Name: using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants
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1. Culture E. coli OP50
<ol>
	<li>Prepare 1 M sodium hydroxide solution by dissolving 4 g of sodium hydroxide in 100 mL water.</li>
	<li>Prepare lysogeny broth (LB) medium by dissolving 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride with 1 L of ultrapure water in a 1 L conical flask. Adjust the pH to 7.0 with 1 M sodium hydroxide solution.</li>
	<li>Aliquot the LB liquid medium from step1.2 into 20 conical flasks (maximum allowable volume: 100 mL) with 50 mL medium in each. Cover the conica...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenerational+comparison+of+developmental+and+reproductive+toxicities+in+zearalenone+exposed+Caenorhabditis+elegans.">Transgenerational comparison of developmental and reproductive toxicities in zearalenone exposed Caenorhabditis elegans.</a> Asian Journal of Ecotoxicology. 11 (4), 61-68 (2016).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+dlegans.">The genetics of Caenorhabditis dlegans.</a> Genetics. 77, 71-94 (1974).</li><li>Emmons, S., Klass, M., Hirsch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+constancy+of+DNA+sequences+during+development+and+evolution+of+the+nematode+Caenorhabditis+elegans.">An analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 1333-1337 (1979).</li><li>Van Gilst, M. 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In order to successfully conduct the described protocol, the following suggestions should be taken into consideration. Perform the overall experimental operations in a sterile environment. Improper operation may result in contamination of the E. coli strains, e.g., fungi and mites may hinder the normal growth of C. elegans and therefore affect the experimental results. In the section describing cultivating C. elegans, observe the growth scale of C. elegans on the NGM agars by naked eye...

The application and waste management of chemicals is highly dependent upon the information of their effects at certain concentrations. Notably, time is another essential element between effects and concentrations. That is to say, chemicals, especially those at low concentrations in the actual environments, need time to provoke measurable effects1. Therefore, researchers arrange different lengths of the exposure duration in animal experiments, and even cover the whole life cycle. For example, mice were exposed to nicotine for 30, 90 or 180 days to study its toxic effects 2. Yet, such exposure durations are still not enough to elucidate the long-term effects of pollutants (e.g., persistent organic pollutants [POPs]) that can last over generations of organisms in the environment. Therefore, studies on effects over generations are gaining more and more attention.
There are two main aspects in generational effect studies. The first one is the trans-generational (TG) effect study which can robustly test whether chemical exposure to parents can result in any consequences on the offspring3. The second one is a multi-generational effect study which is more systematic with considerations in both exposure and residual effects. On the one hand, the multi-generational exposure (MGE) effects are used to illustrate adaptive responses in the animals to the long-term challenging environments. On the other hand, the multi-generational residual (MGR) effects are used to demonstrate the long-term residual consequences after exposure, since maternal exposure is accompanied with embryo exposure to the first offspring and germ-line exposure to the second offspring which makes the third offspring as the first generation completely out of exposure4.
Although mammals (e.g., mice) are model organisms in toxicity studies especially in relation to human beings, their application in studying generational effects is quite time-consuming, expensive and ethically concerning 5. Accordingly, organisms including crustacean Daphnia magna6, insect Drosophila melanogaster7 and zebrafish Danio rerio8, provide alternative choices. Yet, these organisms either lack similarities to human beings, or require specific equipment in studies. 
Caenorhabditis elegans is a small free-living nematode (approximately 1 mm in length) with a short life-cycle (approximately 84 h at 20 &#176;C)9. This nematode shares many biological pathways conservative to human beings, and therefore it has been widely employed to illustrate effects of various stresses or toxicants10. Notably, 99.5% of the nematodes are hermaphrodites making this organisms extremely suitable in studying generational effects, e.g., TG effects of heavy metals and sulfonamides3,11, MGE effects of gold nanoparticles and heavy metals12 and temperature13, MGR effects of sulfonamide14, and both MGE and MGR effects of gamma irradiation15 and lindane4. Furthermore, comparable results were found between the effects of chemicals (e.g., zearalenone) on the development and reproduction of mice and C. elegans16,17, which would provide an advantage to extrapolate effects from this small animal to human beings.
