Ringraziamo i membri del laboratorio O&#39;Halloran per i commenti sul manoscritto. Ringraziamo il Dipartimento dell&#39;Università George Washington di Scienze Biologiche e colombiana Collegio delle Arti e delle Scienze per il finanziamento di D. O&#39;Halloran.

1. Base Local Alignment Search Tool (BLAST): Interfaccia online <ol><li> Clicca su questo link per visitare il sito web del server BLAST 1 presso il National Center for Biotechnology Information (NCBI). - <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (Figura 1). </li><li> Immettere una sequenza di testo formattato FASTA (vedi figura 2 per esempio) nella casella di ricerca. </li><li> Fare clic sul programma BLAST appropriato e banca dati pertinente o le singole specie di interesse da utilizzare nella ricerca e poi cliccare su &quot;BLAST&quot;. Nota: sequenza FASTA formattato inizia con una riga di descrizione indicata da un segno &quot;&gt;&quot;. La descrizione deve seguire immediatamente dopo il segno &quot;&gt;&quot;, la sequenza (ad es. Nucleotidi o amminoacidi) seguire la descrizione sulla riga successiva. L&#39;uscita dalla ricerca BLAST è visto come HTML, testo, XML, o colpire taBles (testo o csv) con il default è impostato su HTML (Figura 3). </li></ol> 2. Base Local Alignment Search Tool (BLAST): eseguibili locali <ol><li> Scaricare le ultime riga di comando eseguibili BLAST BLAST da questo link: <a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/">ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/</a> - </li><li> em&gt; Per gli utenti di PC: fare doppio clic sul file più recente esplosione win32.exe e accettare il contratto di licenza e fare clic su Installa. Nota: La directory di installazione predefinita è C: NCBI-blast-2.2.27 +. </li><li> Configurare la variabile di ambiente PC come segue: <ol><li> Clicca sul PC pulsante &quot;start&quot;, e quindi fare clic destro &quot;computer&quot;, </li><li> Fare clic su &quot;Proprietà&quot; e nella finestra pop-up fai clic sulla scheda &quot;avanzate&quot; </li><li> Fare clic sul pulsante &quot;Variabili d&#39;ambiente&quot; e nel nuovo popup fare clic sul pulsante &quot;nuovo&quot; sotto °e &quot;Variabili utente per utente&quot; sezione </li><li> Nel pop-up aggiungere il nome della variabile &quot;Path&quot; e il valore della variabile &quot;C: NCBI-blast-2.2.27 + bin. Nota: la directory bin contiene il file eseguibile (cioè BLASTP, ecc.).. </li></ol></li><li> em&gt; Per gli utenti Mac: Apri l&#39;applicazione Terminale (per farlo basta aprire &quot;Finder&quot; e cercare &quot;Terminal&quot; e questa verrà visualizzata l&#39;icona di &quot;terminale&quot;). Nel tipo di finestra di terminale: &gt; Ftp <a href="ftp://ftp.ncbi.nih.gov">ftp.ncbi.nih.gov</a> Nota: può anche digitare l&#39;URL utilizzato sopra nell&#39;esempio per PC </li><li> Per accedere al NCBI tipo di sito ftp &quot;anonimo&quot; per nome e password, e poi digitare: &gt; Cd abbattitori / eseguibili / ULTIME </li><li> Elencare gli eseguibili digitando: &gt; LS </li><li> Scarica l&#39;ultima versione digitando il seguente (o qualunque sia l&#39;ultima versione è attualmente): 2; ottenere NCBI-blast-2.2.7-macosx.tar.gz </li><li> Uscire dal sito ftp server NCBI digitando &quot;exit&quot;. </li><li> Decomprimere i file scaricati digitando: &gt; Tar-xzf NCBI-blast-2.2.7-macosx.tar.gz </li><li> Aggiungere il percorso dei binari per l&#39;eseguibile esplosione al vostro percorso in modo che la shell può cercare attraverso questa directory quando alla ricerca di comandi digitando: &gt; PATH = $ PATH: new_folder_location </li><li> Verificare se questo aggiunto il percorso al percorso digitando: &gt; Echo $ PATH </li><li> Scarica un preformattati database BLAST (aggiornati quotidianamente) cliccando qui: <a href="ftp://ftp.ncbi.nlm.nih.gov/blast/db/">ftp://ftp.ncbi.nlm.nih.gov/blast/db/</a> </li><li> Posizionare