Name: systematic approach to identify novel antimicrobial and antibiofilm molecules from plants' extracts and fractions to prevent dental caries
Uploaded: 2021-03-31T12:30:00
Description: Watch this Scientific Journal Video about Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries at JoVE.com

We express our gratitude to N&#250;cleo de Bioensaios, Bioss&#237;ntese e Ecofisiologia de Produtos Naturais (NuBBE) of the Chemistry Institute of UNESP, Araraquara/SP for providing the laboratories for preparing plant material. We also thank the Applied Microbiology Laboratory of the Department of Dental Materials and Prosthodontics, UNESP, Araraquara/SP. This research was supported by a research grant from the S&#227;o Paulo Research Foundation (FAPESP #2013/07600&#8211;3 to AJC) and scholarships plus overhead funds (FAPESP #2017/07408&#8211;6 and FAPESP #2019/23175-7 to SMR; #2011/21440&#8211;3 and #2012/21921&#8211;4 to PCPB). The National Council for Scientific and Technological Development in association with FAPESP provided additional support (INCT CNPq #465637/2014&#8211;0 and FAPESP #2014/50926&#8211;0 to AJC).

1. Collection of Plant Material
<ol>
	<li>Plant material
	<ol>
		<li>Record the access to the plant material on electronic platforms that regulate access to genetic heritage in the country where the collection will take place. For example, in Brazil, register with the National System for the Management of Genetic Heritage and Associated Traditional Knowledge &#8211; SisGen (website https://sisgen.gov.br/paginas/login.aspx).</li>
		<li>Collect samples of the plant material of interest (e.g., leaves, stems, roots, flower...

The main challenges related to the work with natural crude extracts comprise their complex composition and the inadequacies of classic bio-guided isolation studies. Although this process is slow, it is effective and has led to major findings in NP research. To rationalize, prioritization-driven studies are needed to rationalize. Thus, the use of modern chemical profiling approaches for the analysis of CE and dereplication before isolation are important to characterize the studied material and especially useful to avoid r...

