Name: a platform of anti-biofilm assays suited to the exploration of natural compound libraries
Uploaded: 2016-12-27T12:00:00
Description: Watch this Scientific Journal Video about A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries at JoVE.com

The authors thank professor Paul Cos and LMPH, University of Antwerp, Belgium for his support during the filming process in his laboratory. This work was funded by Academy of Finland projects (projects 272266 and 282981) and Svenska Tekniska Vetenskapsakademien i Finland. The technical contributions of MSc Janni Kujala are acknowledged.

1. Growing the Bacteria
<ol>
	<li>Pre-culture the bacteria overnight in a tryptic soy broth (TSB) at 37 &#176;C with 220 rpm shaking (16-18 hr).</li>
	<li>Dilute the pre-culture 100-1,000 times (depending on the growth rate of the bacteria; here, 1,000 times is used for S. aureus) in fresh TSB and let it grow at 37 &#176;C and 200 rpm to reach exponential growth (optical density at 595 nm (OD595) between 0.2 and 0.6). 
	NOTE: This step requires strain-specific optimization.</li>
</ol>
<p class=...

<ol>
	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Dufour, D., Leung, V., L&#233;vesque, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm:+structure,+fuction,+and+antimicrobial+resistance.">Bacterial biofilm: structure, fuction, and antimicrobial resistance.</a> Endodontic Topics. 22, 2-16 (2012).</li><li>Donne, J., Dewilde, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenging+World+of+Biofilm+Physiology.">The Challenging World of Biofilm Physiology.</a> Adv. 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There is no single method that can simultaneously measure the effect of a compound on the viability, biomass, and biofilm matrix. Therefore, there is a need for combining assays in order to detect an effect on the three endpoints, preferably at a primary screening stage.
Resazurin is a very simple staining protocol consisting only of the addition of the redox probe. However, establishing the optimal incubation time of the biofilms with the resazurin is crucial to the success of this assay. In ...

