinc oxide (ZnO) nanoparticles using the Eucommia ulmoides bark-mediated polyisoprene-rich aqueous extract. Meanwhile, the proposed protocol is associated with the preparation of wound healing material by easing the process. In addition, the wound-healing potential of the synthesized nanoparticles (Eu-ZnO-NPs) was evaluated using a simple scratch assay on a human umbilical vein endothelial cell (HUVEC) monolayer. After 24 h of treatment with Eu-ZnO-NPs, the cell proliferation and migration of HUVEC cells were assessed. At the end of the study, cell proliferation and migration were observed in scratched monolayer treated with different concentrations of Eu-ZnO-NPs, whereas poor cell migration and proliferation rates were observed in control cells. Of the chosen concentrations, 20 &#181;g/mL Eu-ZnO nanomaterials showed better cell migration and enhanced wound healing potential.

The present investigation aims to synthesize nanoparticles using the barks of Eucommia ulmoides tree. The bark material was dried completely in a shaded environment (Figure 1). The bark materials were utilized to prepare the hot water aqueous crude extract by heating the samples at 130 &#176;C for 20 min. A slight alteration in the temperature and duration may disrupt the phytocompounds and make them unsuitable for preparing the aqueous extract. The aqueous crude bark extracts of <e...

Formulation of Zinc-Based Nanomaterials using the Eucommia ulmoides Bark Extract and their Wound Healing Potential

Here, we present a protocol to synthesize the zinc oxide (ZnO) nanoparticles using the polyisoprene-rich aqueous extract obtained from Eucommia ulmoides tree bark. The wound-healing potential exhibited by the synthesized ZnO nanoparticles on human umbilical vein endothelial cells (HUVECs) was evaluated using scratch assay, a simple, cost-effective, and efficient method.

The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here ...

formulation-zinc-based-nanomaterials-using-eucommia-ulmoides-bark

Research

JoVE Journal

Biology

263 Views.  Central South University. Here, we present a protocol to synthesize the zinc oxide (ZnO) nanoparticles using the polyisoprene-rich aqueous extract obtained from Eucommia ulmoides tree bark. The wound-healing potential exhibited by the synthesized ZnO nanoparticles on human umbilical vein endothelial cells (HUVECs) was evaluated using scratch assay, a simple, cost-effective, and efficient method. 

Video: Formulation of Zinc-Based Nanomaterials using the Eucommia ulmoides  Bark Extract and their Wound Healing Potential

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

Formulation of Zinc-Based Nanomaterials using the Eucommia ulmoides Bark Extract and their Wound Healing Potential

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Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

Assessment of Bone Fracture Healing Using Micro-Computed Tomography