The authors would like to acknowledge Dr. Jean-Michel An&#233; in the Department of Agronomy at UW-Madison for providing Medicago truncatula samples. This work was supported in part by funding from the National Science Foundation (NSF) grant CHE-0957784, the University of Wisconsin Graduate School and the Wisconsin Alumni Research Foundation (WARF) and Romnes Faculty Research Fellowship program (to L.L.). E.G. acknowledges an NSF Graduate Research Fellowship (DGE-1256259).

1. Instrumentation
<ol>
	<li>MALDI-TOF/TOF MSI. Use a mass spectrometer equipped with a MALDI source for analysis of small molecules (see Table of Materials/Equipment). Perform acquisitions in positive or negative ion mode depending on the analytes of interest. Specify a mass range of interest and collect 500 laser shots/spot at 50 &#181;m intervals in both the x and y dimensions across the surface of the sample to generate ion images. The raster width and number of laser shots can be adjusted to obtain higher spatial resolution and maximum signal intensity respectively. Use DHB matrix peaks or internal standards applied to the slide or directly to the tissue to calibrate the mass spectra.</li>
	<li>High Resolution LC-MS. (see Table of Materials/Equipment) Run sample extracts with LC-MS using either reversed phase (RP) LC with a C-18 column, or normal phase (NP) with a HILIC column depending on the analytes of interest. Use mobile phases and gradients as appropriate for the specific sample type. Perform acquisitions in positive or negative ion modes depending on the sample type.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Trim the root nodule from the plant, leaving 3-4 mm of root attached to the nodule.</li>
	<li>Immediately after dissection, use forceps to place the tissue in a cryostat cup and cover with gelatin (100 mg/ml&#160;in deionized water). It is essential for the tissue to be stuck to the bottom of the cup&#160;with no air bubbles.</li>
	<li>Flash freeze the tissue by placing the cup in a dry ice/ethanol bath until the gelatin hardens and becomes opaque. Store samples at -80 &#176;C until use.</li>
	<li>Remove samples from the -80 &#176;C freezer, cut away the plastic cryostat cup and trim away excess gelatin. Mount the embedded tissue to the cryostat chuck with a dime-sized amount of optimal cutting temperature (OCT) media, while not letting the OCT touch the tissue. Place in cryostat box until the OCT solidifies.</li>
	<li>Allow the chuck and gelatin to equilibrate in the cryostat box (set to -20 or -25 &#176;C) for approximately 15 min.</li>
	<li>Use the cryostat (see Table of Materials/Equipment) to section tissue approximately the thickness of one cell (8-20 &#181;m depending on the tissue type) and thaw mount each slice onto the ITO-coated glass by warming the back (non-ITO-coated side) of an ITO-coated glass slide on the back of your hand. Place the ITO-coated side of the warmed slide near the frozen tissue slice and allow the slice to stick onto the slide. Placing the sections close together on the slide will provide better alignment during MSI.</li>
</ol>
3. Matrix Application
<ol>
	<li>Airbrush Application of MALDI Matrix
	<ol>
		<li>All airbrush procedures should be performed in a fume hood.</li>
		<li>Thoroughly clean the airbrush solution container and nozzle (see Table of Materials/Equipment) with methanol and fill the solution container with DHB matrix solution (150 mg/ml in 50% methanol/0.1% TFA v/v).</li>
		<li>Hold the airbrush approximately 35 cm from the sample and apply 10-15 coats of matrix on the surface of the slide with a duration of 10 sec spray and 30 sec drying time in between each coat.</li>
		<li>Thoroughly clean the airbrush again with methanol when finished to avoid clogging from the matrix solution.</li>
	</ol>
	</li>
	<li>Automatic Sprayer Application of MALDI Matrix
	<ol>
		<li>Follow the start-up instructions provided by the manufacturers of the automatic sprayer system.</li>
		<li>For MSI of metabolites in root nodules using 40 mg/ml (in 50% methanol/0.1% TFA v/v) DHB as the matrix, set the temperature to ~80 &#176;C, velocity to 1,250 mm/min, flow rate to 50 &#181;l/min, and number of passes to 24. For best coverage, it is recommended to rotate the nozzle 90&#176; and/or offset the nozzle 1.5 mm between each pass. Start sprayer method. 