Bacteria can switch between two very different lifestyles, planktonic and sessile, of which a biofilm is the most common example. In biofilms, bacteria form structured communities embedded in a self-produced matrix1. This self-produced matrix is a barrier between the bacteria and their external environment, and it protects the microbial cells, keeping them in close proximity. The composition of the biofilm matrix varies between and even within species, but it mostly consists of a tight network of lipopolysaccharides, extracellular DNA, and proteins. The matrix serves as a physical barrier inhibiting the entrance of harmful agents, but it also protects the biofilm from dehydration and prevents nutrients from escaping the cell2.
Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are purportedly responsible for over 80% of antibiotic-tolerant infections3. They display an inherently high tolerance against external threats: humidity, osmotic pressure, mechanical stress4, heat, UV radiation5, disinfectants, antimicrobial agents, and the host immune system1. For example, the required antimicrobial agent concentration required to kill a biofilm has been shown to be up to 1,000 times higher in comparison to that required to kill planktonic bacteria. The explanation for this higher tolerance seems to be multifactorial. The matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse; they transition between different metabolic states due to an existing gradient of oxygen, nutrients, and metabolites between the inner and outer parts of the biofilm6. Hence, in some biofilm regions, such as the core, bacteria are deprived of oxygen and nutrients and live in a metabolically less active or a completely dormant state7. The completely dormant cells are referred to as persister cells, and they are not susceptible to conventional antimicrobial treatments8. Thus, it is likely that biofilm resilience arises from a combination of the presently suggested and other, yet unknown, mechanisms. Staphylococcus spp. are still among the most problematic gram-positive bacteria, causing severe, often biofilm-related, infections4. It is suggested that up to 99% of all bacteria are associated within biofilms, making it the predominant bacterial lifestyle3. However, all of the currently existing antibiotics have been developed against single-cell (planktonic) bacteria. So far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place.
From a methodological perspective, additional challenges exist, as only a limited number of biofilm methods have been developed by standard-setting organizations, especially those applicable to the high-throughput screening of chemical libraries. All standardized assays (with only one exception) are based upon biofilm reactors, and these methods require large working volumes and large amounts of compounds to be tested, which are usually unavailable during the early investigational stage9-12. The only existing standardized screening-applicable assay is the so-called Calgary Biofilm Device, from which the commercially available minimum biofilm eliminating concentration (MBEC) system was developed13-15. However, the limitation of this assay is that the biofilms are grown on pegs, and not all bacterial species or even strains within the same species are able to form biofilms on this device. Moreover, methods that can be particularly applied to the exploration of natural compounds are needed. Natural products have been the major source for innovation in antimicrobial drug discovery over the past century16. They can provide novel anti-biofilm compounds with unique mechanisms of action that can also be effective against persister cells. Thus, the exploration of natural and naturally inspired libraries has high chances of producing promising and unique anti-biofilm leads.
Here, we present the experimental details of a platform of assays that was developed for the chemical screening of anti-biofilm compounds using three assays to measure the effects on the viability, total biomass, and matrix of Staphylococcus aureus biofilms. The first assay measures biofilm viability, and it is based on resazurin staining. Resazurin is a redox stain that is blue and non-fluorescent in its oxidized state and turns into pink, highly fluorescent resorufin when reduced by the metabolic activity of the bacteria. It is a very simple and fast method suitable for primary screenings17-20. The second assay, based on crystal violet staining, measures total biofilm mass. Crystal violet is a widely used stain for studying bacteria and bacteria in biofilms19,21-23. The assay is based on inexpensive reagents and has a simple absorbance endpoint reading. Finally, the third assay targets the extracellular polymeric substance (EPS)-matrix of the biofilm via wheat germ agglutinin (WGA), which binds specifically to poly-N-acetyl-glucosamine residues (PNAG) present in the matrix of staphylococcal biofilms24. The WGA is conjugated with a fluorophore that can be detected using fluorescence intensity readers25. We present here the rationale and details of the platform we developed, including examples of applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Resazurin</td>
			<td>Sigma Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>HT90132&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W11261</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD BacLight&#160;</td>
			<td>Molecular Probes</td>
			<td>L7012</td>
			<td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone soy agar</td>
			<td>Lab M, Neogen</td>
			<td>LAB011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptine soy broth</td>
			<td>Lab M, Neogen</td>
			<td>LAB004</td>
			<td></td>
		</tr>
		<tr>
			<td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td>
			<td>Thermo Fisher Scientific</td>
			<td>161093</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND caps, strips of 8</td>
			<td>Sigma Aldrich</td>
			<td>BR781413-300EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson CPX series ultrasonic bath</td>
			<td>Sigma Aldrich</td>
			<td>Z769428-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipipette</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomek 3000</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Multiplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC&#160;</td>
			<td>25923</td>
			<td></td>
		</tr>
	</tbody>
</table>

a platform of anti-biofilm assays suited to the exploration of natural compound libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

In the proposed platform, the effects on the viability, biomass, and biofilm matrix are quantified. In the working sequence (Figure 1), one sample plate is stained with resazurin and subsequently with crystal violet to simultaneously evaluate the effects on bacterial biofilm viability and on total biofilm biomass. Both assays can be performed consecutively in the same plate because it was demonstrated earlier that a first staining with resazurin had no statistically signi...

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A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

