Authors are grateful to Thomas Mison for his technical support, and to Rachel Kopec for revising the manuscript.

1. Bacterial Cultures
<ol>
	<li>Prepare a lawn of the bacteria (Bacillus cereus strain ATCC 14579) by spreading 100 &#181;l of an overnight culture of the strain incubated at 30 &#176;C in LB (Luria-Bertani medium), over the surface of a plate of LB agar medium. Incubate the plate overnight at 30 &#176;C.</li>
</ol>
2. ECL: Equivalent Chain Length
<ol>
	<li>Calculate ECL as follows:&#160; <img alt="Equation" src="/files/ftp_upload/54960/54960eq1.jpg" /> with: 
	i, the solute of interest; 
	n, the carbon number of the straight chain saturated fatty acid methyl ester eluting before solute I; 
	n+1, the carbon number of the straight chain saturated fatty acid methyl ester eluting after solute I; 
	Rti, Rtn, Rt(n+1) the retention times of the FAME peaks described above. 
	&#8203;NOTE: Obtain the retention times of straight chain saturated fatty acids methyl esters by injection of a mixture of standards (BAME).</li>
</ol>
3. FAME Preparation and Analysis
<ol>
	<li>In order to obtain lipid fatty acids, harvest bacterial cells by scraping colonies from the agar plate and transfer 40 mg (fresh weight, equivalent to 109 viable cells) of bacteria into a 10 ml glass tube with screw caps and PTFE seals.</li>
	<li>Perform transesterification via the ester link method14,15 detailed below.
	<ol>
		<li>Add 5 ml of 0.2 M KOH in methanol to the fresh bacterial cells and incubate at 37 &#176;C for 1 hr. This reaction consists of alkaline methanolysis, breaking the ester link in the lipid and producing fatty acid methyl esters.</li>
		<li>Add 1 ml of 1 M acetic acid to lower the pH to 7.0. Check the pH with pH test strips.</li>
		<li>Add 3 ml of hexane to extract FAMEs.</li>
		<li>Transfer supernatant (organic phase) into clean tubes and concentrate by evaporation at room temperature under a continuous flow of nitrogen to obtain approximately 200 &#181;l of extract. Transfer the sample into a GC vial with insert.</li>
		<li>Inject extracts into a gas chromatography-mass spectrometry (GC-MS) system.</li>
	</ol>
	</li>
</ol>
4. GC/MS Conditions
<ol>
	<li>Inject FAME samples into a GC-MS instrument equipped with a capillary column ZB-WAX (length, 30 m; diameter, 0.25 mm; film thickness, 0.25 &#181;m).</li>
	<li>Set the injection port (in splitless mode) temperature to 250 &#176;C. Use helium as a carrier gas, with a linear velocity of 37 cm/sec. Hold the oven temperature at 50 &#176;C for 1 min, increase to 190 &#176;C at a rate of 20 &#176;C/min, and increase further to a final temperature of 230 &#176;C at a rate of 2 &#176;C/min.</li>
	<li>For the MS, record the mass spectra by electron ionization (EI) at 70 eV, and set the acquisition of the total ion current between 50 and 400 atomic mass units (amu) (2 scans/sec).</li>
	<li>When required, inject DMOX and picolinyl derivatives under the same condition except the temperature program oven as follows: 
	DMOX: 50 &#176;C (1 min), 20 &#176;C/min until 210 &#176;C and 2 &#176;C/min until 240 &#176;C (5 min); 
	Picolinyl: 6 &#176;C (1 min), 20 &#176;C/min until 220 &#176;C and 2 &#176;C/min until 250 &#176;C (20 min).</li>
</ol>
5. Picolinyl Ester Preparation from FAME16
<ol>
	<li>Evaporate the FAME extract from Section 3 with a flow of nitrogen (at least 10 mg dry material) and dissolve in 1 ml of dry dichloromethane.</li>
	<li>Prepare a 1.0 M solution of potassium tert-butoxide in tetrahydrofuran.</li>
	<li>Add the FAME extract and 0.2 ml 3-pyridinemethanol to 0.1 ml of solution made in step 5.2.</li>
	<li>Heat the solution at 40 &#176;C for 30 min in a closed vial.</li>
	<li>After cooling to room temperature, add purified deionized water (2 ml, see Materials Table) and hexane (4 ml). Mix with a vortex, allow phase to separate, and collect the organic phase.</li>
	<li>Dry it by adding anhydrous sodium sulfate until the organic phase is perfectly clear. Transfer it into a clean tube. Then evaporate to 200 &#181;l. Transfer the sample into a GC vial with insert.</li>
</ol>
6. DMOX Preparation from FAME17
<ol>
	<li>Evaporate the FAME extract from Section 3 with a flow of nitrogen (at least 10 mg dry material).</li>
	<li>To the FAME dry extract, add 250 mg of 2-amino-2-methyl-1-propanol. Flush the vessel with nitrogen, add a stopper, and place it in a heating block overnight at 190 &#176;C.</li>
	<li>After cooling to room temperature, add 3 ml dichloromethane to the tube, and 5 ml purified deionized water (See Materials Table).</li>
	<li>Shake for phase separation and then remove the aqueous phase.</li>
	<li>Wash the organic phase with 5 ml water. Shake for phase separation and then remove the aqueous phase.</li>
	<li>Dry by adding anhydrous sodium sulfate until the organic phase is perfectly clear and transfer it into a clean tube. Evaporate under a stream of nitrogen until reaching a volume of 200 &#181;l. Transfer the sample into a GC vial with insert.</li>
</ol>

