Name: describing a transcription factor dependent regulation of the microrna transcriptome
Uploaded: 2016-06-15T13:00:00
Description: Watch this Scientific Journal Video about Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome at JoVE.com

This study was supported by a grant from the CLL Global Research Foundation. The University of Texas MD Anderson Cancer Center is supported in part by the National Institutes of Health through a Cancer Center Support Grant (P30CA16672).

1. Identify Transcription Factor Binding Sites in the Promoter of MicroRNA Genes Using Data Mining Approach
<ol>
	<li>Use the University of California Santa Cruz (UCSC) genome browser to extract chromatin immunoprecipitation (ChIP) sequencing data generated as part of the Encyclopedia of DNA element (ENCODE) project.
	<ol>
		<li>Open the table browser in the UCSC genome browser.</li>
		<li>Use the following specifications to extract the table: Clade: (Mammals), genome: (Human), Assembly: (Feb2009(GRch37/hg18)), group: ...

<ol>
	<li>Bentwich, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hundreds+of+conserved+and+nonconserved+human+microRNAs.">Identification of hundreds of conserved and nonconserved human microRNAs.</a> Nat Genet. 37, 766-770 (2005).</li><li>Hata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+microRNAs+in+cardiovascular+biology+and+disease.">Functions of microRNAs in cardiovascular biology and disease.</a> Annu Rev Physiol. 75, 69-93 (2013).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Piriyapongsa, J., Jordan, I. K., Conley, A. B., Ronan, T., Smalheiser, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+sites+are+highly+enriched+within+microRNA+precursor+sequences.">Transcription factor binding sites are highly enriched within microRNA precursor sequences.</a> Biol Direct. 6, 61 (2011).</li><li>Rozovski, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transducer+and+activator+of+transcription+(STAT)-3+regulates+microRNA+gene+expression+in+chronic+lymphocytic+leukemia+cells.">Signal transducer and activator of transcription (STAT)-3 regulates microRNA gene expression in chronic lymphocytic leukemia cells.</a> Mol Cancer. 12, 50 (2013).</li><li>Turner, M. J., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+microRNA+expression+in+C.+elegans:+promoting+better+understanding.">Transcriptional control of microRNA expression in C. elegans: promoting better understanding.</a> RNA Biol. 6, 49-53 (2009).</li><li>Cui, Q., Yu, Z., Pan, Y., Purisima, E. O., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+preferentially+target+the+genes+with+high+transcriptional+regulation+complexity.">MicroRNAs preferentially target the genes with high transcriptional regulation complexity.</a> Biochem Biophys Res Commun. 352, 733-738 (2007).</li><li>Yamakuchi, M., Lowenstein, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-34,+SIRT1+and+p53:+the+feedback+loop.">MiR-34, SIRT1 and p53: the feedback loop.</a> Cell Cycle. 8, 712-715 (2009).</li><li>Baer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+promoter+DNA+hypermethylation+and+hypomethylation+is+associated+with+aberrant+microRNA+expression+in+chronic+lymphocytic+leukemia.">Extensive promoter DNA hypermethylation and hypomethylation is associated with aberrant microRNA expression in chronic lymphocytic leukemia.</a> Cancer Res. 72, 3775-3785 (2012).</li><li>Hazan-Halevy, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+is+constitutively+phosphorylated+on+serine+727+residues,+binds+DNA,+and+activates+transcription+in+CLL+cells.">STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells.</a> Blood. 115, 2852-2863 (2010).</li><li>Melo, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TARBP2+mutation+in+human+cancer+impairs+microRNA+processing+and+DICER1+function.">A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function.</a> Nat Genet. 41, 365-370 (2009).</li><li>Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+STAT+family+proteins:+lessons+from+knockout+mice.">Functional roles of STAT family proteins: lessons from knockout mice.</a> Stem Cells. 17, 138-146 (1999).</li><li>Frank, D. A., Mahajan, S., Ritz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+lymphocytes+from+patients+with+chronic+lymphocytic+leukemia+contain+signal+transducer+and+activator+of+transcription+(STAT)+1+and+STAT3+constitutively+phosphorylated+on+serine+residues.">B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues.</a> J Clin Invest. 100, 3140-3148 (1997).</li><li>Calin, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling+reveals+distinct+signatures+in+B+cell+chronic+lymphocytic+leukemias.">MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias.</a> Proc Natl Acad Sci U S A. 101, 11755-11760 (2004).</li><li>Consortium, 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=A+user's+guide+to+the+encyclopedia+of+DNA+elements+(ENCODE).">A user's guide to the encyclopedia of DNA elements (ENCODE).</a> PLoS biology. 9, e1001046 (2011).</li><li>Miranda, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pattern-based+method+for+the+identification+of+MicroRNA+binding+sites+and+their+corresponding+heteroduplexes.">A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes.</a> Cell. 126, 1203-1217 (2006).</li><li>Consortium, 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=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489, 57-74 (2012).</li><li>Lau, J. C., Hanel, M. L., Wevrick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+and+imprinted+epigenetic+modifications+of+the+human+NDN+gene.">Tissue-specific and imprinted epigenetic modifications of the human NDN gene.</a> Nucl Acids Res. 32, 3376-3382 (2004).</li><li>Corcoran, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+mammalian+microRNA+promoters+emerge+from+polymerase+II+chromatin+immunoprecipitation+data.">Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data.</a> PLoS One. 4, e5279 (2009).</li><li>Bhattacharyya, M., Feuerbach, L., Bhadra, T., Lengauer, T., Bandyopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+transcription+start+site+prediction+with+multi-objective+feature+selection.">MicroRNA transcription start site prediction with multi-objective feature selection.</a> Stat Appl Genet Mol Biol. 11, (2012).</li></ol>

