Gli autori sono grati a Thomas Mison per il suo supporto tecnico, e di Rachel Kopec per la revisione del manoscritto.

1. colture batteriche <ol><li> Preparare un prato di batteri (Bacillus cereus ceppo ATCC 14579) diffondendo 100 ml di una coltura durante la notte del ceppo incubato a 30 ° C in LB (Luria-Bertani medio), sulla superficie di una piastra di terreno LB agar. Incubare la piastra per una notte a 30 ° C. </li></ol> 2. ECL: lunghezza della catena Equivalente <ol><li> Calcolare ECL come segue: <img alt="Equazione" src="/files/ftp_upload/54960/54960eq1.jpg" /> con: i, il soluto di interesse; n, il numero di carbonio del rettilineo catena di acido grasso saturo estere metilico eluendo prima soluto I; n + 1, il numero di carbonio del rettilineo estere metilico catena satura di acido grasso eluendo dopo soluto I; Rti, Rtn, Rt (n + 1) i tempi di ritenzione dei picchi FAME sopra descritti. NOTA: ottenere i tempi di ritenzione di esteri acidi grassi saturi a catena metilici rettilinei mediante iniezione di una miscela di standard (BAME). </li></ol> 3. FAME preparazione e l&#39;analisi <ol><li> Per ottenere acidi grassi lipidi, cellule batteriche raccolta raschiando colonie dalla piastra di agar e trasferire 40 mg (peso fresco, equivalente a 10 9 cellule vitali) di batteri in una provetta di vetro da 10 ml con tappo a vite e guarnizioni PTFE. </li><li> Eseguire transesterificazione tramite il metodo di collegamento estere 14,15 descritto di seguito. <ol><li> Aggiungere 5 ml di 0,2 M KOH in metanolo alle cellule batteriche fresche e incubare a 37 ° C per 1 ora. Questa reazione è costituito metanolisi alcalina, interrompendo il collegamento di estere nel lipidico e produrre esteri metilici degli acidi grassi. </li><li> Aggiungere 1 ml di 1 M di acido acetico per abbassare il pH a 7,0. Controllare il pH con le strisce reattive pH. </li><li> Aggiungere 3 ml di esano per estrarre FAMEs. </li><li> Trasferire il surnatante (fase organica) in provette pulite e concentrato mediante evaporazione a temperatura ambiente sotto un flusso continuo di azoto per ottenere circa 200 microlitridi estratto. Trasferire il campione in una fiala GC con inserto. </li><li> Iniettare estratti in una spettrometria di massa cromatografia gas sistema (GC-MS). </li></ol></li></ol> 4. GC / MS Condizioni <ol><li> Iniettare campioni FAME in uno strumento GC-MS equipaggiato con una colonna capillare ZB-WAX (lunghezza 30 m, diametro, 0,25 mm, spessore del film, 0,25 micron). </li><li> Impostare la porta di iniezione (in modalità splitless) temperatura a 250 ° C. Utilizzare l&#39;elio come gas di trasporto, con una velocità lineare di 37 cm / sec. Mantenere la temperatura del forno a 50 ° C