The earliest models of medicine used in societies were based on natural products (NPs). Since then, humans have been searching for new chemicals in nature that can be transformed into drugs1. This search caused a continuous improvement of technologies and methods for ethnobotanical screening1,2,3. NPs offer a rich source of structurally diverse substances, with a wide range of biological activities useful for developing alternative or adjuvant therapies. However, the inherent complex plant matrix makes the isolation and identification of the active compounds a challenging and time-consuming task4.
NPs-based drugs or formulations can be used to prevent and/or treat several conditions affecting oral, including dental caries4. Dental caries, one of the most prevalent chronic diseases globally, derives from the interaction of sugar-rich diet and microbial biofilms (dental plaque) formed on the tooth surface that leads to demineralization caused by organic acids derived from microbial metabolism, and if not treated, leads to teeth loss5,6. Although other microorganisms may be associated7, Streptococcus mutans is a critical cariogenic bacterium because it is acidogenic, aciduric, and an extracellular matrix builder. This species encodes multiple exoenzymes (e.g., glycosyltransferases or Gtfs) that use sucrose as a substrate8 to build the extracellular matrix rich in exopolysaccharides, which are a virulence determinant9. Also, the fungus Candida albicans can drive up the production of that extracellular matrix7. Albeit fluoride, administered in various modalities, remains the basis for preventing dental caries10, new approaches are needed as adjuvants to increase its effectiveness. In addition, the available anti-plaque modalities are based on the use of broad-spectrum microbicidal agents (e.g., chlorhexidine)11. As an alternative, NPs are potential therapies for controlling biofilms and preventing dental caries12,13.
The further advance in the discovery of new bioactive compounds from plants includes necessary steps or approaches such as: (i) the use of reliable and reproducible protocols for sampling, considering that plants often show intraspecific variability; (ii) the preparation of comprehensive extracts and their respective fractions in small scale; (iii) the characterization and/or dereplication of their chemical profiles thought the acquisition of multidimensional data such as GC-MS, LC-DAD-MS, or NMR, for example; (iv) the use of viable and high-yield models to assess bioactivity; (v) the selection of potential new hits based on multivariate data analysis or other statistical tools; (vi) to perform the isolation and purification of the targeted compounds or promising candidates; and (vii) the validation of the corresponded biological activities using the isolated compounds2,14.
Dereplication is the process of rapidly identifying known compounds in crude extract and allows differentiating novel compounds from those that have already been studied. Besides, this process prevents isolation when bioactivity has already been described for certain compounds, and it is particularly helpful to detect &#8220;frequent hitters&#8221;. It has been used in different untargeted workflows ranging from major compound identification or the acceleration of activity-guided fractionation up to the chemical profiling of collections of extracts. It can be fully integrated with metabolomic studies for the untargeted chemical profiling of CE or the targeted identification of metabolites. All of this ultimately leads to prioritizing extracts before the isolation procedures1,15,16,17.
Therefore, in the present manuscript, we describe a systematic approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions. It includes four multidisciplinary steps: (1) collection of plant material; (2) preparation of crude extracts (CE) and fractions (CEF), followed by their chemical profile analysis; (3) bioassays; and (4) biological and chemical data analyses (Figure 1). Thus, we present the protocol developed to analyze of the antimicrobial and antibiofilm activities of Casearia sylvestris extracts and fractions against Streptococcus mutans and Candida albicans13, as well as the procedures for the phytochemical characterization and data analysis. For simplicity, the focus here is to demonstrate the approach for screening natural compounds using the bacterium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61773/61773fig01v2.jpg" /> 
Figure 1: Flow-chart of the Systematic Approach to identify active molecules from plants extracts and fractions. <a href="https://www.jove.com/files/ftp_upload/61773/61773fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>96-well microplates&#160;</td>
			<td>Kasvi</td>
			<td>Flat bottom</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated carbon</td>
			<td>LABSYNTH</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical mill</td>
			<td>Ika LabortechniK</td>
			<td>Model A11 Basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood agar plates</td>
			<td>Laborclin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column C18</td>
			<td>Phenomenex Kinetex</td>
			<td>150 &#215; 2.1 mm, 2.6 &#181;m, 100&#194;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Vehicle solution</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate reader</td>
			<td>Biochrom Ez</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J. T. Baker</td>
			<td>For extraction and fractionation steps, and mobile phase composition</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>Vehicle solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>J. T. Baker</td>
			<td>Fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>La Jolla</td>
			<td>GraphPad Prism7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>J. T. Baker</td>
			<td>Fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>J. T. Baker</td>
			<td>For extraction step</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer (a freeze dryer)</td>
			<td>Savant</td>
			<td>Modulyo</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Millipore</td>
			<td>LAC</td>
			<td>0.22 &#181;m x 13 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Quimis</td>
			<td>Model G816&#8201;M20</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamide solid phase extraction cartridge</td>
			<td>Macherey-Nagel</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Merck</td>
			<td>40&#8211;63&#8201;&#956;m, 60&#8201;&#194;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Synth</td>
			<td>0,89% in water</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid phase extraction cartridges (SPE)</td>
			<td>Macherey-Nagel</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC-DAD</td>
			<td>Dionex</td>
			<td>Ultimate 3000 RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>UNIQUE</td>
			<td>Model USC 2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic approach to identify novel antimicrobial and antibiofilm molecules from plants' extracts and fractions to prevent dental caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

We provide an example of using a systematic approach to screen the biological activity of plant extracts and fractions to identify potentially active molecules for possible new anti-caries therapies: antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans13.
Background 
Complex interactions between specific oral microorganism...