The mechanism underlying the RNA polymerase II- dependent transcription of protein coding genes has been extensively studied. While these elements make up only 1% - 2% of the human genome, evidence from the ENCODE project suggest that over 80% of the human genome may undergo transcription 17 and what regulates the transcription of the non-coding DNA elements remains largely unknown 6.
Several studies, which indicated that Pol II is also responsible for the transcription o...

MicroRNAs are endogenous small non coding regulatory RNAs that typically function as negative regulators of mRNA expression at the posttranscriptional level. Approximately 1,000 non-coding 20 to 25 nucleotide long microRNAs are found in the human genome 1,2. MicroRNAs regulate gene expression through canonical base pairing between the seed sequence of the microRNA and its complementary seed match sequence, which is commonly located at the 3&#39; untranslated region (UTR) of the target mRNAs. Collectively, microRNAs regulate more than 30% of protein coding genes 3, but only little is known about the transcription from DNA of microRNAs. It has been suggested that the regulation of microRNA transcription is similar to that of mRNA 4,5. In particular, similar to its activity in promoting transcription of protein coding genes, transcription factors are thought to activate transcription of microRNAs 6. Transcription factor-microRNA interplay has been reported as a modulator factor of gene expression 7, and may also form feed-back and feed-forward loops. For example, Yamakuchi et al. reported a feedback loop in which p53 induces the expression of microRNA34a, which in turn inhibits translation of the p53 repressor SIRT and thereby increasing p53 activity 8.
Whereas specific examples of transcription factor dependent expression of microRNAs have been reported, an accepted method which provides information on how a transcription factor of interest regulates the expression of the microRNA-transcriptome is lacking. The purpose of the protocol suggested herein is to provide an in-depth description of transcription factor-dependent regulation of the microRNA-transcriptome. By combining publically available data, bioinformatics tools and using microarray technology, researchers who follow this algorithm would be able to capture on a genomic scale how any transcription factor in any cell type of interest regulates the expression of the microRNA-transcriptome and to explore a putative contribution of the transcription factor-mRNA in regulating microRNA expression.

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			<td height="21" style="height:21px;width:363px;">Lipofectamin 2000</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:119px;">11668027</td>
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			<td height="21" style="height:21px;">0.45 um syringe filter&#160;</td>
			<td style="width:204px;">Thermo Scientific (Nalgene)</td>
			<td>190-2545</td>
			<td></td>
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			<td height="21" style="height:21px;">Amicon ultracentrifugal filter device with threshold of 100kDa&#160;</td>
			<td style="width:204px;">Merck Millipore</td>
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			<td height="21" style="height:21px;">Polybrene&#160;</td>
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			<td height="21" style="height:21px;">TRIzol reagent&#160;</td>
			<td style="width:204px;">Life Tachnologies (Invitrogen)</td>
			<td>15596-026</td>
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			<td height="21" style="height:21px;width:363px;">293 Cell line human</td>
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describing a transcription factor dependent regulation of the microrna transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

STAT3 is a transcription factor which typically induces the transcription of genes that have anti apoptotic and proliferative effects 12 . Whether STAT3 also affect the non-coding RNA transcriptome is currently unknown. In all CLL cells STAT3 is constitutively phosphorylated on serine 707 residues 10,13. Phosphoserine STAT3 shuttles to the nucleus, binds to DNA, and activates genes known to be activated by tyrosine pSTAT3 in other cell types 10. Because CL...