per 1 min, aumentare a 190 ° C ad una velocità di 20 ° C / min, e aumentare ulteriormente ad una temperatura finale di 230 ° C ad una velocità di 2 ° C / min. </li><li> Ai MS, registrare gli spettri di massa per ionizzazione elettronica (EI) a 70 eV, e impostare l&#39;acquisizione della corrente ionica totale tra 50 e 400 unità atomiche di massa (amu) (2 scans / sec). </li><li> Quando necessario, iniettare DMOX e picolinyl derivati ​​sotto la stessa condizione eccet forno programma di temperatura come segue: DMOX: 50 ° C (1 minuto), 20 ° C / min fino 210 ° C e 2 ° C / min fino a 240 ° C (5 min); Picolinyl: 6 ° C (1 minuto), 20 ° C / min fino a 220 ° C e 2 ° C / min fino a 250 ° C (20 min). </li></ol> 5. Picolinyl Ester Preparazione a partire da 16 FAME <ol><li> Evaporare l&#39;estratto FAME dalla Sezione 3 con un flusso di azoto (almeno 10 mg materiale secco) e sciogliere in 1 ml di diclorometano anidro. </li><li> Preparare una soluzione di ter di potassio terz.butossido in tetraidrofurano 1.0 M. </li><li> Aggiungere l&#39;estratto FAME e 0,2 ml di 3-pyridinemethanol a 0,1 ml di soluzione fatte nella fase 5.2. </li><li> Riscaldare la soluzione a 40 ° C per 30 min in una fiala chiusa. </li><li> Dopo raffreddamento a temperatura ambiente, aggiungere acqua deionizzata purificata (2 ml, vedi Materiali Tavolo) ed esano (4 ml). Mescolare con un vortice, permettono di fase per separare e raccogliere la fase organica. </li><li> Dry aggiungendo solfato di sodio anidro fino alla fase organica è perfettamente chiaro. Trasferire in una provetta pulita. Poi evaporare a 200 l. Trasferire il campione in una fiala GC con inserto. </li></ol> 6. DMOX Preparazione a partire da 17 FAME <ol><li> Evaporare l&#39;estratto FAME dalla Sezione 3 con un flusso di azoto (materiale secco, di almeno 10 mg). </li><li> Per l&#39;estratto secco FAME, aggiungere 250 mg di 2-ammino-2-metil-1-propanolo. Lavare il recipiente con azoto, aggiungere un tappo, e metterlo in un blocco di riscaldamento durante la notte a 190 ° C. </li><li> Dopo raffreddamento a temperatura ambiente, aggiungere 3 ml diclorometano nel tubo, e 5 ml di acqua deionizzata purificata (vedi Materiali Tabella). </li><li> Agitare per separazione di fase e quindi rimuovere la fase acquosa. </li><li> Lavare la fase organica con acqua 5 ml. Agitare per separazione di fase e quindi rimuovere la fase acquosa. </li><li> Lavaggio aggiungendo solfato di sodio anidro fino alla fase organica è perfettamente limpida etrasferirlo in una provetta pulita. Evaporare sotto corrente di azoto fino a raggiungere un volume di 200 microlitri. Trasferire il campione in una fiala GC con inserto. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