<ol>
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Third edition. , (2015).</li><li>Sasser, M. <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+Bacteria+by+Gas+Chromatography+of+Cellular+Fatty+Acids.">Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids.</a> MIDI Technical note. 101, 1-6 (1990).</li><li>Spitzer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+analysis+of+fatty+acids+by+gas+chromatography+-+Low+resolution+electron+impact+mass+spectrometry+of+their+4,4-dimethyloxazoline+derivatives+-+A+review.">Structure analysis of fatty acids by gas chromatography - Low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives - A review.</a> Prog Lipid Res. 35 (4), 387-408 (1996).</li><li>Harvey, D. J., Christie, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in lipid methodology. Volume 1. , 19-80 (1992).</li><li>Diomande, S. E., Nguyen-The, C., Guinebreti&#232;re, M. -. H., Broussolle, V., Brillard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+fatty+acids+in+Bacillus+environmental+adaptation.">Role of fatty acids in Bacillus environmental adaptation.</a> Front Microbiol. 6, (2015).</li><li>Brillard, J., 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+Bacillus+cereus+Genes+Specifically+Expressed+during+Growth+at+Low+Temperatures.">Identification of Bacillus cereus Genes Specifically Expressed during Growth at Low Temperatures.</a> Appl Environ Microbiol. 76 (8), 2562-2573 (2010).</li><li>de Sarrau, B., 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=Influence+of+Anaerobiosis+and+Low+Temperature+on+Bacillus+cereus+Growth,+Metabolism,+and+Membrane+Properties.">Influence of Anaerobiosis and Low Temperature on Bacillus cereus Growth, Metabolism, and Membrane Properties.</a> Appl Environ Microbiol. 78 (6), 1715-1723 (2012).</li><li>Diomand&#233;, S. 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E., Dick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fatty+acid+methyl+ester+(FAME)+methods+for+characterizing+microbial+communities.">Comparison of fatty acid methyl ester (FAME) methods for characterizing microbial communities.</a> Soil Sci Soc Am J. 64 (5), 1659-1668 (2000).</li><li>Destaillats, F., Angers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+methodology+for+the+synthesis+of+FA+picolinyl+esters+from+intact+lipids.">One-step methodology for the synthesis of FA picolinyl esters from intact lipids.</a> J Am Oil Chem Soc. 79 (3), 253-256 (2002).</li><li>Fay, L., Richli, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+double-bonds+in+polyunsaturated+fatty-acids+by+gas-chromatography+mass-spectrometry+after+4,4-dimethyloxazoline+derivatization.">Location of double-bonds in polyunsaturated fatty-acids by gas-chromatography mass-spectrometry after 4,4-dimethyloxazoline derivatization.</a> J Chromatogr. 541 (1-2), 89-98 (1991).</li><li>Zhang, J. Y., Yu, Q. T., Liu, B. N., Huang, Z. 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K., Mikolajczak, K. L., Earle, F. R., Wolff, I. 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The authors declare that they have no competing financial interests.

The FAs chromatogram profiles shown in Table 1 correspond to B. cereus ATCC 14579 grown on an agar plate surface. Similar profiles were obtained when the bacterium was grown in aerated liquid media at the same temperature8. In the case of bacteria grown in liquid media, the bacterial biomass is collected by centrifugation of the growth medium and can be washed according to previously described protocols depending on the growth conditions8,19. The identification of the vario...

