Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Caution: Please consult the safety data sheets (SDS) of all involved chemicals before use. The drug screening experiment involves the synthesis and handling of nanomaterials, which may have additional hazards compared to their bulk counterpart. Please ensure all necessary control measures to be practiced throughout the experiment, including the use of engineering controls (fume hood) and personal protective equipment (PPE, e.g., safety length pants, closed-toe shoes, chemical resistant gloves, and safety goggles).
1. Preparation of Chemical Reagents for Drug Screening
<ol>
	<li>Precursors for Au NCs synthesis
	<ol>
		<li>Dissolve 30 mg of gold (III) chloride solution (99.99% trace metals basis, 30 wt.% in dilute HCl) in 6.9 ml of ultrapure water to prepare 15 mM of gold (III) chloride solution. Caution: gold chloride solution is corrosive and irritant. Wear proper PPE to avoid direct contact with eye and skin.</li>
		<li>Dissolve 74 mg of HSA or BSA in 1 ml of ultrapure water to prepare 74 mg/ml of protein stock solution.</li>
	</ol>
	</li>
	<li>Drug solutions
	<ol>
		<li>Dissolve the desired amount of drug, e.g., 1.9 mg for (a) ibuprofen, 2.8 mg for (b) warfarin, 2.3 mg for (c) phenytoin, and 1.5 mg for (d) sulfanilamide in 20 &#181;l of DMSO to prepare 450 mM of drug stock solutions. 
		Note: DMSO is selected as the solvent because these drugs are hydrophobic and have poor solubility in water.</li>
	</ol>
	</li>
	<li>Other reagents
	<ol>
		<li>Dissolve 600 mg of NaOH pellets in 10 ml of ultrapure water to prepare 1.5 M of NaOH solution. Dissolve 2.4 g of urea in 2 ml of ultrapure water to prepare 20 M of urea solution.</li>
	</ol>
	</li>
</ol>
2. Synthesis of Protein-Templated Au NCs
<ol>
	<li>HSA-templated Au NCs (HSA-Au NCs)
	<ol>
		<li>Place two glass vials, each containing a micro magnetic stir bar in it, on two separate temperature controllable magnetic stirrers.</li>
		<li>Set the temperature of one magnetic stirrer to 60 &#176;C, and label the glass vial on top of it as &#8220;60 &#176;C&#8221;; while the other magnetic stirrer is kept at room temperature (RT) where the glass vial on top of it is labeled as &#8220;RT&#8221;.</li>
		<li>Add 200 &#181;l of HSA solution, 200 &#181;l of ultrapure water, and 200 &#181;l of gold (III) chloride solution to each vial under constant stirring (set as 360 rpm unless otherwise specified) to allow the encapsulation of Au ions inside the protein template.</li>
		<li>2 min later, add 20 &#181;l of NaOH solution to each vial so as to activate the reducing capability of HSA in forming Au NCs and start to record the reaction time as 0 min.</li>
		<li>Every 20 min, draw 50 &#181;l of solutions from each of the sample to a 384-well black plate and measure the emission spectrum with a microplate reader. The typical scanning setup: &#955;ex = 370 nm, &#955;em = 410 &#8211; 850 nm.</li>
		<li>Stop the magnetic stirrer after 100 min.</li>
		<li>Cool down the glass vials under running water in a sink. Caution: The glass vials are hot. Wear heat resistant gloves to avoid burning.</li>
		<li>Plot the photoemission spectra at all time for each sample to acquire the formation kinetics of Au NCs at different temperature conditions.</li>
	</ol>
	</li>
	<li>BSA-templated Au NCs (BSA-Au NCs)
	<ol>
		<li>Place a glass vial containing a micro magnetic stir bar on the top of a magnetic stirrer. Leave the temperature as room temperature.</li>
		<li>Mix 200 &#181;l of BSA solution, 200 &#181;l of urea, and 200 &#181;l of gold (III) chloride solution in the glass vial under constant stirring.</li>
		<li>2 min later, add 20 &#181;l of NaOH solution to activate the reducing capability of BSA to form Au NCs and start to record the reaction time as 0 min. Measure the photoemission spectrum of the reaction mixture hourly. 