I profili cromatogramma AF indicati nella tabella 1 corrispondono a B. cereus ATCC 14579 cresciuta su una superficie della piastra di agar. Profili simili sono stati ottenuti quando il batterio è stato coltivato in terreni liquidi aerato alla stessa temperatura 8. Nel caso dei batteri coltivati in terreno liquido, la biomassa batterica viene raccolto per centrifugazione del mezzo di crescita e può essere lavato secondo protocolli precedentemente descritti a seconda delle condizioni...

Acidi grassi gascromatografia estere metilico (FAME) (GC) è un metodo essenziale per la caratterizzazione dei lipidi. Si separa rapidamente e quantifica i vari acidi grassi (FAS) di un campione dopo una breve fase di estrazione. Derivati ​​di esteri metilici sono altamente volatili, stabile ed inerte verso la colonna cromatografica, evitando così i picchi di decantazione. La loro identificazione è piuttosto semplice quando il campione è costituito da FAs ben noti in quanto i profili cromatografici o sono pubblicati o rispetto agli standard. Inoltre, l&#39;iniezione ripetuta di standard di calibrazione per la quantificazione di vari FAs non è necessario, data la loro pressoché costante risposta al rilevamento a ionizzazione di fiamma (FID) 1. Oltre a FID, spettrometria di massa (MS) di rilevamento fornisce una serie complementare di informazioni per confermare FAMEs. Tuttavia, quando FAMEs pagano usando ionizzazione elettronica (EI), gli spettri risultanti non sempre consentono °e l&#39;identificazione di FA struttura fine. Per esempio, la posizione ramificazione (cioè, un gruppo metile ramificato) è difficile da prevedere perché gli ioni diagnostici sono difficili da rilevare 1 e il cambiamento caratteristico in abbondanza ione bersaglio dipende dalla macchina, evitando l&#39;uso di librerie di spettri di massa 2. Un&#39;altra sfida consiste nell&#39;individuare la posizione di doppio legame, perché EI provoca la migrazione doppio legame. Così, isomeri FA con diverse posizioni doppio legame non possono essere differenziati per la loro spettri di massa. Fortunatamente, altri strumenti sono stati sviluppati per l&#39;identificazione FA. Per esempio, la presenza e la posizione di ramificazione o di doppi legami in FAs ipotizzabile calcolando la lunghezza della catena equivalente (ECL) 3. Altri metodi di derivatizzazione comportano diversi spettri di massa, dipende dalla posizione di un doppio legame o un gruppo metile ramificato. 4,4-dimetil derivati ossazolinici (DMOX) 4 permettono di easIdentificazione y della posizione di doppi legami di acidi grassi monoinsaturi. 3-pyridylcarbinyl estere (picolinyl estere) i derivati permettono l&#39;identificazione univoca della posizione di metile ramificata FAs 5. Combinando ritenzione cromatografica (ECL) e spettrometria di massa (DMOX e picolinyl) informazioni consente l&#39;identificazione della maggior parte delle federazioni senza la necessità di utilizzare complessi metodi di purificazione, come richiesto per la risonanza magnetica nucleare (NMR), il metodo incontestabile per la caratterizzazione strutturale 1 . I batteri del genere Bacillus, che include alcuni agenti patogeni umani ed animali, sono in grado di colonizzare estremamente diverse nicchie e sono quindi ampiamente distribuiti nell&#39;ambiente 6. Tra genere Bacillus, composizione FA è influenzata dalla nicchia ecologica della specie con modulazioni nei modelli FA di adattarsi ad un&#39;ampia gamma di cambiamenti ambientali (ad esempio, il terreno di coltura, la temperatura,pH, ecc) 7-9. A causa della relativa omogeneità del modello FA attraverso specie del genere Bacillus durante la crescita in condizioni standardizzate, determinazione della composizione FA è uno dei criteri essenziali usati per definire le specie Bacillus. Un attributo unico del genere Bacillus è l&#39;abbondanza del FAS a catena ramificata contenenti 12-17 atomi di carbonio 10-12 con il rapporto tra ISO e anteiso isomeri di essere un fattore determinante di adattamento alle condizioni ambientali. Specie Bacillus adattano anche alle fluttuazioni ambientali alterando la percentuale di acidi grassi insaturi. In alcune specie, come il Bacillus cereus, due desaturasi di acidi grassi creano doppi legami in diverse posizioni della catena alchilica 13 con diversi ruoli in adattamento 9. L&#39;esempio del genere Bacillus illustra l&#39;importanza di individuare con precisione la posizione doppio legame e FA ramificazione. Collezionarevamente, l&#39;identificazione di modelli di Bacillus FA ha diverse applicazioni utili. Qui, proponiamo un nuovo approccio GC-MS per l&#39;identificazione modello Bacillus FA che supera i limiti intrinseci di una classica analisi GC-MS. Questo approccio innovativo può essere utilizzato direttamente sul materiale biologico grezzo, e consiste in una combinazione di tecniche esistenti: informazioni sui tempi di ritenzione (ECL) e spettri di massa dei diversi derivati ​​AF (FAME, DMOX e picolinyl-estere). Usiamo la seguente nomenclatura FA. i, a, e n indicano iso, anteiso metil ramificata, e acidi grassi a catena lineare, rispettivamente. FAs insaturi sono stati nominati da C: d dove C è il numero di atomi di carbonio dell&#39;acido grasso e d è il numero di doppi legami. Δ x indica la posizione del doppio legame, in cui il doppio legame è situato il legame carbonio-carbonio xth, contando dal terminale acido carbossilico.

