Authors are thankful to Dale Leitman for initial training in use of transient transfection assays to determine estrogenic activity of primate plant foods. Thanks to Bradford Westrich and C. Eric Johnson for helping to set up laboratory equipment and training students in extraction methods. Finally, thank you to Indiana University for funding this research.

1. Preparation of plant materials
<ol>
	<li>Freeze dry plant items that were collected fresh using a lyophilizer.
	<ol>
		<li>To protect samples from light, cover chambers with aluminum foil during drying process.</li>
		<li>To ensure that samples are completely dry, lyophilize until chambers no longer feel cold to touch and plant materials no longer lose mass when weighed.</li>
		<li>Store dried plants in sterile low residue bags in absence of light until grinding.</li>
	</ol>
	</li>
	<li>Finely grind samples using a grinding mill with 0.85 mm mesh screen.
	<ol>
		<li>Store the ground samples in the bags in the absence of light until extraction.</li>
	</ol>
	</li>
</ol>
2. Extraction of plant secondary metabolites
<ol>
	<li>To extract the secondary plant metabolites, use a ratio of 1 g of dried sample to 10 mL of HPLC grade methanol.
	<ol>
		<li>Weigh sample on an analytical balance and add it to an appropriately sized Erlenmeyer flask (125 &#8211; 250 mL). Then add appropriate volume of methanol. Record the mass of sample extracted.</li>
		<li>Cover the plant-methanol solution with aluminum foil, and then set to rotate at 100 rpm speed at room temperature (RT) for 3 days on an orbital shaker, allowing the potentially estrogenic compounds to dissolve into the methanol.</li>
		<li>Decant the supernatant into a drip filtration system using filter paper (125 mm).</li>
		<li>Using a rotary evaporator, dry the plant extract until the sample is thickened, but pourable, in a 300 mL round-bottom flask. Pour sample into a 50 mL round-bottom flask, rinsing the large flask with a small amount of methanol. Continue to dry the sample in the small flask until the methanol is completely evaporated.</li>
		<li>Weigh the sample residue using an analytical balance. Record residue mass.</li>
		<li>Dissolve the plant extract in dimethyl sulfoxide (DMSO) at a concentration of 0.1 g of extract to 2 mL of DMSO. Vortex until homogenized.</li>
		<li>Store the plant extract-DMSO solution at 4 &#176;C in amber glass vials until the assay.</li>
	</ol>
	</li>
</ol>
CAUTION: Plants can produce unknown biologically active chemicals, and DMSO is a vehicle that can transport them across cell membranes. Use appropriate personal protective equipment and care when handling these samples.
3. Human estrogen receptor &#946; transfection assay12
NOTE: Aseptic technique and a laminar flow hood is required for Day 1 of the assay protocol.
<ol>
	<li>Prepare dilutions of 17&#946;-Estradiol for the standard curve.
	<ol>
		<li>Transfer the Cell Recovery Medium and Compound Screening Medium (CSM) from the freezer storage and thaw in a 37 &#176;C water bath.</li>
		<li>Label microcentrifuge tubes Intermediate 1 and 2 (INT1, INT2) and 1-8.</li>
		<li>Fill INT1 with 995 &#956;L of CSM, INT2 with 615 &#956;L of CSM, tube 1 with 900 &#956;L of CSM, and tubes 2-8 with 600 &#956;L of CSM. Set tube 8 aside.</li>
		<li>Transfer 5 &#956;L of 100 &#956;M 17&#946;-Estradiol Stock into INT1. Discard the tip. Vortex.</li>
		<li>Before each transfer, rinse pipette 3 times, and then transfer 10 &#956;L from INT1 into INT2. Discard the tip.</li>
		<li>Rinse pipette 3 times, and then transfer 100 &#956;L from INT2 into tube 1. Discard tip. Transfer 300 &#956;L from tube 1 into tube 2. Repeat for tubes 3 through 7. Discard 300 &#956;L from tube 7 into waste container. Tube 8 is a Zero and does not receive estradiol. Final concentrations of plated standards are: 400, 133.3, 44.44, 14.815, 4.938, 1.646, 0.5487, and 0 pM estradiol.</li>
	</ol>
	</li>
	<li>Prepare sample compounds.
	<ol>
		<li>Vortex samples.</li>
		<li>Take 4 &#956;L of each plant sample in DMSO and add to 496 &#956;L of CSM to yield a 0.8% DMSO solution.</li>
	</ol>
	</li>
	<li>Rapidly thaw Reporter Cells.
	<ol>
		<li>Retrieve the tube of Cell Recovery Medium from the 37 &#176;C water bath. Disinfect the outside surface using 70% ethanol.</li>
		<li>Retrieve Reporter Cells from -80 &#176;C storage and thaw by transferring 10 mL of the pre-warmed CRM into the tube of frozen cells.</li>
		<li>Close the tube of Reporter Cells and transfer to a 37&#176;C water bath for 5-10 min.</li>
		<li>Retrieve the tube of Reporter Cell Suspension from the water bath. Invert the tube of cells several times gently to break up aggregates of cells and produce a homogenous suspension. Clean the surface of the tube with 70% ethanol.</li>
	</ol>
	</li>
	<li>Assay plating
	<ol>
		<li>Dispense 100 &#956;L of the Reporter Cell Suspension into each well using a multichannel pipette.</li>
		<li>Dispense 100 &#956;L of samples in triplicate into appropriate assay wells.</li>
		<li>Transfer the plate into a 37 &#176;C, humidified 5% CO2 incubator for 22-24 h.</li>
	</ol>
	</li>
	<li>Thaw Detection Substrate and Detection Buffer in a dark refrigerator overnight to prepare for Day 2.</li>
	<li>Just prior to the end of the plate incubation, remove Detection Substrate and Detection Buffer from refrigerator and place in low light area until equilibrated to RT. Once at RT, invert each tube gently several times to thoroughly mix solutions.
	<ol>
		<li>Immediately before the incubation is complete, pour the entire contents of the Detection Buffer into the tube of Detection Substrate to create Luciferase Detection Reagent. Mix gently so as not to produce foam.</li>
		<li>Once the incubation is complete, invert the plate to discard content into an appropriate waste container. Gently tap the plate on a clean absorbent paper towel to remove the last droplets from the wells.</li>
		<li>Add 100 &#956;L of the Luciferase Detection Reagent to each well. Allow the assay plate to rest at RT for 15 min. Do not shake the plate.</li>
	</ol>
	</li>
	<li>Quantify luminescence using a 96-well plate-reading luminometer.</li>
</ol>