Watch this Scientific Journal Video about Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome at JoVE.com

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

The overall goal of this methodology is to describe how a transcription factor of interest regulates the microRNA transcriptome. This method can help answer key questions on whether transcription factors that modulate transcription of protein coding genes are also involved in transcription of microRNAs. The main advantage of this methodology is that it integrates data mining, laboratory data, and bioinformatic analysis to provide a comprehensive description of microRNA genes transcriptions that is dependent on the activity of transcription factor of interest.Though this method can provide insight into the interaction of microRNA gene transcription and activity of the transcription factor status, it is important to remember that this is a descriptive method and confirmation of such an association should be done using additional assays. To begin, open a genome browser, such as the University of California Santa Cruz genome browser, in order to extract chromatin immunoprecipitation sequencing data. Once on the site, go to the top menu, select Tools and open the Table Browser.Enter the specifications, as shown here, into their respective boxes, and then select Get Output to extract the table. Save the output as a text file. Then, import the file into a spreadsheet.Once in the spreadsheet program, sort the name column and filter the results for a transcription factor of interest, such as STAT2. Then, copy the list of microRNA promoters based on their H3K4me3 epigenetic signature and paste the list into a txt file. Then, use the supplemental code written in SQL and provided in the supplementary data files to map the collected data and determine the median binding affinity.First, add 1.5 million HEK293 cells to a 10 centimeter dish and add DMEM supplemented with 10%FBS. Culture the cells for 24 hours until they reach about 50%confluency. Next, replace the media.Then, add a transvection agent combined with five micrograms of a green fluorescence protein Lentivirus containing the targeted shRNA and five micrograms of packaging vectors in accordance with the manufacturers instructions. As a control, transvect an identical plate of cells using scrambled shRNA and the packaging vectors. Incubate the transvection mix on the cells for 16 hours and then change the media to 10 milliliters of fresh DMEM media supplemented with 10%FBS.48 hours post-transvection, remove the media from the cells. Then, centrifuge for five minutes in order to collect infectious supernatant. Filter the supernatant through a 0.45 micron syringe filter to remove any floating cells.Next, add the media into an ultracentrifigal filter device with a threshold of 100 kilodaltons. Centrifuge the media to concentrate the Lentivirus until the volume is less than 250 microliters. Transfer the remaining liquid into a cryotube and store the concentrated virus at minus 80 degrees Celsius until it is needed.When ready to continue, remove the frozen Lentivirus from the minus 80 freezer and bring the vial to room temperature. Transfer 100 microliters of viral supernatant to a fresh 1.5 milliliter microfuge tube and add 900 microliters of medium without serum. Next, add 10 nanograms per milliliter hexadimethrine bromide to the one milliliter virus suspension.Mix the tube gently, and let the mixture stand for five minutes. Harvest five million cells for each planned transduction and then gently re-suspend the cell pellet in 0.5 milliliters of media containing virus. Transfer the cells to a 12-well plate and incubate them for four to 24 hours.Then, add an additional 0.5 milliliters of medium containing 20%FBS. At 48 to 72 hours post-transduction, harvest the cells and stain them with propidium iodide according to the manufacturer's instructions. After staining, protect the cells from light and use facts to estimate transvection efficiency by measuring the rates of GFP positive, propidium iodide negative cells.Then, collect the sorted GFP positive cell population and extract their proteins. Finally, use western blotting to determine levels of the targeted transcription factor before and after infecting the cells with the designated shRNA. After the transvection of CLL cells with STAT3 shRNA, the levels of STAT3 mRNA and STAT3 protein both significantly decreased.In addition, a microRNA array of the CLL cells depicted 23 microRNAs whose expression differed significantly between CLL cells that were trasnfected with STAT3 shRNA and CLL cells that were transvected with an empty vector. The results of the microRNA array were validated for seven of the genes, shown here, using quantitative RT-PCR. Following this procedure, other methods like chromatin immunoprecipitation and electromobility shift assay and a difference assay can be performed in order to validate that this transcription factor binds and activates the promoter of a specific mirror gene.After watching this video, you should have a good understanding of how to integrate bio informatics analysis, publicly available data and silencing a transcription factor using shRNA approach to find how this transcription factor regulates the expression of microRNA genes.

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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...