<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

Le specie Bacillus contengono ramificata catena e acidi grassi insaturi (FAS) con diverse posizioni del ramo di metile (ISO o anteiso) e del doppio legame. Cambiamenti nella composizione FA giocano un ruolo cruciale nell&#39;adattamento dei batteri al loro ambiente. Queste modifiche comportano una variazione del rapporto di iso rispetto anteiso ramificato AF, e nella proporzione di insaturo FAs rispetto al satura AF, con doppi legami creati in posizioni specifiche. Identificazione precisa del profilo di FA è necessario comprendere i meccanismi di adattamento di specie Bacillus. Molte delle federazioni da Bacillus non sono disponibili in commercio. La strategia proposta nel presente documento identifica FAs combinando le informazioni sul tempo di ritenzione (calcolo della lunghezza della catena equivalente (ECL)), con gli spettri di massa di tre tipi di derivati ​​FA: gli esteri metilici degli acidi grassi (FAME), 4,4-dimetil ossazolinici derivati ​​(DMOX), e3-pyridylcarbinyl estere (picolinyl). Questo metodo può identificare FAS senza la necessità di purificare l&#39;ignoto AF. Confrontando i profili cromatografici di FAME preparato da Bacillus cereus con una miscela commerciale di standard consente l&#39;identificazione di catena lineare saturo AF, il calcolo della ECL, e ipotesi sull&#39;identità dell&#39;altro AF. FAMEs di ramificata saturi Federcalcio, iso o anteiso, visualizzare un cambiamento negativo costante nella ECL, rispetto alla FAS lineari saturi con lo stesso numero di atomi di carbonio. FAMEs di insaturo AF possono essere rilevati dalla massa dei loro ioni molecolari, e determinano uno spostamento positivo nella ECL rispetto ai corrispondenti federazioni saturi. La posizione ramificazione del FAS e la posizione del doppio legame insaturi FAs possono essere identificati dagli spettri di massa a ionizzazione di elettroni picolinyl e DMOX derivati, rispettivamente. Questo approccio identifica tutto il ramo saturo sconosciutaEd FAs, insaturi a catena lineare FAs e AF ramificati insaturi dall&#39;estratto cereus B..

La strategia di identificazione FA da cellule batteriche è presentato in Figura 1. Ogni passo fornisce informazioni spettrali complementari o informazioni su di ritenzione cromatografica. Fase 1 consiste preliminare identificazione FA utilizzando una soluzione standard. Step 2 permette l&#39;interpretazione di FAME EI spettri e la loro ECL, al fine di tentare di identificare i prodotti. Fase 3 identifica la posizione esatta ramificazione nella catena ramificata-FAS. Inf...

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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).

Identificazione di acidi grassi a<em&gt; Bacillus cereus</em

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

identification-of-fatty-acids-in-bacillus-cereus

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

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.

Un sensore polianilina a base di acidi nucleici

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

Ricerca

Biochimica

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.

Espressione proteica ricombinante, cristallizzazione e studi biofisici di a<em&gt; Bacillus</em&gt; -conservato nucleotide pirofosforilasi, BcMazG

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.

Fluorescenza a tensione di serraggio in<em&gt; Xenopus</em&gt; Oociti che utilizzano aminoacidi innaturali fluorescenti

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.

Preparazione dei campioni per l'identificazione basati sulla spettrometria di massa di RNA-binding Regions

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.

Acido grasso 13C Isotopologue profilatura fornisce approfondimenti trasferimento trofico carbonio e metabolismo dei lipidi dei consumatori invertebrati marini

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.

Identificazione della chinasi Cyclin-dipendente 1 siti di fosforilazione specifico da una chinasi In Vitro test

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.

Identificazione delle regioni di proteina funzionale attraverso la proteina chimerica costruzione

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.

Identificazione dei recettori Orexin ed Endocannabinoidi nei pesci zebra per adulti utilizzando i metodi di immunoperossiasi e immunofluorescenza

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.

Screening cinetico dell'attività nucleana utilizzando sonde con acido Nucleic

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).

Entità DI legame SUMO (SUBE) come strumenti per l'arricchimento, l'isolamento, l'identificazione e la caratterizzazione del proteoma SUMO nel cancro al fegato

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

Identificazione di acidi grassi a

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