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+plant+defensive+chemistry+on+nutrient+availability+predict+reproductive+success+in+a+mammal.">The effects of plant defensive chemistry on nutrient availability predict reproductive success in a mammal.</a> Ecology. 90 (3), 711-719 (2009).</li><li>Wasserman, M. D., Steiniche, T., Despr&#233;s-Einspenner, M. -. L., Lambert, J. E., Rothman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Diet &amp; Nutrition. , (2020).</li><li>Benavidez, K. M., Chapman, C. A., Leitman, D. C., Harris, T. R., Wasserman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intergroup+variation+in+oestrogenic+plant+consumption+by+black-and-white+colobus+monkeys.">Intergroup variation in oestrogenic plant consumption by black-and-white colobus monkeys.</a> African Journal of Ecology. , (2019).</li><li>Bennetts, H. W., Underwood, E. J., Shier, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+breeding+problem+of+sheep+on+subterranean+clover+pastures+in+Western+Australia.">A specific breeding problem of sheep on subterranean clover pastures in Western Australia.</a> Australian Veterinary Journal. 22 (1), 2-12 (1946).</li><li>Tubbs, C. W., 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=Estrogenicity+of+captive+southern+white+rhinoceros+diets+and+their+association+with+fertility.">Estrogenicity of captive southern white rhinoceros diets and their association with fertility.</a> General and Comparative Endocrinology. 238, 32-38 (2016).</li><li>Shen, 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=Observation+of+the+influences+of+diosgenin+on+aging+ovarian+reserve+and+function+in+a+mouse+model.">Observation of the influences of diosgenin on aging ovarian reserve and function in a mouse model.</a> European Journal of Medical Research. 22 (1), 42 (2017).</li><li>Bou&#233;, S. 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The authors have nothing to disclose.

The ER&#946;&#160;reporter assay developed to individually screen pharmaceutical agents is also suitable for screening plant foods for phytoestrogens biologically active through the ER&#946;. Important considerations in the protocol include treating the plant samples with care: fresh plant material needs to be dried swiftly to prevent molding or other biological degradation, and it needs to be kept away from light to prevent photolysis of the compounds18. The assay protocol12</su...