		As a side note, the particular sprayer system used here heats the nozzle for faster evaporation of the solvent. As the solvent evaporates, the concentration of the matrix quickly increases. The matrix applied to the sample with the airbrush and the automatic sprayer have comparable concentrations.</li>
		<li>Follow the shut-down instructions provided by the manufacturers of the automatic sprayer system.</li>
	</ol>
	</li>
	<li>Sublimation Application of MALDI Matrix
	<ol>
		<li>Weigh out 300 mg DHB into the bottom of the sublimation chamber (see Table of Materials/Equipment).</li>
		<li>Stick the glass slide to the cold finger (top portion of the sublimation chamber) with the tissue sections facing down with double-sided, conductive tape. Cover the entire back of the slide with the double sided tape for even conductivity, producing even matrix deposition.</li>
		<li>Clamp the top and bottom halves of the sublimation chamber together with the C-clamp. Connect the vacuum and add ice and cold water to the top reservoir.</li>
		<li>Place sublimation chamber in a heating mantle that is at room temperature.</li>
		<li>Turn on the vacuum pump. Wait 15 min and turn on the heating mantle. The heating mantle should reach 120 &#176;C over the course of 10 min.</li>
		<li>After 10 min, turn off the heat, close valve to vacuum (so the inside of the chamber remains under vacuum) and turn off the vacuum pump.</li>
		<li>Allow the chamber to come to room temperature, open the valve releasing the vacuum pressure and remove the sample. The size of the sublimation chamber will determine the amount of matrix sublimed to the glass slide. The larger sublimation chambers (the size of a 400 ml beaker) will use approximately 300 mg of DHB while the smaller chambers (the size of a 150 ml beaker) will use approximately 100 mg of DHB and will require cutting the glass slide so that it fits in the chamber.</li>
	</ol>
	</li>
</ol>
4. Image Acquisition
<ol>
	<li>Mark a + pattern on each corner of the sample with a WiteOut correction fluid pen to be used as &#34;teach points&#34;. Place the glass slide into the MALDI slide adapter plate and take an optical image of the sample using a scanner.</li>
	<li>Set up an image acquisition file using the software provided by the instrument company with a raster step size of 50 &#181;m and a laser diameter equal to or smaller than the raster step size. On this particular instrument, the minimum laser setting gives a laser diameter of approximately 10 &#181;m and small laser setting has a 40-50 &#181;m diameter.</li>
	<li>Load the optical image into the software and align the plate with the optical image.</li>
	<li>Calibrate the instrument before beginning the acquisition using common matrix cluster ions, internal standards, or a calibration mix.</li>
	<li>Specify the areas of tissue to be analyzed with MSI, including a spot of pure matrix on the slide to be used as a &#34;blank&#34;.</li>
	<li>Begin acquisition.</li>
</ol>
5. Image Generation
<ol>
	<li>Open the imaging file in the commercially available software provided by the vendor and extract the ion images. Other open-source software is available for MSI data processing19.</li>
</ol>
6. Metabolite Identification
<ol>
	<li>Select a specific m/z of interest from the mass spectrum using the vendor specific software (see Table of Materials/Equipment). An analyte can be distinguished from a matrix ion when an analyte peak is selected from the mass spectrum and ion images are generated specifically localized to the tissue section.</li>
	<li>Generate a list of analytes of interest and perform MS/MS experiments. See Table 1 and Table 2 in Representative Results for sample lists of analytes.</li>
	<li>Perform targeted LC-MS analysis on a high resolution mass spectrometer (or high resolution MALDI-MS if available) to obtain accurate mass measurements of the analytes of interest and also perform LC-MS/MS of the target analytes to obtain characteristic fragmentation patterns.</li>
	<li>Perform database searching to determine putative identifications for the targeted analytes. Examples of metabolite databases include: METLIN, ChemSpider, PubChem, KEGG, and HMDB.</li>
	<li>To confirm the putative identifications from accurate mass database searching, match the MS/MS from the targeted analytes to MS/MS spectra of standards, literature, and/or fragmentation prediction software.</li>
</ol>