		Note: The photoemission spectrum of BSA-Au NCs is measured less frequently because the formation rate of BSA-Au NCs is slower than that of HSA-Au at the same temperature due to less effectiveness of urea to unfold the protein.</li>
		<li>Stop the magnetic stirrer after 7 hr. Plot the photoemission spectra at all time to see the formation kinetics of Au NCs by urea denaturation.</li>
	</ol>
	</li>
</ol>
3. Small Molecular Drug Screening
<ol>
	<li>Screen relative binding affinity of different small molecular drugs to HSA
	<ol>
		<li>Place five glass vials, each containing a magnetic stir bar in it, on top of a temperature controllable multipoint magnetic stirrer. Label these vials as a, b, c and d respectively for each drug, namely ibuprofen, warfarin, phenytoin, and sulfanilamide. Label the one with pure HSA as control.</li>
		<li>Add 200 &#181;l of HSA to each vial. Add 1 &#181;l of drug solution to the four corresponding vials. Add 1 &#181;l of DMSO to the control. Switch on the stirrer and set the spin speed to 360 rpm and incubate for 1 hr to allow the drug binding to HSA to complete.</li>
		<li>1 hr later, add 200 &#181;l of Milli-Q water and 200 &#181;l of gold (III) chloride solution to each vial under constant stirring. Set the temperature to 60 &#176;C under constant stirring for 10 min. Add 20 &#181;l of 1.5 M NaOH to each vial and start to record the reaction time as 0 min.</li>
		<li>Quickly draw 50 &#181;l of solution from each vial to a 384-well black plate and measure the emission spectrum (results labeled as 0 min). Record the emission spectra of each sample every 10 min. Collect at least four spectra at four different time, that is 0, 10, 20, and 30 min.</li>
		<li>Repeat steps 3.1.1 - 3.1.4 several times to obtain results with a consistent trend. Stop the magnetic stirrer.</li>
		<li>Plot the time-resolved photoemission spectra for each sample (a - d, and control). Identify the peak intensity of all samples and plot against the time to compare the formation kinetics of Au NCs in different drug-loaded protein templates.</li>
	</ol>
	</li>
	<li>Measure the binding constant of a specific drug to HSA
	<ol>
		<li>Repeat steps 3.1.1 &#8211; 3.1.4. Replace the drug solution with ibuprofen solutions at four different concentrations (including a control sample using HSA only without drug loading). Label the glass vial according to the drug concentration correspondingly.</li>
		<li>10 min later, cool down the glass vials under running water in a sink. Caution: The glass vials are hot. Wear heat resistant gloves to avoid burning.</li>
		<li>Draw 50 &#181;l of the above solutions from each vial to a 384-well black plate and measure the emission spectrum.</li>
		<li>Repeat steps 3.2.1 &#8211; 3.2.3 twice more to obtain another two sets of results at different drug concentrations.</li>
		<li>Stop the magnetic stirrer. Plot the photoemission spectra and analyze the results.
		<ol>
			<li>For the raw data obtained in each individual batch, plot the photoemission spectra of each sample in software such as OriginPro software and find out the peak intensity.</li>
			<li>Calculate and plot the relative fluorescence intensity [(I0 - I) /I0] against the drug concentration, where I and I0 refer to the fluorescence intensity of Au NCs formed in drug-loaded HSA and pure HSA, respectively.</li>
			<li>Calculate the binding constant by fitting the data to a single site binding model using the Michaelis-Menten equation: Y = Rmax &#215; C/(KD + C), where Rmax indicates the maximum response signal, C is the concentration of ligand and KD is the binding constant. Perform this in software such as OriginPro. Select the menu Analysis Fitting Nonlinear Curve Fit, then select Hill function from Growth/Sigmoidal category. On the Settings: Function Selection page, click Fit to show up the fitted results.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

