This work was supported by the Science and Technology Department of Jiangxi Province (20203BBFL63065) and the General Project of Science and Technology Research Project of Jiangxi Education Department (GJJ211430). We would like to thank Editage (www.editage.cn) for English language editing.

NOTE: Here, we present a protocol for assessing the response characteristics and internal mechanisms of pepper seed germination and seedling growth under different mixed salt stresses, which can serve as a reference method for seed salt tolerance evaluation.
1. Experimental preparation
<ol>
	<li>Prepare crop seeds for cultivars-Hongtianhu 101 with strong salt tolerance and Xinxiang 8 with low tolerance.</li>
	<li>Prepare 0.2% KMnO4 solution as a seed disinfection reagent. First, weigh 4.0 g of KMnO4, and then add 2,000 mL of distilled water. 
	NOTE: Potassium permanganate is usually unstable due to its strong oxidation; accordingly, it is prepared immediately before use.</li>
	<li>Prepare the mixed salts using seven salts, including sodium carbonate, sodium bicarbonate, sodium chloride, calcium chloride, magnesium chloride, magnesium sulfate, and sodium sulfate13. Add the same molar amount of each, which successively account for 14.8%, 11.7%, 8.2%, 15.5%, 13.3%, 16.7%, and 19.8% of the total mass ratio of the mixed salts, respectively.</li>
	<li>Prepare Petri dishes (single use) and filter paper (medium speed qualitative filter paper), both with a diameter of 9 cm. 
	&#8203;NOTE: The material of the Petri dish can be changed; however, the diameter of the Petri dish and filter paper must be the same.</li>
</ol>
2. Seed soaking and preparation for germination
<ol>
	<li>For seed optimization, select pepper seeds with consistent size and full particles from each variety, with an average diameter of 4.2 mm and 3.7 mm for Hongtianhu 101 and Xinxiang 8 seeds, respectively. Calculate the total number of seeds selected according to the test workload.</li>
	<li>For seed disinfection, soak selected pepper seeds in 0.2% KMnO4 solution for 15 min and then rinse five times with distilled water.</li>
	<li>For seed soaking, transfer the sterilized seeds to distilled water and allow them to soak for 24 h. Rinse the seeds several times with distilled water and dry for further use. 
	&#8203;NOTE: The soaking time of seeds for different crops may vary.</li>
</ol>
3. Seed germination and seedling growth
<ol>
	<li>Prepare six concentrations of the mixed salts: 0 (control), 3, 5, 10, 15, and 20 g/L. Measure the conductivity of the salt solution using a conductivity meter; the solution conductivity EC values are 0.092, 3.05, 4.73, 8.33, 11.53, and 15.22 ms/cm, respectively.</li>
	<li>For seed preparation, evenly place 40 pepper seeds in a Petri dish with two layers of filter paper. Prepare the seeds for six experimental treatments and repeat each treatment five times.</li>
	<li>For seed germination, add a suitable amount of the six mixed salt concentrations to the Petri dish to ensure the filter paper is kept moist. Place the seeds in an air incubator at 28 &#176;C and 80% air humidity for germination in the dark.</li>
	<li>After seed germination, allow the seedlings to continue to grow in light (light intensity of approximately 450 Lux; light cycle of 12/12h) in the incubator for 14 days after sowing. The temperature and humidity at the seedling growth stage must be the same as those used at the germination stage.</li>
	<li>Replenish the solution in the culture dish every 12 h to retain a moist filter paper, and completely wash the filter paper every 24 h with the corresponding concentration of the mixed salt solution to keep a constant mixed salt concentration in the Petri dish. 
	&#8203;NOTE: The amount of salt solution added to wet seeds can be adjusted according to the seed germination and growth stages. Many methods are available for maintaining a constant concentration of salt solutions in culture dishes. In addition to the methods described in this experiment, the strategy of adding distilled water by weight can be used.</li>
</ol>
4. Measurement and calculation of indicators
<ol>
	<li>Determination of the seed germination indexes
	<ol>
		<li>Determine the germination rate daily after sowing, with the radicle breaking seed coat reaching half the seed diameter length as the germination marker.</li>