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The authors declare that they have no competing financial interests.

As discussed above, sample preparation is the most critical step in the MSI workflow. Embedding the tissue unevenly will cause sectioning to be difficult or not possible in some cases. The section size and adequate equilibration time are crucial to maintaining the tissue integrity and avoiding folding and tears. Selection of matrix and application technique will play a role in determining the types of analytes to be detected, the spatial resolution, and reproducibility of the results. Using a combination of matrices or a...

The growing field of metabolomics has many important biological applications including biomarker discovery, deciphering metabolic pathways in plants and other biological systems, and toxicology profiling4,6-10. A major technical challenge when studying biological systems is to study metabolomic pathways without disrupting them11. MALDI-MSI allows for direct analysis of intact tissues that enables sensitive detection of analytes in single organs12,13 and even single cells14,15.
Sample preparation is a crucial step in producing reproducible and reliable mass spectral images. The quality of the images greatly depends upon factors such as tissue embedding medium, slice thickness, MALDI matrix, and matrix application technique. For imaging applications, ideal section thickness is the width of one cell (8-20 &#181;m depending on the sample type). MALDI requires deposition of an organic, crystalline matrix compound, typically a weak acid, on the sample to assist analyte ablation and ionization.16 Different matrices provide different signal intensities, interfering ions, and ionization efficiencies of different classes of compounds.
The matrix application technique also plays a role in the quality of mass spectral images and different techniques are appropriate for different classes of analytes. Three matrix application methods are presented in this protocol: airbrush, automatic sprayer, and sublimation. Airbrush matrix application has been widely used in MALDI imaging. The advantage of airbrush matrix application is that it is relatively fast and easy. However, the quality of the airbrush matrix application greatly depends on the skill of the user and tends to be less reproducible and cause diffusion of analytes, especially small molecules17. Automatic sprayer systems have similar mechanics to airbrush matrix application, but have been developed to remove the variability seen with manual airbrush application, making the spray more reproducible. This method can sometimes be more time-consuming than traditional airbrush matrix application. Both manual airbrush and automatic sprayer systems are solvent-based matrix application methods. Sublimation is a dry matrix application technique that is becoming more and more popular for mass spectral imaging of metabolites and small molecules because it reduces analyte diffusion; however, it lacks the solvent necessary to extract and observe higher mass compounds18.
Confident identification of metabolites typically requires accurate mass measurements to obtain putative identifications followed by tandem mass (MS/MS) experiments for validation, with MS/MS spectra being compared to standards, literature, or theoretical spectra. In this protocol high resolution (mass resolving power of 60,000 at m/z 400), liquid chromatography (LC)-MS is coupled to MALDI-MSI to obtain both spatial information and confident identifications of endogenous metabolites, using Medicago truncatula roots and root nodules as the biological system. MS/MS experiments can be performed directly on the tissue with MALDI-MSI or on tissue extracts with LC-MS and used for the validation of metabolite identifications.
This protocol provides a simple method to map endogenous metabolites in M. truncatula, which can be adapted and applied to MSI of small molecules in various tissue types and biological systems.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gelatin</td>
			<td style="width:181px;">Difco</td>
			<td style="width:163px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=214340">214340</a></td>
			<td style="width:611px;">heat to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat- HM 550</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:163px;">956564A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">indium tin oxide (ITO)-coated glass slides&#160;</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25-75-0.8 mm (width-length-thickness)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-Dihydroxybenzoic acid (DHB) matrix&#160;</td>
			<td>ICN Biomedicals</td>
			<td><a href="http://www.labsource.com/Catalog/Item.aspx?ItemID=1787195">PI90033</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Airbrush</td>
			<td>Paasche Airbrush Company</td>
			<td>TG-100D</td>
			<td>coupled with 75 ml steel container</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Automatic matrix sprayer system- TM-Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td style="width:163px;">HTX.TMSP.H021-U</td>
			<td>Specific start-up and shut-down instructions will be given when the instrument is installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sublimation apparatus&#160;</td>
			<td>Chemglass Life Science</td>
			<td style="width:163px;"><a href="http://www.chemglass.com/product_view.asp?pnr=CG-3038">CG-3038-01</a></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>Ultimate Pressure = 1x10-4 Torr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrafleXtreme MALDI-TOF/TOF</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td style="width:163px;">276601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">FlexImaging</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td>269841</td>
			<td>One example of &#34;vender specific software&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MALDI LTQ Orbitrap</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMASZ</td>
			<td>High resolution MALDI instrument for accurate mass measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Q Exactive</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>High resolution LC-MS instrument for accurate mass measurements</td>
		</tr>
	</tbody>
</table>

maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

An experimental overview of MSI is shown in Figure 1. At the very beginning of the experiment, sample preparation is a critical step. Nodules are trimmed from the plant root and embedded in gelatin. The tissue must be pressed flat against the cryostat cup, with no bubbles, while it is being frozen; this will ensure easier and proper alignment of the tissue while it is being sectioned. When the tissue is being sliced, it is important to cut the tissue at the proper thickness; too thin of sections will tea...

Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

maldi-mass-spectrometric-imaging-for-investigation-metabolites

Research

JoVE Journal

Biology

18.8K Views.  University of Wisconsin- Madison.  Масс-спектрометрическое томография (MSI) представляет собой мощный инструмент, который может использоваться, чтобы обнаружить и идентифицировать различные химические вещества в здоровых тканей, сохраняя соединений в их родных средах, которые могут обеспечи?...

Video: MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...