Plants are a necessary source of food for many animals, providing calories and nutrients critical to survival, reproduction, growth, development, and behavior1. Plants produce thousands of chemicals, many as adaptations for their own growth, stomatic maintenance, and reproduction. Other compounds, deemed plant secondary metabolites (PSMs), have functions that are less clear, though some are toxic and likely used as a defense against herbivory and parasitism (e.g., alkaloids, tannins)2,3. Some of these chemicals have the ability to affect long term physiological processes in animals, such as endocrine functioning, although why these endocrine-active phytochemicals interact with the vertebrate endocrine system is still unclear2,4.
Phytoestrogens, the most well studied endocrine-active phytochemicals, are polyphenolic PSMs that structurally and functionally mimic estrogens, directly interacting with the hypothalomo-pituitary gonadal axis of the vertebrate endocrine system5. Ingestion of phytoestrogens in the human diet is associated with protection against some cancers, heart disease, and menopausal symptoms, though other effects include fertility problems. In fact, the physiological effects of these compounds were discovered in the 1940s when infertility in sheep was attributed to their grazing on phytoestrogen-rich clover (Trifolium subterrareum)6. When ingested, phytoestrogens can pass into cells and mimic the effects of estrogen. While phytoestrogens had negative effects on sheep fertility, the relationship between phytoestrogens and physiology is not simple. Like sheep, southern white rhinoceros display sensitivity to estrogenic compounds in feed derived from high quantities of soy and alfalfa. Daughters of females fed this diet during pregnancy are less likely to reproduce7. However, other studies have shown that phytoestrogens may have positive effects as well, including maturation of ovarian follicles in older mice8, prevention of certain cancers, antioxidant activity, and antiproliferative effects9.
The breadth of effects of phytoestrogens are not surprising given that estrogens affect a wide array of biological functions, including growth,&#160;development, and regulation of the reproductive and central nervous systems10. Although there are many mechanisms of action, phytoestrogens often have the ability to modify, enhance, or disrupt estrogen signaling through their ability to act as ligands for the intranuclear estrogen receptors alpha and beta (ER&#945; and ER&#946;). Many phytoestrogens have a phenolic ring structure similar to estrogens that allows them to bind estrogen receptors. Those with agonistic estrogenic activity function like estrogen, forming an activated ER-ligand complex that can dimerize and bind to an estrogen response element (ERE) and trigger gene transcription11. Thus, estrogens and phytoestrogens regulate cell activity and system functions through their actions as transcription factors.
Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses Chinese hamster ovary CHO cells engineered to highly express ER&#946;, which have been transfected with the firefly (Photinus pyralis) luciferase gene linked to an ERE promoter12. When estrogenic compounds are present, they bind to the ER, dimerize, and bind to the ERE, leading to transcription of the luciferase gene. Upon addition of a substrate solution, the luciferase catalyzes a reaction leading to photon emission. Therefore, positive samples produce light and negative samples do not.
This commercially available assay eliminates the need for laboratories to transfect the mammalian cells with the reporter gene and estrogen receptor13,14, which was unstable and variable in efficacy. The assay provides a stable transfection platform that allows for quickly and simply determining whether a plant has estrogenic activity via receptor binding.
We test the hypothesis that soybeans have higher estrogenic activity than all other foods given their known concentrations of estrogenic isoflavones15 using human foods from local grocers.

<table border="0" cellpadding="0" cellspacing="0" style="width:721px;" width="722">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1000 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">37 &#8451; water bath</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">37 &#8451;, humidified 5% CO2 incubator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% ethanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">analytical balance</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">cell culture-rated laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">dimethyl sulfoxide</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">disposable media basin, sterile</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">drip filtration system</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Erlenmeyer flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>125 mL and 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HPLC grade methanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Human ER&#946; Reporter Assay System, 1 x 96-well format assays</td>
			<td style="width:80px;">Indigo Biosciences</td>
			<td style="width:119px;">IB00411</td>
			<td>Assay kit - analyzes 24 samples plus standard curve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">lyophilizer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">multi-channel pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">orbital shaker</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">plate-reading luminometer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>ex. Bioteck Synergy HTX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">rotory evaporator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">round bottom flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>50 mL and 300 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">sterile microcentrifuge tubes or sterile multi-channel media basins</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sterile tips</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>200 &#181;L and 1000 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Whatman grade 1 paper</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">whirl-pak bags</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>sterile polyethylene bags</td>
		</tr>
	</tbody>
</table>

screening for phytoestrogens using a cell-based estrogen receptor  &#946; reporter assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

Twenty-two extracts of fruits and vegetables commonly found in human diets were screened for the presence of estrogenic compounds. A variety of foods were assayed, including legumes, such as soybeans, snow peas, and snap peas, as the pea family is a known source of phytoestrogens16, as well as figs, dates, corn, carrots, apples, bananas, strawberries, tomato, kale, and cabbage. Endocrine disrupting compounds are found in common substances (e.g., plastics and pesticides) and some are biologically a...

Watch this Scientific Journal Video about Screening for Phytoestrogens using a Cell-based Estrogen Receptor  ÃŽÂ² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

screening-for-phytoestrogens-using-cell-based-estrogen-receptor

Research

JoVE Journal

Biochemistry

4.7K Views.  Indiana University. We have optimized a commercially available estrogen receptor β reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Video: Screening for Phytoestrogens using a Cell-based Estrogen Receptor  β Reporter Assay

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...