The, overall, goal of this assay platform is to provide a meaningful screening work flow combining three, relevant, end points, viability, biomass, and biofilm matrix, for identification and characterization of new, anti-biofilm compounds. This platform can help answers key questions in the field of antibiofilm agent discovery, by allowing an early distinction of short versus long term chemotherapeutic effects in a quick, explaining compatible manner. The main advantage of this developed platform is that it combines different assays to provide a more complete picture of the anti-biofilm effects of chemical libraries, especially of natural compounds.To begin the experiment, prepare untreated, control samples with 200 microliters of 10 to the sixth CFU per milliliter bacterial culture per well of a sterile, 96 microwell plate. Include media controls without bacterial culture. Next, on the same 96 well plates, prepare samples with four microliters of 50x antibiotic stock solution and 196 microliters of the bacteria culture per well, then, incubate the cultures.Continue with resazurin staining by using a multichannel pipette to transfer the entire planktonic solution, carefully, without touching the biofilms or creating air bubbles, to a separate, clean, 96 well plate. Again, using a multichannel pipette, wash the biofilms, once, with sterile PBS by adding 200 microliters per well and remove the solution carefully. Add 200 microliters of 20 micromolar resazurin solution, per well, of the biofilm plate and incubate the biofilm, in darkness, at 200 rpm for 20 minutes, until the untreated biofilm controls are evenly pink.Then, measure the fluorescence with a plate reader. Remove the resazurin stain carefully from the wells. Fix the biofilms with 200 microliters of methanol, for 15 minutes.After fixation, remove the methanol and let the plate air dry for 10 minutes. Next, use a multichannel pipette to carefully add 190 microliters of 0.02%crystal violet solution to the biofilm. Avoid touching the sides of the wells, with the stain, while pipetting, and prevent the formation of air bubbles, and do not press the pipette to complete blowout.Then, incubate the plate for five minutes at room temperature. With a multichannel pipette, remove the stain carefully. Wash the biofilm with 200 microliters of deionized water.Let the wells dry for five minutes at room temperature, and dissolve the remaining stain in 33%acetic acid. Incubate the plate for one hour at room temperature. After incubation, read the absorbance at 595 nanometers.Measure the turbidity at 595 nanometers of the prepared, planktonic solution plate. This can be done during the incubation of the crystal violet stained plate. Next, stain the planktonic bacteria, with resazurin, by adding 10 microliters of the resazurin stock, per well.Pipette the solution to mix it well. Incubate the plate, in darkness, at room temperature, 200 rpm, for approximately five minutes, until the untreated controls are evenly pink. Then, measure the fluorescence.Obtain one of the parallel sample plates for this staining, and use a multichannel pipette to remove the planktonic solution from the wells. Wash the wells, once, with steril PBS carefully, without touching the biofilms. Add 200 microliters of wheat germ agglutinin, or WGA solution, per well, to be stained.Then, incubate the plate. After incubation, wash the cells with PBS, three times, to remove the unbound stain. Let the plate dry for 15 minutes at room temperature.Visualize the samples with a fluorescence microscope and an FITC filter. Dissolve the bound stain in 200 microliters of 33%acetic acid per well. Seal the wells with strip caps and sonicate them using a water bath sonicator.After sonication, incubate the plate, for one hour, at 37 degrees Celsius. After one hour, repeat sonication while keeping the wells sealed between the sonication steps. Measure the fluorescence with a plate reader.Again, take one of the parallel sample plates for this staining, and using a multichannel pipette, remove the planktonic solution from the wells. Then, wash the wells once with sterile PBS. Add six microliters of the staining solution per well, and incubate the plate in darkness for 15 minutes.Before microscopy, remove the excess liquid, manually, using a multichannel pipette. Capture the images using a fluorescence microscope with a FITC filter, for green fluorescence, to visualize viable cells and a TRITC filter, for red fluorescence, to visualize dead cells. Use one of the parallel sample plates for this staining, and remove the planktonic solution, from the wells, and wash the wells once with sterile PBS, using a multichannel pipette.Next, add 200 microliters of the staining solution, per well, and incubate them, for 15 minutes, in darkness. Finally, measure the fluorescence with a plate reader. Two screenings were run to demonstrate the performance of the platform as a percent of the untreated biofilm control.The screening of a cinchona alkaloid derivative library identified one, active compound. The screening of a library of abietane-type diterpenoids and derivatives, identified five active compounds. Penicillin G and Ciprofloxacin both significantly decrease the viability, biomass, and biofilm matrix PNAG content prior to biofilm formation.However, this was not the case when post-exposure was used. The most prominent result was the increase, of the biofilm matrix, to over 200%when preformed biofilms were treated with high concentrations of Penicillin G.This fluorescent image confirms that Penicillin G kills about 50%of the preformed, Staph aureus, biofilm, bacterial population. The average of the green to red fluorescence ratios for the untreated biofilm wells indicate a predominance of live cells.While Penicillin treated wells have about 56%live cells. The cells remaining alive, after Penicillin treatment, produced a significantly higher amount of extracellular polymeric substance, or EPS, when compared with untreated biofilms. While attempting this procedure, it's important to remember to pay attention to the manual handling of the pipette to avoid contamination of bacteria and stain between wells.Following this platform, antibiofilm compounds effective against Staphylococcus aureus can be quickly identified and characterised. If other bacterial species are used, other optimization of the staining protocols will be needed, but the rational of the platform will remain the same.

a-platform-anti-biofilm-assays-suited-to-exploration-natural-compound

Research

JoVE Journal

Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...