Name: a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke
Uploaded: 2012-01-02T12:00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

This work was supported by the National Institute of Health, grant No. NIH/NCRR P41-RR 016292 (for ACERT).

1. Materials
All solvents used in this work were reagent grade. The spin trap, including N-tert-butyl-&alpha;-phenylnitrone (PBN) and the standard spin label 2,2,6,6-tetramethyl-1-piperinyoxyl (TEMPO) were obtained from Sigma and were used as supplied. The plant antioxidants were obtained commercially from Swanson, Inc. USA.
2. Preparation of cigarette smoke and analysis of free radicals
<ol>
	<li>To introduce antioxidants into the filter, the antioxidants pycnogenol and grape seed extract were first dissolved in 95% ethanol, while lycopene was dissolved in acetone. The solvent volumes were different depending on the antioxidant solubility.</li>
	<li>The amount of antioxidants of 0.4 mg/filter was used. They were then coated with 10 mg of activated carbon. For this purpose activated carbon was stirred for ~12 hrs with the antioxidant solution in anaerobic conditions, filtered and dried under vacuum.</li>
	<li>Antioxidants were then introduced into the conventional acetate filter (CA filter). For this purpose, the filter was cut into two pieces. The coated plant antioxidants were inserted between two pieces of the filter and wrapped with a piece of tape to form a filter - antioxidant -filter sandwich (Fig. 1A). This combined filter was subsequently attached to cigarette rods containing tobacco. The control filter was made in the same way as the antioxidant filter except no antioxidant was added.</li>
	<li>Prior to the smoking simulation, the research cigarettes were unpacked and kept in a constant humidity environment (20 &deg;C, 60% relative humidity) using a saturated NaBr solution for a minimum of 2 days.</li>
	<li>Smoking simulation for routine analysis was performed at ambient temperature using a single-port smoking device which, as shown in Fig.1, consists of a water aspirator or GAST DOA-P104-BN Vacuum Pump/Air Compressor (Benton Harbor, Mich) connected to the spin-trapping assembly through a T-junction with one end open. Puffs were performed by plugging the open end which remained open between puffs. The gas flow was checked by flow meter and set at ~2.2 SCFH = 17.5 mL/s by adjusting a valve placed between the pump and the vent.</li>
	<li>The research cigarettes were smoked under the condition of puff volume of 35 mL for a duration of 2 seconds repeated every 60 seconds, similar to1. Smoking simulation for quantitative estimation of gas-phase free radicals was performed in conjunction with the spin trapping system as shown in Fig. 1.</li>
	<li>Ten intense puffs (35 mL/puff) were taken for each cigarette. Gas-phase free radicals were collected by passing the MCS through a Cambridge filter pad and then introduced into the spin trapping solution (0.05 M PBN in benzene, 2.0 mL).</li>
	<li>After the last puff, the bubbled trapping solution was readjusted to its initial volume (2.0 mL) with the same benzene. An aliquot was transferred into a ~250 mm long, 3 mm ID glass tube sealed at one end.</li>
	<li>The trapping solution was deoxygenated using a freeze-pump-thaw procedure. It was frozen under liquid nitrogen, and a vacuum was applied. Then it was thawed under argon atmosphere, allowing trapped gas bubbles to escape, and frozen again. After this cycle was repeated three times, the tube was flame-sealed under vacuum and used in further ESR measurements. This step is required because oxygen, which is well soluble in benzene, broadens ESR lines of organic radicals. Deoxygenation dramatically improves the signal-to-noise ratio.</li>
	<li>X-Band ESR spectra were recorded on a Bruker EMX spectrometer at a frequency of 9.34 GHz under standard conditions. The spectrometer settings used in most experiments were: center field 3312.5G, scan width 80G, modulation amplitude 0.5G, time constant 82 microseconds, scan time 40 sec.</li>
	<li>The spin trap adducts are relatively stable at the experiment conditions, which in some cases required 25 accumulations, which typically took ~20 min. However, after 12 hrs the intensity of the ESR signals in benzene solution decreased by a factor of ~5.</li>
	<li>To quantify the concentration of the trapping adduct, its initial first-derivative ESR spectrum was integrated. The resulting absorption spectrum shows a broad singlet background, most likely due to soot/tar products contained in the smoke. 
	 