		<li>Calculate the germination rate, germination potential, relative salt rate, germination index, and seed germination vigor index using the following formulas: 
		Germination rate (%) = (number of normal germinated seeds on day 7 after sowing/number of tested seeds) &#215; 100 
		Germination potential (%) = (number of normal germinated seeds on day 3 after sowing/number of tested seeds) &#215; 100 
		Relative salt rate (%) = (control germination rate - treatment germination rate)/control germination rate &#215; 100 
		calculated using the seed germination rate on day 7 after sowing 
		Germination index (GI) = &#8721; [Gt/Dt] 
		where Gt refers to the seed germination number at a period of time (t) after sowing and Dt refers to the corresponding germination days 
		Seed germination vigor index (VI) = GI x S 
		&#8203;where S is the root length</li>
	</ol>
	</li>
	<li>Determination of the seedling growth index
	<ol>
		<li>On day 14 after sowing, randomly select 10 representative seedlings from each Petri dish and measure the root length and hypocotyl length.</li>
		<li>Use a knife to divide the pepper seedlings into two parts: radicle and aboveground parts. Remove the water from the seedlings by wiping, and weigh the seedlings separately to determine the fresh weight.</li>
	</ol>
	</li>
	<li>Determine the antioxidant enzyme activity, malondialdehyde (MDA) level, and proline (Pro) content in pepper as follows.
	<ol>
		<li>To preserve the pepper seedlings, select representative whole pepper seedlings (approximately 24.0 g) from each treatment on day 14 after sowing. After removing the surface water, immediately freeze the seedlings in liquid nitrogen for 1 min and store them in a refrigerator at an ultra-low temperature (-80 &#176;C). 
		NOTE: The sample number of pepper seedlings stored in the ultra-low temperature refrigerator should be sufficient, in case some indicators need to be retested.</li>
		<li>Retrieve approximately 1.0 g of seedling sample from each treatment collected in triplicate. Place the seedling sample in a centrifuge tube, add liquid nitrogen, and grind the sample using a grinding rod to determine the physiological indexes of the seedlings. The determined indexes and the measurement scheme are shown below.</li>
		<li>Determine the seedling protective enzyme activity (peroxidase [POD], catalase [CAT], superoxide dismutase [SOD]), malondialdehyde (MDA), and proline (Pro) contents using a commercially available kit (spectrophotometry-based) for each factor14. 
		NOTE: Earlier observations revealed no difference in salt stress between the 15 and 20 g/L mixed salt concentrations. As a result, only five salt concentrations (0, 3, 5, 10, and 15 g/L) are measured.</li>
	</ol>
	</li>
	<li>Comprehensive evaluation of salt tolerance using the membership function method 
	NOTE: The membership function uses a fuzzy mathematics method, which converts qualitative evaluation into quantitative evaluation15, to evaluate a variety of physiological indexes affected by salt damage.
	<ol>
		<li>Calculate the value of the membership function using the following formula by Zhoubin Liu et al.15: 
		Ri = (Xi - Xmin)/(Xmax - Xmin) 
		If a trait is negatively correlated with salt tolerance, calculate the inverse membership function using: 
		Ri = 1 - (Xi - Xmin)/(Xmax - Xmin) 
		&#8203;Accumulate the membership values of each physiological index, where Xi is the measured value of a certain trait, Xmax and Xmin are the maximum and minimum values for Xi, respectively, and Ri is the membership value of that trait.</li>
		<li>Include the following relevant indicators: seed germination characteristics (germination potential, germination rate, germination index, and seed germination vigor index), seedling growth characteristics at the germination stage (root length, hypocotyl length, root fresh weight, and fresh weight aboveground), MDA, Pro, and protective enzyme activity (CAT, POD, SOD) for membership function value calculation. The membership function values are obtained from each indicator.</li>
	</ol>
	</li>
	<li>Use spreadsheet and SPSS software (version 22.0) to analyze and process the test data and apply the least significant difference (LSD) method for multiple comparisons to identify significant differences. Use Pearson&#39;s correlation analysis to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress.</li>
</ol>