il database nella cartella &quot;db&quot;. </li><li> em&gt; Su un PC: aprire un prompt di MS-DOS (per fare ciò cliccate &quot;start&quot; e digitare &quot;cmd&quot; nella barra di ricerca) e cambiare la directory della cartella NCBI-blast digitando: C: Users&gt; cd .. [mossesu una cartella] C: &gt; cd NCBI-blast-2.2.27 + Questo cambierà la directory: C: NCBI-blast-2.2.27 +&gt; </li><li> Creare il database utilizzando il comando &quot;makedb&quot; seguente: &gt; Makedb-in db / briggsae.fasta-dbtype prot-out db / briggsae Nota: Nell&#39;esempio riportato di seguito (Figura 4) il database è denominato &quot;briggsae&quot; ed è composto da un gruppo di linkage dall&#39;organismo Caenorhabditis briggsae. </li><li> Creare una sequenza proteica query denominata &quot;test&quot; inserendo una sequenza di testo formattato proteina FASTA nella cartella &quot;db&quot;. </li><li> Interrogare il database tramite una ricerca BLASTP digitando il seguente comando: &gt; BLASTP-query db / test.txt-db db / briggsae-out text.txt </li><li> em&gt; Su Mac: scaricare un database per le ricerche Blast locali mediante l&#39;accesso al sito ftp NCBI secondo le istruzioni di cui sopra (punto 2.4) e l&#39;Tipo n: &gt; Lcd .. / databases / </li><li> Scarica il genoma o la sequenza di interesse digitando: &gt; Get NC_ [adesione #]. Fna Nota: &quot;. Fna&quot; si riferisce alla sequenza nucleotidica FASTA formattato e &quot;FAA.&quot; Si riferisce alle sequenze amminoacidiche FASTA formattata. </li><li> Digitare &quot;quit&quot; per uscire dal sito ftp. </li><li> Rendere il database digitando: &gt; Makeblastdb-in db / mouse.faa-out mouse dbtype prot </li><li> Inserire una sequenza di interrogazione VELOCE formattata nella cartella &quot;bin&quot; e interrogare il database con il seguente comando: &gt; BLASTP-query &quot;il vostro query.fasta&quot;-db &quot;database&quot;-out results.txt </li></ol> 3. Generazione di allineamenti multipli di sequenza <ol><li> Clicca su questi link per accedere a più di allineamento (MSA) programmi Sequenza comunemente utilizzati: ClustalW 6 <a href="http://www.clustal.org/">http://www.clustal.org/</a> Kalign 7 <a href="http://msa.sbc.su.se/cgi-bin/msa.cgi">http://msa.sbc.su.se/cgi-bin/msa.cgi</a> MAFFT 8,9 <a href="http://mafft.cbrc.jp/alignment/software/">http://mafft.cbrc.jp/alignment/software/</a> MUSCLE 10 <a href="http://www.drive5.com/muscle/">http://www.drive5.com/muscle/</a> T-Caffè 11 <a href="http://www.tcoffee.org/Projects/tcoffee/">http://www.tcoffee.org/Projects/tcoffee/</a> PROBCONS 12 <a href="http://toolkit.tuebingen.mpg.de/probcons">http://toolkit.tuebingen.mpg.de/probcons</a> </li><li> Clicca su questo link - <a href="http://tcoffee.crg.cat/apps/tcoffee/do:regular">http://tcoffee.crg.cat/apps/tcoffee/do:regular</a> - e l&#39;ingresso FASTA dati di sequenza formattati nella casella di ricerca Nota: Un esempio di output T-caffè può essere visto in Figura 5, residui simili sono codificati a colori. </li><li> Scarica il Clustal MSA come una versione a riga di comando (ClustalW) o v graficaersione (ClustalX) cliccando questo link: <a href="http://www.clustal.org/clustal2/">http://www.clustal.org/clustal2/</a> - poi clicca sul file eseguibile appropriato (cioè win, Linux, Mac OS X). </li><li> Carica dati FASTA testo formattato sequenza e allineare (Figura 6). </li></ol> 4. Determinazione modelli best-fit di Evolution <ol><li> Clicca qui per scaricare il programma ProtTest 13: <a href="http://darwin.uvigo.es/our-software/">http://darwin.uvigo.es/our-software/</a> </li><li> Una volta ProtTest è stato scaricato, fare doppio clic sul file ProtTest.jar </li><li> Una volta ProtTest viene lanciato, fate clic su &quot;select file&quot; e caricare i dati di sequenza (Figura 7). </li><li> Poi cliccare su &quot;start&quot; e il programma