Both TG and MG effect studies are time consuming and need careful design and performance. Notably, differences existed in life-stage choices, exposure conditions and generation separation methods in the aforementioned studies. Such differences hindered the direct comparison among the results and hampered further interpretation of the results. Therefore, it is imperative to establish uniform protocols to guide TG and MG effects studies, and also to provide a bigger picture to reveal similar patterns of various toxicants or pollutants in long-term consequences. The over goal of the present protocols will demonstrate clear operation processes in studying trans- and multi-generational effects with C. elegans. The protocols will benefit researchers that are interested in studying the long-term effects of toxicants or pollutants.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="684">
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		<tr height="37">
			<td height="37" style="height:37px;width:191px;">&#160;agar powder</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td style="width:123px;">9002-18-0</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">79nnHT Fast Real-Time PCR System</td>
			<td>&#160;Applied Biosystems&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">96-well sterile microplate</td>
			<td style="width:200px;">Costar&#65292;Corning&#65292;America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Autoclave sterilizer</td>
			<td style="width:200px;">Tomy, Tomy Digital Biology, Japan</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Biosafety cabinet</td>
			<td style="width:200px;">LongYue, Shanghai longyue instrument equipment co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">calcium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">centrifuge&#160; 5417R</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Centrifuge tubes</td>
			<td style="width:200px;">Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">cholesterol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">57-88-5</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Dimethyl sulfoxide</td>
			<td style="width:200px;">VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">disodium hydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">7558-79-4</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">ethanol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Filter</td>
			<td style="width:200px;">Thermo, Thermo Fisher Scientific, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">incubator</td>
			<td style="width:200px;">YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">inoculating loop</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">K2HPO4&#8226;3H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>16788-57-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">kraft paper</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Mcroplate Reader</td>
			<td style="width:200px;">Boitek, Boten apparatus co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MgSO4&#8226;7H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Microscopes XTL-BM-9TD</td>
			<td style="width:200px;">BM, Shanghai BM optical instruments manufacturing co. Ltd, China&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petri dishes</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Pipette</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Potassium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">potassium dihydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Qiagen RNeasy kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">QuantiTect SYBR Green RT-PCR kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">RevertAid First Strand cDNA Synthesis Kit</td>
			<td style="width:200px;">Thermo Scientific, Wilmington, DE, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium hydroxide</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">sodium hypochlorite solution</td>
			<td style="width:200px;">Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China</td>
			<td style="width:123px;">7681-52-9</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">tryptone</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>73049-73-7</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">yeast extract</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>119-44-8</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
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using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Here, we describe protocols for studying effects of chemicals over generations using C. elegans in trans-generational (TG), multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. Our own research results are presented as examples. One study presents the TG effects of heavy metals on locomotion behavior3. The other two studies present MGE and MGR effects of sulfomethoxazole and lindane on the reproduction and biochemical and...

