Name: methods for comparing nutrients in beebread made by africanized and european honey bees and the effects on hemolymph protein titers
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Description: Watch this Scientific Journal Video about Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers at JoVE.com

The authors thank Mona Chambers, Geoffrey Hidalgo and Maurissa Two Two for their technical assistance. We also thank Dr. Earl White for amino acid analysis, Dr. Roger Simonds for analysis of pollen for pesticides, and Drs. Stanley S. Schneider, Vanessa Corby-Harris and Kirk Anderson for their reviews and discussion of earlier versions of the manuscript and many helpful suggestions.

1. Obtaining Beebread from AHB and EHB Colonies
<ol>
	<li>Place pollen traps on honey bee colonies and collect pollen. Grind the pollen into a fine powder (similar to pollen shed from anthers) using a coffee grinder.</li>
	<li>Establish 5 colonies each of AHB and EHB in an enclosed flight area (EFA) so that bees forage only on the pollen provided. To prevent workers from drifting between EHB and AHB colonies, divide the EFA into separate sections so that bees cannot cross between them. Place individual EHB or AHB colonies with 3,500-4,000 worker bees, wax comb with nectar, honey, immature brood and empty comb in each section of the EFA. 
	NOTE: The colonies do not have stored pollen when established. The rate that pollen is stored can be increased by not including a laying queen in the colonies.</li>
	<li>Feed ground pollen to colonies by placing a tray with the pollen in each section of the EFA. Spreading about 60 g of pollen on each tray so that foraging bees can collect it as corbicular loads and store the pollen in their colonies as beebread. Continue providing fresh pollen on each tray daily for 3 weeks.</li>
	<li>Refer to beebread from the European colonies as European beebread (EBB), and from Africanized colonies as Africanized beebread (ABB).</li>
</ol>
2. Feeding Bees in Cages
<ol>
	<li>Place frames of sealed worker brood from AHB and EHB colonies in separate emergence cages in an environmental room set at 32-34 &#176;C and 40% relative humidity..</li>
	<li>When the workers emerge and are about 24 hr old, establish 12 Plexiglas bioassay cages (dimensions = 11.5 x 7.5 x 16.5 cm3) and add either 100 newly emerged EHB or 100 newly emerged AHB worker bees to each cage. Place a section of comb with a known number of either EBB or ABB cells (24-30 cells per cage) in each cage to generate the following treatment combinations: AHB fed ABB, EHB fed ABB, AHB fed EBB and EHB fed EBB. (4 treatments; 6 cages per treatment; 24 cages in total).</li>
	<li>Add vials of water and a 50% honey and water solution formulated by volume to each cage. Refill the honey and water vials daily for the 11 day study period.</li>
</ol>
3. Sampling Worker Bees and Beebread and Estimating Consumption
<ol>
	<li>Sample 10 newly emerged EHB and AHB workers prior to placing them in the cages. Refer to these as Day 0 bees and have them serve as a baseline for hemolymph protein concentrations.</li>
	<li>Remove 10 bees from each cage after they fed on EBB or ABB for 4, 7, and 11 days.</li>
	<li>Place the live bees in individual microcentrifuge tubes and set on ice packs. Select a subsample of four bees for analysis of hemolymph protein concentration.</li>
	<li>After sampling bees on Day-11, count the number of comb cells that still contain beebread. This is a relative measure of beebread consumption.</li>
	<li>Remove the remaining beebread from the cells in each cage and store in separate microcentrifuge tubes according to cage. Keep the beebread samples at -80 &#176;C until analyzed for pH, soluble protein concentration, and amino acid content.</li>
</ol>
4. Estimating pH of Pollen and Beebread
<ol>