	After subtraction of this background the separated triplet of the trapping adduct was integrated one more time (Fig. 2).</li>
</ol>
3. Representative Results
Most of the free radicals in burning cigarette-produced smoke (gas phase) are instantaneous and unstable. In order to observe these radicals a spin trap technique is employed. It captures gas-phase free radicals by transforming them into a spin adduct which is more stable and can be detected by ESR (Fig.1). In the present study, the spin-trap solution of 0.05 M PBN was used to collect smoke gas phase free radicals, which are a mixture of oxygen- and carbon- centered radicals which are difficult to separate15. In our case, however, the observed hyperfine splitting constants aN = 13.7G and aH = 1.95G are very similar to the corresponding values for trapping adducts of alkoxyl free radicals (RO.)2, suggesting that they are the major product. We showed that weak ESR signals and low reproducibility observed in our initial measurements (Fig. 2) were due to the moisture in the smoke flow. To resolve this problem, we added a liquid nitrogen (LN2) trap between the Cambridge filter and the spin trap solution. The LN2 trap removed water from the MCS flow by quick freezing and capturing it on the inner wall of the glass tube. This greatly improved ESR signals and allowed for highly reproducible results (Fig. 3).
The amount of trapped free radicals was determined using a reference sample. For control samples without antioxidants, the typical adduct concentration in benzene estimated by comparison of its double integral spectrum with a double integral spectrum for known concentration of TEMPO was 1.24 &mu;M (Fig.2). Since the amount of air passed through each cigarette during smoking was ~350 ml, this gives an estimate for the free radical concentration in the gas phase of MCS of ~7.1X10-9M, and a total number of radicals trapped from the gas phase of a whole cigarette of ~1.5X1015. An estimate for the total amount of free radicals in the smoke of a whole cigarette, including both gas and particulate phase, was ~1016 free radicals9.
Different levels of the scavenging effect of plant antioxidants on gas-phase free radicals in main stream cigarette smoke were observed. Their scavenging rates were presented in Fig. 4. Lycopene and grape seed extract showed the highest rates while lower rate was observed for pycnogenol (Fig.4).
<img alt="Figure 1" src="/files/ftp_upload/3406/3406fig1.jpg" /> 
Figure 1. A diagram of improved smoking simulation design for collecting gas-phase free radicals in mainstream cigarette smoke (MCS) using spin trap. MCS was drawn by a water aspirator through CA filter, then Cambridge filter (yellow filter) and passed through a liquid nitrogen trap to remove H2O. The gas-phase products finally went to spin trap and bubbled through the spin trap solution. Plant antioxidant was placed between two pieces of conventional acetate filters (Enlarged in circle A) attached to cigarette to scavenge free radicals in MCS.
<img alt="Figure 2" src="/files/ftp_upload/3406/3406fig2.jpg" /> 
Figure 2. A quantitative estimate for the spin-trapping adduct concentration in benzene requires subtracting a broad background signal from the first integral spectrum. The hyperfine splitting parameters for the spin-trapping adduct are aH = 1.95 G, aN = 13.7G.
<img alt="Figure 3" src="/files/ftp_upload/3406/3406fig3.jpg" /> 
Figure 3. Passing through LN2 trap substantially improves the quality of ESR signals obtained by spin-trapping of MCS.
<img alt="Figure 4" src="/files/ftp_upload/3406/3406fig4.jpg" /> 
Figure 4. Effect of natural antioxidants on the concentration of free radicals in MCS. The relative signal intensities are: control - 100%, pycnogenol - 55%, grape seed extract - 12%, lycopene - 10%.