There are several critical steps that need to be highlighted in this method. In the protocol of screening the relative binding affinity of different small molecular drugs, Steps 3.1.2, 3.1.3, and 3.1.4 are critical to obtain good results showing consistent trend for the relative binding strength. In these steps, the actions of adding chemicals and drawing reaction solutions for measurement should be as quickly as possible to minimize the time lag effect and the same sequence of adding chemicals and drawing reaction solut...

Serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) are the most abundant protein in plasma and play a vital role in maintaining the osmotic pressure of the blood compartment. They are also recognized as carrier proteins for small molecules of low water solubility, such as steroids, fatty acids, thyroid hormones, and a wide variety of drugs. The binding property (e.g., binding sites, binding affinity or strength) of these molecules to serum albumins forms an important topic in pharmacokinetics.1-4 Several analytical methods have been developed to study the binding properties of different drugs to serum albumins, such as X-ray crystallography,5,6 nuclear magnetic resonance (NMR),7-11 and surface plasmon resonance (SPR),12,13 etc. However, these methods are constrained by either a tedious and time-consuming analyzing process (e.g., growth of single crystal for X-ray crystallographic study), requirement of specialized and expensive equipment (SPR), or in need of costly isotope labeling (NMR) for detection. It is therefore highly desirable to develop alternative ways for small molecular drug screening in a fast, straight-forward, and cost-efficient manner.
Gold nanoclusters (Au NCs) are a special type of nanomaterial, which contain several to tens of metal atoms with sizes smaller than 2 nm.14-17 They have attracted extensive research interests due to their discrete and size-dependent electronic structure,18,19 and molecular-like absorptions and emissions.20-23 Such unique materials properties, in particular the strong fluorescence, have found diverse applications such as sensing and imaging in biological systems. 24-32 Ultrasmall fluorescent Au NCs can be synthesized using functional proteins, such as serum albumins, as template. 33 In a typical protein-templated synthesis of Au NCs, a certain amount of Au salts are first encapsulated inside the protein and subsequently reduced by the protein itself. The reducing ability of the protein is attributed to constituent functional amino acid residues (e.g., tyrosine) that can be activated by increasing the solution pH to alkaline. Unfolding of protein structure is considered as a critical step for the formation of Au NCs. This is because in an unfolded protein, more reducing functional groups can be exposed to the encapsulated Au salts. Protein unfolding can be achieved by heat treatment or exposure to denaturing agents. Introduction of small molecular drugs can also affect the unfolding process, i.e. modifying the midpoint denaturation temperature and the enthalpy of unfolding. 34,35 The effect of all these factors, in turn can be reflected by the formation kinetics of fluorescent Au NCs and manifested in the fluorescence intensity of resultant Au NCs.36
This video demonstrates the method of drug screening by synthesizing Au NCs in drug-loaded albumin proteins at a higher temperature (60 &#176;C) or in the presence of denaturing agents (e.g., urea). The fluorescence intensity of resultant Au NCs is the signal readout. First, Au NCs are synthesized in HSA and BSA templates treated at 60 &#176;C or in the presence of urea to show how protein unfolding (induced by heat treatment or denaturants) affects the formation kinetics of Au NCs. Second, Au NCs are synthesized in protein templates preloaded with different drugs, and the drug loading effect on the relative fluorescence intensities of resultant Au NCs is studied, which provide the measure of relative binding strength. Finally, the Au NC-drug screening protocol is modified for quantitative measurement of drug-protein binding constant (KD) by varying the drug content preloaded in the protein of a fixed concentration.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

Protein unfolding is an important procedure for the formation of protein-templated Au NCs because more reactive functional groups (e.g., tyrosine residues) of a protein can be exposed to reduce the encapsulated Au ions and thus accelerate the formation rate of Au NCs. Heating and external denaturing agents are two common means to promote the protein unfolding process. Figure 1 demonstrates the effect of heating and adding external denaturing agents on the formation kinetics of Au NCs, using HSA ...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Research

JoVE Journal

Bioengineering

9.9K 조회수.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

비디오: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...