	<li>Take six random 0.3 g samples of the pollen fed to bees in the EFA and dissolve it in 300 &#181;l of distilled water. Measure the pH using a waterproof double junction pH spear with an accuracy of +0.01.</li>
	<li>Take 0.3 g sample of beebread that remained after the 11-day feeding period in each cage. Dissolve the beebread in 300 &#181;l of distilled water and measure pH as described for pollen (4.1).</li>
</ol>
5. Protein Analysis
<ol>
	<li>Take six samples of the pollen and a sample of EBB and ABB from each cage. Store samples at -20 &#176;C until analyzed for soluble protein concentration.</li>
	<li>Mix 20 mg of either pollen or beebread with 1,000 &#181;l of 0.1 M phosphate buffer solution (PBS).</li>
	<li>Vortex the mixture for 10 sec and centrifuge at 571.2 x g for 1 min.</li>
	<li>Remove a 10 &#181;l sample of the supernatant and place in wells of a 96 well flat bottom EIA/RIA polystyrene plate. Replicate each sample in three wells.</li>
	<li>Draw hemolymph from bees collected from each cage by inserting a 20 &#956;l capillary tube (that had been heated and pulled to a needle-sharp point) into the right lateral portion of the thorax near the point of attachment of the wings. Collect additional hemolymph, if needed, by inserting the same tube into the membrane between the abdominal tergites.</li>
	<li>Add 1 &#956;l of hemolymph to 9 &#956;l of 0.1 M PBS. Store the hemolymph solution at -20 &#176;C until analysis for soluble protein.</li>
	<li>Determine total soluble protein concentrations in pollen, beebread, and hemolymph samples using a commercial Bradford protein assay kit. Follow the manufacturer&#8217;s instructions.</li>
	<li>Establish a standard curve to estimate soluble protein concentration in the samples by measuring protein absorbance with known protein concentrations in bovine serum albumin (BSA). Measure protein absorbance at 595 nm using a spectrophotometer.</li>
</ol>
6. Amino Acid Analysis
<ol>
	<li>Pool individual samples from comb cells of each colony to create a representative sample of EBB and ABB for analysis.</li>
	<li>Take a 50 mg pollen or beebread sample weighed into autosampler vials, and add 1 ml of distilled water to the vial, along with 100 &#181;l of a 50 ng/&#956;l internal standard solution consisting of d4-alanine, d23-lauric acid, 13C6-glucose and d39-arachidiac acid.</li>
	<li>Cap the sample and sonicate for 5 min.</li>
	<li>Condition an HLB cartridge by adding 1 ml of methanol, equilibrated by adding 1 ml of distilled water followed by the addition of 1 ml of the beebread or pollen sample. Wash the cartridge with 1 ml of 5.0% MeOH/H2O and elute with 1 ml of 80% MeOH/H2O.</li>
	<li>Evaporate the sample to dryness under a stream of nitrogen. Reconstitute the sample with 50 &#181;l of Pyridine and 100 &#181;l of N,O-Bis (trimethylsilyl)trifluoroacetamide + Trimethylchlorosilane (BSTFA + TMCS).</li>
	<li>Cap and incubate the sample at 70 &#176;C for 30 min.</li>
	<li>Allow the sample to cool and transfer it to a clean autosampler vial.</li>
	<li>Cap and place the sample into a Mass Selective Detector interfaced to a gas chromatograph to analyze the samples both for volatile compounds and organic acids. Separate the sugar and organic acids following TMS derivatization with BSTFA + TMCS using a column (30 m x 0.25 mm i.d.) with a 1.0 &#181;m film thickness.</li>
	<li>Set the column oven at 50 C for 2 min, then increase the temperature linearly to 290 &#176;C at 5 C/min. and hold for 7 min. Set the GC injector and GC/MS interface to 250 &#176;C and 290 &#176;C, respectively.
	<ol>
		<li>Use Helium as a carrier at a flow rate of 1.0 ml/min. Set the MS source temperature to 230 &#176;C.</li>
	</ol>
	</li>
	<li>Tune and calibrate the mass spectrometer daily with Perfluorotributylamine (PFTBA). Use a 1 &#181;l injection of PFTBA in the full scan (35-700 amu) positive ion mode to obtain data on the presence and concentrations of amino acids.</li>
</ol>