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The authors declare no conflicts of interest.

This research method comprises four key steps that affect the accuracy of the experimental results. First, owing to the poor dissolution of mixed salts caused by the increased solute content in high salt concentration solutions, and the low solubility of reagents such as calcium chloride, which are more difficult to solubilize in water, the weighed reagents must be fully ground in a mortar. Further, the reagents must be dissolved via ultrasonic waves before determining the capacity. Second, the configured salt s...

Salinity is a major limiting factor&#160;for crop productivity worldwide1. At present, nearly 19.5% of the world&#39;s irrigated land and 2.1% of dry land are affected by salinity, and approximately 1% of agricultural land degenerates into saline-alkali land every year. By 2050, 50% of arable land is expected to be affected by salinization2,3. In addition to natural factors, such as natural rock weathering and salty rainwater near or around the coast, rapid surface evaporation, low rainfall, and unreasonable agricultural management methods have exacerbated the process of soil salinization. Soil salinization inhibits the growth of plant roots and reduces the absorption and transportation of water and nutrients from the plant roots to the leaves. This inhibition results in physiological water shortages, nutritional imbalances, and ion toxicity, which lead to reduced crop productivity and a complete loss of crop yield. The salinization of cultivated land is gradually becoming one of the most critical abiotic stress factors affecting global agricultural food production4. Salt stress reduces the arable land available for agriculture, which may result in a significant imbalance between the supply and demand of future agricultural products. Therefore, exploring the effects of soil salinization on crop growth and physiological and biochemical mechanisms is conducive for breeding salt-tolerant varieties, the sustainable utilization of saline soil, and the safety of agricultural products.
Pepper (Capsicum annuum L.) is planted worldwide owing to its high nutritional and medicinal value. For example, capsaicin is an alkaloid responsible for the spicy flavor of pepper. Capsaicin can be used for pain relief, weight loss, improving cardiovascular, gastrointestinal tract, and respiratory systems, and in several other applications5. Pepper is also rich in bioactive substances, especially different antioxidant compounds (carotenoids, phenolics, and flavonoids) and vitamin C6. Currently, pepper is reported to be the vegetable crop with the largest cultivation area in China, with an annual planting area of more than 1.5 x 106 ha, thereby accounting for 8%-10% of the total vegetable planting area in China. The pepper industry has become one of the largest vegetable industries in China and has the highest output value7. However, pepper cultivation is often subjected to a variety of biological (pests and fungi) and abiotic stresses, especially salt stress, which has a direct negative impact on seed germination, growth, and development, resulting in the reduction of pepper fruit yield and quality8.
Seed germination is the first stage of interaction between plants and the environment. Seed germination is highly sensitive to fluctuations in the surrounding media, especially soil salt stress, which may exert reversed effects on physiology and metabolism, and eventually disorder the normal growth, development, and morphogenesis of crops9. In previous studies, pepper seed germination and seedling growth under salt stress were extensively investigated; however, most studies used NaCl as the only salt for stress induction10,11,12. However, soil salt damage is mainly due to Na+, Ca2+, Mg2+, Cl-, CO32-, and SO42- ion toxicity generated by the dissociation of sodium, calcium, and magnesium salts. Owing to the synergy and antagonism between ions, the effects of mixed salt and single salt on crop growth and development may be quite different. However, the corresponding characteristics of pepper seed germination and growth in mixed salt are still unclear. Therefore, two pepper varieties with remarkable differences in salt tolerance are used as materials in this study. Analyzing the effects of different salt concentrations on pepper seed germination, growth, and physiological and biochemical indexes after equimolar mixing of seven salts can reveal the response mechanism of pepper seed germination to salinity stress. It can also provide a theoretical basis for cultivating strong pepper seedlings, as well as high yield and high-quality cultivation in saline cultivated land.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride</td>
			<td>Shanghai Experiment Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal machine</td>
			<td>Shanghai Luxianyi Centrifuge Instrument Co., Ltd., China</td>
			<td>TGL-16M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductivity meter</td>
			<td>Shanghai Instrument&#38;Electronics Science Instrument Co., Ltd., China</td>
			<td>DDSJ-308F</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature and humidity box</td>
			<td>Ningbo Laifu Technology Co., Ltd.,China</td>
			<td>PSX-280H</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital display vernier caliper</td>
			<td>Deli Group Co., Ltd.,China</td>
			<td>DL90150</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Mettler Toledo Instruments (Shanghai) Co., Ltd.,China</td>
			<td>ME802E/02</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Hangzhou Fuyang North Wood Pulp and Paper Co., Ltd.,China</td>
			<td>GB/T1914-2017</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding rod</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Hongtianhu&#160; 101</td>
			<td>Seminis Seed (Beijing) Co., Ltd.,China</td>
			<td>11933955/100147K1-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice machine</td>
			<td>Shanghai Kehuai Instrument Co., Ltd., China</td>
			<td>IM150G</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Tianjin Kermel Chemical Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Tianjin Kermel Chemical Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Jiangsu Yizhe Teaching Instrument Co., Ltd.,China</td>
			<td>I-000163</td>
			<td></td>
		</tr>
		<tr>
			<td>Pocket knife</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium permanganate (KMnO4</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water equipment</td>
			<td>Sichuan Youpu Ultrapure Technology Co., Ltd.,China</td>
			<td>UPT-I-20T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate&#160;</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Test kit</td>
			<td>Suzhou Keming, Biotechnology Co., Ltd, Suzhou.,China</td>
			<td>Spectrophotometer method</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low temperature freezer</td>
			<td>SANYO Techno Solution TottoriCo.,Ltd.</td>
			<td>MDF-382</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet visible spectrophotometer</td>
			<td>Shanghai Precision Scientific Instrument Co., Ltd., China</td>
			<td>&#160;760CRT</td>
			<td></td>
		</tr>
		<tr>
			<td>Xinxiang 8</td>
			<td>Jiangxi Nongwang High Tech Co., Ltd.,China</td>
			<td>GPD Pepper 2017(360013)</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of effect of compound salt stress on seed germination and salt tolerance analysis of pepper (capsicum annuum l.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Seed germination characteristics 
As the mixed salt concentration increases, the germination potential and germination index of Hongtianhu 101 and Xinxiang 8 decreases significantly. Both cultivars have a sharp decline in salt concentrations from 0-3 g/L, and a slow and steady decline for salt concentrations from 3-20 g/L (Figure 1A,B). The germination rate of the two varieties gradually decreases as the mixed salt concentrations increase, and the relat...

Watch this Scientific Journal Video about Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L. at JoVE.com

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L.

The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 ...

analysis-effect-compound-salt-stress-on-seed-germination-salt

Research

JoVE Journal

Biology

4.2K Views.  Gannan Normal University. The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties. 

Video: Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L.

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...