Fatty acid methyl ester (FAME) gas chromatography (GC) is an essential method for lipid characterization. It rapidly separates and quantifies the various fatty acids (FAs) of a sample after a short extraction step. Derivatives of methyl esters are highly volatile, stable and inert toward the chromatographic column, thereby avoiding tailing peaks. Their identification is rather straightforward when the sample consists of well-known FAs because the chromatographic profiles are either published or compared to standards. In addition, the repeated injection of calibration standards for quantification of various FAs is not required, given their almost constant response to flame ionization detection (FID)1.
In addition to FID, mass spectrometry (MS) detection provides a complementary set of information to confirm FAMEs. However, when FAMEs are charged using electron ionization (EI), the resulting spectra do not always allow for the identification of FA fine structure. For instance, branching position (i.e., a branched methyl group) is difficult to predict because the diagnostic ions are difficult to detect1 and the characteristic change in target ion abundance is machine-dependent, preventing the use of mass spectra libraries2. Another challenge lies in identifying the double bond position because EI causes double bond migration. Thus, FA isomers with varying double bond positions cannot be differentiated by their mass spectra. Fortunately, other tools have been developed for FA identification. For instance, the presence and the position of branching or of double bonds in FAs can be conjectured by calculating the equivalent chain length (ECL)3.
Other derivatization methods result in different mass spectra, dependent on the location of a double bond or a branched methyl group. 4,4-Dimethyl oxazoline derivatives (DMOX)4 allow for easy identification of the position of monounsaturated fatty acid double bonds. 3-pyridylcarbinyl ester (picolinyl ester) derivatives allow for the unambiguous identification of the location of methyl branched FAs5. Combining chromatographic retention (ECL) and mass spectra (DMOX and picolinyl) information allows for the identification of most FAs without the need to use complex methods of purification, as required for nuclear magnetic resonance (NMR) spectrum, the uncontestable method for structural characterization1.
Bacteria of the genus Bacillus, which include some human and animal pathogens, are able to colonize highly diverse niches and are therefore widely distributed in the environment6. Among the Bacillus genus, FA composition is influenced by the ecological niche of the species with modulations in FA patterns to adapt to a wide range of environmental changes (e.g., growth medium, temperature, pH, etc.)7-9. Because of the relative homogeneity of the FA pattern across species of the genus Bacillus during growth in standardized conditions, determination of FA composition is one of the essential criteria used to define the Bacillus species. A unique attribute of the Bacillus genus is the abundance of branched-chain FAs containing 12-17 carbons10-12 with the ratio between iso and anteiso isomers being a key determinant of adaptation to environmental conditions. Bacillus species also adapt to environmental fluctuations by altering the proportion of unsaturated fatty acids. In some species, such as Bacillus cereus, two fatty acid desaturases create double bonds in different positions of the alkyl chain13 with different roles in adaptation9. The example of the Bacillus genus illustrates the importance of precisely identifying the double bond position and FA branching. Collectively, identification of Bacillus FA patterns has several useful applications. Herein, we propose a novel GC-MS approach for Bacillus FA pattern identification that overcomes the inherent limitations of a classical GC-MS analysis.
This innovative approach can be used directly on raw biological material, and consists of a combination of existing techniques: information on retention times (ECL) and mass spectra of different FAs derivatives (FAME, DMOX and picolinyl-ester).
We use the following FA nomenclature. i, a, and n indicate iso, anteiso methyl branched, and straight-chain fatty acid, respectively. Unsaturated FAs were named by C:d where C is the number of carbon atoms in the fatty acid and d is the number of double bonds. &#916;x indicates the position of the double bond, where the double bond is located on the xth carbon-carbon bond, counting from the carboxylic acid end.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GC/MS</td>
			<td>Shimadzu</td>
			<td>QP2010</td>
			<td></td>
		</tr>
		<tr>
			<td>capillary column ZB WAX</td>
			<td>Phenomenex</td>
			<td>7HG-G007-11</td>
			<td>30m x 0.25mm x 0.25&#181;m</td>
		</tr>
		<tr>
			<td>Methanol Lichrosolv</td>
			<td>VWR</td>
			<td>1.06018.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Aldrich</td>
			<td>P1767</td>
			<td></td>
		</tr>
		<tr>
			<td>THF</td>
			<td>Hipersolv Chromanorm</td>
			<td>28559.320</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Hipersolv Chromanorm</td>
			<td>23373.320</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Hipersolv Chromanorm</td>
			<td>24575.320</td>
			<td></td>
		</tr>
		<tr>
			<td>3-pyridinemethanol</td>
			<td>Aldrich</td>
			<td>P6-680-7</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium tertiobutoxide</td>
			<td>Aldrich</td>
			<td>156671</td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-2-methyl-1-propanol</td>
			<td></td>
			<td>A-9879</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Academic</td>
			<td>Millipore</td>
			<td>ZMQS50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Acid Methyl Ester (BAME) Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47080-U Supelco</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of fatty acids in bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

The strategy of FA identification from bacterial cells is presented in Figure 1. Each step provides complementary spectral information or information about chromatographic retention. Step 1 consists of preliminary FA identification using a standard solution. Step 2 allows for the interpretation of FAME EI spectra and their ECL, in order to tentatively identify the products. Step 3 identifies the exact branching location in branched chain-FAs. Finally, step 4 identifies th...

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Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

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9.5K Views.  INRA, Université d'Avignon. We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl). 

Video: Identification of Fatty Acids in Bacillus cereus

A Polyaniline-based Sensor of Nucleic Acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...