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The authors have nothing to disclose.

Using the methods described above, we found that the beebread made by AHB was consumed in greater amounts by both AHB and EHB. Though EHB and AHB consumed similar amounts of each type of beebread, AHB had higher hemolymph protein titers. Findings based on our methods were similar to previous reports where hemolymph protein levels in AHB were higher than in EHB though both were fed the same diets 26 . By measuring consumption of EBB and ABB which were consumed at different rates by both EHB and AHB, it was determined that hemolymph protein concentration in each subspecies could not be raised by increasing food consumption. There seems to be a plateau for hemolymph protein concentration in workers of nurse bee age and that the set point for the plateau is higher in AHB than EHB.
There are several important conditions for establishing colonies for beebread production that will optimize the rate of pollen storage. First, the colonies need frames with open brood. Without open brood to feed, workers will not collect much pollen. Secondly, the colony must be queenless so no additional brood is produced. Brood rearing requires large amounts of pollen, and only excess pollen is stored. In the small colonies established in EFA, there would be little pollen to be stored as beebread if brood areas were expanding so colonies need to be queenless. Finally, for beebread to be made, pollen must be collected as corbicular loads and stored in comb cells. If the pollen is collected in pollen traps, it must be ground to a fine powder before presenting it to the bees so they can collect it as corbicular loads.
The methods to measure the consumption of beebread generated qualitative rather than absolute estimates. The only consumption that was counted was when cells were completely emptied of bee bread. A more accurate estimate of total bee bread consumption might be obtained by removing the bee bread from the cells and making it into a patty that could be weighed before and after the study period. However, we wanted to keep the bee bread in the cells so that the bees could feed on it as they would in a colony and perhaps continue processing it during the study period. The difference in weight of the comb sections before and after the study was not used as an estimate of consumption because the weight might have increased because bees put the diluted honey fed to them in some cells.
The workers also might have added some of the diluted honey to the bee bread. For these reasons, cells containing approximately equal amounts of bee bread before and after the feeding period were counted and generated a qualitative measurement. Still, there was a striking difference between the two types of beebread in the number of empty ABB cells counted compared with EBB after 11 days.
Determining when stored pollen becomes beebread can be difficult because bees continually add pollen to cells. The colonies used for producing beebread were established with frames of open brood so bees would collect pollen. However, the colonies were queenless so there were larvae to feed for only about 9 days after the colony was established. For the remainder of the 3 week period when colonies were in the EFA, the pollen that the bees collected was stored and being converted to beebread. Keeping the stored pollen in the comb cells for an additional 11 days when feeding it to bees in cages also may have continued the processing of pollen to beebread. The conversion of pollen to bee bread takes about 7 days 8. The beebread fed to EHB and AHB had lower pH and reduced protein concentrations compared with the pollen fed. Similar findings of changes in pollen after conversion beebread have been reported by others 7,10,27. Our results differed from previous reports however, in that there were differences in concentrations of certain amino acids between beebread and pollen. The changes in both protein and amino acid concentrations could be due to the activity of proteolytic enzymes, the source of which might be the bees themselves or the microbial communities established in the beebread 7,8,28,29 .
The methods used to measure protein concentration were similar to those previously described to determine the effects of dietary protein on Africanized and European bees 26. As an extension of the methods, we were able to estimate soluble protein in the pollen and beebread. Those methods generated similar findings to previous reports 7,10,27. Our findings provide additional evidence that AHB more efficiently assimilate dietary protein than EHB, and that this could be a key factor in the ecological dominance of AHB in most regions where it has immigrated and become established 30-32.

Nutrition plays a fundamental role in the health and vigor of honey bee colonies and in their establishment as populations. Nutrients from food provide energy and the biochemical components needed for brood rearing, thermoregulation, foraging and immune response. For honey bee colonies, the nutrients needed to grow colony populations and maintain their health come from nectar and pollen. Nectar provides carbohydrates and pollen supplies the remaining dietary requirements such as protein, lipids, vitamins and minerals 1.
Subspecies of honeybees can differ in nutritionally based colony-level parameters such as worker longevity, brood rearing, and mechanisms of social immunity 2-6. These differences might be linked to how food, particularly pollen is processed by the colony and digested in individuals. Pollen is stored in comb cells and through microbially mediated lactic acid fermentation is chemically changed 7-10. The fermented pollen is called beebread. There might be genotypic differences among subspecies in their ability to acquire nutrients from the pollen they collect and store and this could affect colony growth and survival. Genotypic effects on food processing and nutrient acquisition have been documented in other organisms causing some individuals to obtain more nutrients and calories than others while consuming the same foods 11-14.
Here we describe methods used to compare the composition and consumption of beebread made by different subspecies of honey bees. Methods to measure the resulting hemolymph protein titers in worker bees also are described. Previous studies on the nutritional composition of beebread were done with European honey bees (EHB) 10,15,16. However, there may be differences in beebread made by bees of different subspecies even when they feed on the same pollen. EHB and AHB were compared because these subspecies have distinct behavioral and physiological differences that might be related to food processing and nutrient acquisition 17. Some of the most notable differences are that AHB collect and consume more pollen than EHB and seem to convert it more readily into brood 18. AHB colonies have higher swarming rates than EHB and abscond when food resources become limited 19-23. Absconding is rare in EHB. AHB also have higher metabolic rates than EHB 24. The nutritional basis for the colony-level differences between EHB and AHB might be related to the rate of pollen collection and also to its nutrient content (e.g., amino acids and protein) after it is converted to beebread. Beebread consumption and the resulting protein acquisition also might play a role in the colony-level differences between EHB and AHB. Using the developed methods, EHB and AHB made beebread from the same pollen source. The beebread was then fed back to the bees of each subspecies and could determine if bees acquire protein from beebread in a manner distinctive to their subspecies or to the source of the beebread.