<ol>
	<li>Baum, S. L., Anderson, I. G. M., Baker, R. R., Murphy, D. M., Rowlands, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spin+resonance+and+spin+trap+investigation+of+free+radicals+in+cigarette+smoke:+development+of+a+quantification+procedure.">Electron spin resonance and spin trap investigation of free radicals in cigarette smoke: development of a quantification procedure.</a> Analytica Chimica. Acta. 481, 1-13 (2003).</li><li>Bluhm, A. L., Weinstein, J., Sousa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+tobacco+smoke.">Free radicals in tobacco smoke.</a> Nature. 229, 500-500 (1971).</li><li>Church, D. F., Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-radical+chemistry+of+cigarette+smoke+and+its+toxicological+implications.">Free-radical chemistry of cigarette smoke and its toxicological implications.</a> Environ. Health. Perspect. 64, 111-126 (1985).</li><li>Deliconstantinos, G., Villiotou, V., Stavrides, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenging+effects+of+hemoglobin+and+related+heme+containing+compounds+on+nitric+oxide,+reactive+oxidants+and+carcinogenic+volatile+nitrosocompounds+of+cigarette+smoke.+a+new+method+for+protection+against+the+dangerous+cigarette+constituents.">Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. a new method for protection against the dangerous cigarette constituents.</a> Anticancer. Res. 14, 2717-2726 (1994).</li><li>Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+carcinogens+and+lung+cancer.">Tobacco smoke carcinogens and lung cancer.</a> Journal of the National Cancer Institute. 91, 1194-1210 (1999).</li><li>Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., Akimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+chemistry+of+cigarette+smoke+and+its+implication+in+human+cancer.">Free radical chemistry of cigarette smoke and its implication in human cancer.</a> Anticancer. Res. 17, 433-437 (1997).</li><li>Nair, A. K., Brandt, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+smoking+on+health+care+costs.">Effects of smoking on health care costs.</a> Journal of Oklahoma State Medical Association. 93, 245-250 (2000).</li><li>Nishizawa, M., Kohno, M., Nishimura, M., Kitagawa, A., Niwano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Peroxyradicals+in+Cigarette+Smoke+and+the+Scavenging+Effect+of+Shikonin,+a+Naphthoquinone+Pigment.">Presence of Peroxyradicals in Cigarette Smoke and the Scavenging Effect of Shikonin, a Naphthoquinone Pigment.</a> Chem. Pharm. Bull. 53, 796-799 (2005).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+the+involvement+of+free+radical+reactions+in+chemical+carcinogenesis.">Cigarette smoke and the involvement of free radical reactions in chemical carcinogenesis.</a> Br. J. Cancer. 8, 19-23 (1987).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+radicals+and+the+role+of+free+radicals+in+chemical+carcinogenicity.">Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity.</a> Environ. Health. Perspect. 105, 875-882 (1997).</li><li>Rozzi, N. I., Singh, R. K., Vierling, R. A., Watkins, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+of+lycopene+from+tomato+processing+byproducts.">Supercritical fluid extraction of lycopene from tomato processing byproducts.</a> J. Agric. Food. Chem. 50, 2638-2643 (2002).</li><li>Shopland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+use+and+its+contribution+to+early+cancer+mortality+with+a+special+emphesis+on+cigarette+smoking.">Tobacco use and its contribution to early cancer mortality with a special emphesis on cigarette smoking.</a> Environmental. Health. Perspectives. 103, 131-142 (1995).</li><li>Shrikhandle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wine+products+with+health+benefits.">Wine products with health benefits.</a> Food Res. Int. 33, 469-474 (2000).</li><li>Valavanidis, A., Haralambous, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+by+electron+paramagnetic+resonance+of+free+radical+species+in+the+mainstream+and+sidestream+smoke+of+cigarettes+with+conventional+acetate+filters+and+'bio-filters'.">A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'.</a> Redox. Rep. 6, 161-171 (2001).</li><li>Valavanidis, A., Vlachogianni, T., Fiotakis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke:+involvement+of+reactive+oxygen+species+and+stable+free+radicals+in+mechanisms+of+oxidative+damage,+carcinogenesis+and+synergistic+effects+with+other+respirable+particles.">Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.</a> International Journal of Environmental Research and Public Health. 6, 445-462 (2009).</li></ol>

No conflicts of interest declared.

A reliable estimate of the effect of different free-radical scavengers in tobacco smoke requires a reproducible technique for quantitative detection of free radicals. Previously1, it has been shown that small volumes of higher concentration spin trap solutions in non-polar solvents are most effective at trapping free radicals from tobacco smoke. Cigarette smoke always contains water vapor from combustion of organic compounds and the residual moisture in tobacco which can end up in the trapping solvent. This admixture of water in the PBN spin trap solution substantially decreases the lifetime of the spin trapping adducts and the intensity of their ESR signals. Removing this moisture by simple passing MCS through a U-shaped tube cooled with liquid nitrogen dramatically improved the quality of ESR spectra in our experiments, even though some fraction of gas-phase radicals might be also trapped on the frozen surface. 
Using this technique we compared relative efficiency of different radical-scavenging natural compounds introduced into cigarette filters. We found that lycopene and grape seed extract immediately after incorporating into cigarette filters are able to scavenge up to 90% of free radicals from the gas phase MSC. Such high scavenging capacity ranks these cheap readily available natural compounds among the most efficient reported free radical protectors like haemoglobin and shikonine8,14. Yet, in our experiments cigarette filters loaded with studied natural antioxidants lost a noticeable part of their scavenging capacity after a week of storage at room temperature. Solving this problem can stimulate future application of lycopene and grape seed extract in commercial cigarette filters.

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

a-protocol-for-detecting-scavenging-gas-phase-free-radicals

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Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
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Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

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A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

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We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

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Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

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An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

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This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...