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Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

The presented protocol will facilitate studies on the trans and the multi-generational effects of proteins that can exist for a long time in the environment. We employed a free-living nematode with 99.5 as hermaphrodites to save time and effort in studying trans and multi-generational effects. This technique can be used to demonstrate the long-term impacts of end drugs because the nematode in the present protocol shares many conservative mechanisms or pathways with humans.This method can provide insight to the fields of pharmaceutical synthesis and environmental management because they need feedback of potential hazards of chemicals. This method can be applied to Daphnia magna with more focus on aquatic toxicity. Also Drosphila melanogaster to further explore influence on gender.He or she would be stuck with idea that studying effects over generations is time and energy-consuming. Don't get frightened at first impression. Following the protocol will earn you fruitful outcome.To culture E.coli, pipette 200 microliters from the bacterial suspensions into 50 milliliters of LB medium in a flask. Or use an inoculating loop to pick a small colony from NGM agars and place it in the LB medium. To culture C.elegans, under a microscope, count the nematodes on the stacking NGM agars prepared earlier.When the number reaches close to 2, 000, use a sterile tip to cut 1/6 of the NGM agar and transfer it onto newly prepared NGM agars with bacterial lawn. Keep the NGM agars upside down in a 22 degree Celsius incubator for the subsequent culture. After about 36 hours, the nematodes become gravid.Use two milliliters of sterile water to flush off the gravid nematodes and newly-produced eggs on each NGM agar into sterile centrifuge tubes. Place the tubes on a rack for 30 minutes to settle down the nematodes, and then discard 85%of the supernatants by pipetting. It's very important to judge whether the nematodes are gravid enough for synchronization.Otherwise, the age-synchronization operation would be in vain. Mix the pellets with seven volumes of sodium hypochlorite solution and shake the centrifuge tubes every two minutes for five to eight times to lyse the larvae and adult nematodes. Centrifuge the tubes at 700 times G for three minutes at 20 degrees Celsius and then discard the supernatants by pipetting.Re-suspend the pellets in five volumes of sterile water to wash the age-synchronized eggs. Centrifuge at 700 times G for three minutes at 20 degrees Celsius. Repeat the wash by sterile water, twice.Next, add one volume of sterile water into the tubes to re suspend the age-synchronized eggs. Distribute the egg suspensions by pipetting fifty micro liters onto each new NGM agars with bacterial lawn. Keep the NGM agars top side up in a 20 degree Celsius incubator for 30 minutes, allowing the water to evaporate or be absorbed by the bacterial lawn.Then, turn over the NGM agars, upside down, for the subsequent culture, and mark the time as the egg time. 36 hours after the egg time, the nematodes reach the L3 larvae stage. Use two milliliters of sterile water to flush the nematodes off each NGM agar into centrifuge tubes.After settling for 30 minutes, replace 85%of the supernatants with K Medium to digest the food in the guts for two hours. Then, discard the supernatants. Use K Medium to adjust the nematode suspensions to about 200 nematodes per 100 microliters for subsequent experiments.To avoid edge effects, in the middle area of a 96-well sterile microplate, add 100 microliters of prepared control or chemical solutions into 10 wells, as replicates in six groups. Add 100 microliters of the prepared nematode K Medium to each of the 60 wells. Mark the time as T0.Perform the exposure for 24 to 96 hours.After the exposure, use a pipette to collect nematodes from five wells in each group, into six 1.5 milliliter centrifuge tubes. Settle the nematodes for 30 minutes and then pipette to discard the supernatants. Wash the nematodes at the bottom with one milliliter of sterilized water.Settle the nematodes for 30 minutes. Discard the supernatants, and use the nematodes at the bottom for indicator measurements of the exposed parent generation, marked as F0.Collect the nematodes from the remaining five wells in each group. Settle and wash the nematodes as previously.Next, add 100 microliters of sterile water to re-suspend the nematodes in each tube, and transfer the nematode suspensions evenly onto three newly prepared NGM agars with bacterial lawn. Incubate the nematodes for 36 hours to become gravid, and perform age-synchronization, as previously. After incubating the synchronized eggs on NGM agars with bacterial lawn for 36 hours, flush the nematodes into six centrifuge tubes.After settling and washing with one milliliter of sterilized water, use the nematodes at the bottom for indicator measurements of the offspring generation marked as T1.Mix 99 milliliters of the warm NGM agars with one milliliter of control or chemical solutions. After preparing the agars with bacteria suspensions according to the manuscript, in the bio-safety cabinet, set aside the upper lids of the petri dishes and expose the bacterial lawn to UV light for 15 minutes. Pipette the age-synchronized eggs onto the UV-treated agars.Mark the start of the exposure to the parent generation, F0, as day zero. Incubate the agars for three days at 20 degrees Celsius. Then, use the mature nematodes to measure effects in F0.Also, on day three, use a glass rod fitted with a man-made fiber wire bent into a ring to pick F0 mature nematodes, and slide onto the surface of new UV-treated NGM agars.On day four, pick out and discard the mature F0 nematodes from NGM agars. On day six, use the F1 mature nematodes that have experienced exposure for three days for indices measurement. In this protocol, the trans-generational effects of cadmium, copper, lead, and zinc on the body-bending frequency showed that the inhibitions in the nematode parent, F0, after prenatal exposure were less than in their progeny, T1.The multi-generational residual effects of sulfamethoxazole on the nematode lifespan and reproduction showed that the reproduction was significantly affected in germ-line exposure, and the toxicities persisted in non-exposed generations from T3 to T6 generations.Multi-generational exposure and multi-generational residual effects of lindane on the nematode biochemical and genetic indices showed obesogenic effects with disturbances in the insulin signal regulation. Moreover, the changes between sgk-1 and akt-1 signaling indicated that nematodes from different exposure generations showed different response strategies for tolerance and avoidance. Check the living status of the nematodes before each operation, and modify following steps accordingly.Other effects, for example, obesogenic effects of chemicals, can be included to further answer the increasing prevalence of obesity over generations. Based on the method, further mechanisms can be explored. For example, the heritable epigenetic signals between generations.The chlorine solutions have strong oxidatative potentials, and avoid direct contact of skin to it.

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Research

JoVE Journal

Environment

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...