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methods for comparing nutrients in beebread made by africanized and european honey bees and the effects on hemolymph protein titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Beebread was stored in -80 &#176;C for less than a month before being analyzed for pH and protein concentration, and for about 4 months before amino acid analysis. Beebread differed from the pollen in pH and protein concentration (Figure 1). The pH of beebread was lower than the pollen as was the protein concentration. Both EHB and AHB consumed more ABB than EBB (Figure 2).
Levels of soluble protein in the hemolymph of AHB were significantly higher than EHB regardless of the type of beebread they consumed (Figure 3). These differences in hemolymph protein levels occurred even though EHB and AHB consumed similar amounts of each type of beebread. The age of the bees at the time of sampling significantly affected soluble protein concentrations in the hemolymph. Protein concentrations were significantly lower in day-4 bees compared with day-7 or 11 which did not differ.
Of the 10 amino acids that are essential for honey bees, all but histidine were detected in the pollen. In most cases, amino acid concentrations measured in beebread were higher than in the pollen (Figure 4). For example, concentrations of leucine and threonine were about 60% higher in beebread compared with pollen, and valine concentrations were about 25% higher. Alanine, aspartic acid, glutamine, and methionine levels also were higher in beebread than in pollen. Amino acids concentrations did not differ greatly between ABB and EBB with the exception of phenylalanine and cysteine. Phenylalanine levels were about twice as high in ABB compared with either EBB or pollen. Cysteine concentrations were lower in EBB compared with ABB or pollen. Tryptophan was the only amino acid present in higher concentrations in pollen than in EBB or ABB. Concentrations of proline in pollen and ABB were higher than in EBB.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig1highres.jpg" width="600" /> 
Figure 1: Comparisons of pH (A) and soluble protein concentrations (B) in pollen and the beebread made by European (EHB) or Africanized (AHB) honey bees. The pH of pollen was significantly higher than the beebread as determined by analysis of variance (F2,12 = 3725, p &#60;0.0001) followed by a Tukeys W- multiple comparison test. The protein concentration in pollen was significantly higher than in beebread made by EHB (EBB) or AHB (ABB) (F2,27 = 16.49; p &#60;0.0001). Means followed by the same letter are not significantly different at the 0.05 level.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig2highres.jpg" width="600" /> 
Figure 2: The average percentage of cells containing beebread that were completely consumed over an 11 day interval by caged bees. The beebread was made by either European (EHB) or Africanized (AHB) bees using the same pollen source. Means were estimated from five cages of each treatment; those with the same letter are not significantly different at the 0.05 level as determined by a one way analysis of variance (F3,16 = 7.3, p = 0.003) and Tukey&#8217;s W test. This Figure has been modified from 25.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig3highres.jpg" width="600" /> 
Figure 3: The average concentration of protein in hemolymph from European (EHB) or Africanized (AHB) honey bees fed beebread made by European (EBB) or Africanized (ABB) bees for 4, 7, and 11 days. A repeated measures analysis of variance indicated significant differences among the 4 treatment groups (F3,20 = 19.7, p &#60;0.001). Levels of soluble protein in AHB fed ABB were significantly higher than EHB fed ABB (p = 0.008) or EBB (p = 0.018). The age of the bees at the time of sampling significantly affected soluble protein concentrations in the hemolymph. Levels were significantly lower in day-4 bees compared with day-7 (p &#60;0.0001) or 11 (p = 0.001). Day 7 and day11 bees did not differ (p = 0.149). This figure has been modified from25 .
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig4highres.jpg" width="600" /> 
Figure 4: Concentrations of amino acids (&#181;g per gram of pollen or beebread) in pollen or the beebread made from it. EBB is beebread made by European bees and ABB was made by Africanized bees. Tryptophan, cysteine, phenylalanine and proline were plotted separately for purposes of clarity in presenting their amounts. This figure has been modified from 25.

Watch this Scientific Journal Video about Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers at JoVE.com

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

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Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...