Watch this Scientific Journal Video about Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries at JoVE.com

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

The presented protocol is used for correctly screening and analyzing bioactive candidates in natural products to control oral biofilms. It can also be adapted for applications in other biofilm research fields. This most starch screening and analysis method make it possible to remove bioactive components simultaneously.Dental caries is a biofilm diet derived, highly prevalent, chronic disease. This protocol can help to identify natural products to control biofilms and consequently, dental caries. Begin by preparing the extraction solvent with a hydroalcoholic mixture.You sample concentrations between 50 and 100 milligrams per milliliter of the extraction solvent and perform 15-minute ultrasound assisted extractions in microtubes. Repeat the extraction three times. After each extraction step, centrifuge the sample to decap the solid residue and remove the supernatant.Combine the supernatants from each extraction step and filter them. Save the aliquots for chemical analysis and bioassays. For fractionation, dilute the crude extract sample to obtain a 100 milligram per milliliter solution, using the initial extraction solvent.Then, transfer one milliliter of this sample to a preconditioned solid-phase extraction cartridge with one gram of Absorbent, and a capacity of six milliliters. Perform fractionation, using around three dead volumes of each extraction LUN, and collect one fraction by LUN composition. Save aliquots for chemical analysis and bioassays.Remove the solvent under vacuum, nitrogen flow, or lyophilization, and register the weight and yield. Reconstitute the dry matter with the best possible solvents. Calculate the solvent concentration for the stock solution, using the formula in the text manuscript.Reactivate the microbial strain of S.mutans UA 159 on blood agar, and culture it in liquid culture medium. Perform a one to 20 dilution of the initial culture using the same culture medium, and incubate it until it reaches the mid-log growth phase. Prepare the inoculum with a defined population in TYEG for anti-microbial assays and TYE with 1%sucrose for biofilm assays.For anti-microbial activity, define the concentration of the sample for analysis, and add it to a 96-well plate. Include a set of controls in each plate, as described in the text manuscript. Using TYEG, adjust the volume to 100 microliters, inoculate 100 microliters of microbial culture, and incubate the plate for 24 hours at 37 degrees Celsius in 5%carbon dioxide.Analyze the bacterial growth according to turbidity by visual inspection of the wells. Inoculate an aliquot of the desired dilution in specific agar plates in duplicates, and incubate the plates. After the incubation, perform colony counts.Weigh hydroxyapatite beads in microtubes and sterilize them. Then wash the beads, using adsorption buffer Ab.Add 500 microliters of saliva into the microtubes to form a salivary film, and incubate at 37 degrees Celsius with 24 rotations per minute for 40 minutes. Remove the saliva supernatant and wash the beads three times with Ab to prepare them for downstream assays.Add 500 microliters of the sample or the control into each microtube containing beads with salivary pellicle, and incubate them at 24 rotations per minute and 37 degrees Celsius for 30 minutes. Wash the beads three times with Ab.Add 500 microliters of microbial culture to each microtube, and incubate the tubes in similar conditions for one hour. Then wash the unbound cells three times with Ab buffer.Resuspend each sample with one milliliter of Ab buffer, and centicade at seven Watts for 30 seconds. Serially dilute aliquots of each suspension, tenfold, and plate them on specific agar plates. Incubate the plates for 48 hours at 37 degrees Celsius in 5%carbon dioxide, and count the colonies.For data analysis, input the raw data for the bioassays into a spreadsheet, and calculate the log of microbial growth inhibition by each treatment, and the log percentage of microbial growth inhibition, compared with the control. Correct the optical density of the readings, and calculate the percentage of biomass inhibition. The chemical profile of biological screening for the quantity of Clerodane-type diterpenes and glycosylated flavonoids in three varieties of C.sylvestris extracts, namely sylvestris, intermediate, and lingua, are shown here.After analysis of the raw data, four extracts showed a favorable response. The chromatographic data of these four extracts show the simultaneous presence of Clerodane-type diterpenes and glycosylated flavonoids. In addition, they include the same biome and variety.Calculated percent CFU of treated planktonic cells, percent biomass of the treated biofilms, and percent CFU of the treated biofilm are shown here. No crude extract significantly affected the removal of S.mutans cells adhered to the salivary pellicle. The adhesion of S.mutans cells to the glucan matrix was weakened by three of the crude extracts.Since each plant species requires optimized and specific chemical analysis methods, the best solvent fraction and analytical method must be selected from previous reports or defined by experimental design. It is critical to prepare formulations with the most active crude extract and fractions and test them on complex models to prevent the development of biofilms and cavities.

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Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...