inizierà (Figura 8). Nota: Al termine della corsa (figura 8), il programma indicherà il miglior modello basato su criteri es<html> &quot;Miglior modello in base ai AIC: WAG + I + G&quot; </li></ol> 5. La deduzione sequenza in base filogenesi da massima verosimiglianza o inferenza bayesiana <ol><li> Scaricato PhyML 4 qui: <a href="https://code.google.com/p/phyml/">https://code.google.com/p/phyml/</a> </li><li> Lanciare l&#39;eseguibile con un doppio clic l&#39;applicazione appropriata (cioè phyml di Windows, phyml Linux, ecc.) E la finestra di interfaccia pop-up (Figura 9). </li><li> Caricare la sequenza di input come una sequenza PHYLIP formattato digitando: &gt; &quot;Nome file&quot;. PHY Nota: Per la conversione tra formati sequencer, utilizzare il programma &quot;Readseq&quot; web disponibile all&#39;indirizzo - <a href="http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi">http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi</a> . </li><li> Lanciare il programma digitando &quot;Y&quot;. </li><li> Scarica MrBayes 5 qui: rceforge.net / download.php &quot;&gt; http://mrbayes.sourceforge.net/download.php </li><li> Per avviare il programma, fare clic sul file eseguibile e leggere NEXUS formattato dati di sequenza nel programma digitando: &gt; Execute &quot;nome file&quot;. Nex </li><li> Impostare il modello evolutivo. </li><li> Selezionare il numero di generazioni per eseguire digitando: &gt; Mcmcp ngen = 1000000 [imposta il numero di generazioni 1000000] &gt; Coppa Burnin = 10000 [imposta il Burnin 10000] </li><li> Salvare le lunghezze filiali nel file risultati digitando: &gt; Mcmcp savebrlens = yes </li><li> Eseguire l&#39;analisi digitando: &gt; MCMC </li><li> Riassumere gli alberi utilizzando il comando &quot;SUMT&quot;. </li></ol> 6. Visualizzazione filogenesi <ol><li> Visualizzare un elenco di programmi di visualizzazione albero qui: <a href="http://www.treedyn.org/overview/editors.html">http://www.treedyn.org/overview/editors.html</a> </li><li> Scarica il TreeView 14 progrsono qui: <a href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html">http://taxonomy.zoology.gla.ac.uk/rod/treeview.html</a> </li></ol>

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	<li>Altschul, S. F., Carroll, R. J., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weights+for+data+related+by+a+tree.">Weights for data related by a tree.</a> J. Mol. Biol. 207 (4), 647-653 (1989).</li><li>Akaike, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+statistical+model+identification.">A new look at the statistical model identification.</a> IEEE Trans. Automat. Contr. 19 (6), 706-723 (1974).</li><li>Schwarz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+dimension+of+a+model.">Estimating the dimension of a model.</a> Ann. Stat. 6 (2), 461-464 (1978).</li><li>Guindon, S., Gascuel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+fast,+and+accurate+algorithm+to+estimate+large+phylogenies+by+maximum+likelihood.">A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.</a> Syst. Biol. 52 (5), 696-704 (2003).</li><li>Huelsenbeck, J. P., Ronquist, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRBAYES:+Bayesian+inference+of+phylogenetic+trees.">MRBAYES: Bayesian inference of phylogenetic trees.</a> Bioinformatics. 17 (8), 754-755 (2001).</li><li>Thompson, J. D., Higgins, D. G., Gibson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLUSTAL+W:+Improving+the+sensitivity+of+progressive+multiple+sequence+alignment+through+sequence+weighting,+position-specific+gap+penalties+and+weight+matrix+choice.">CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.</a> Nucleic Acids Res. 22 (22), 4673-4680 (1994).</li><li>Lassmann, T., Sonnhammer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kalign--an+accurate+and+fast+multiple+sequence+alignment+algorithm.">Kalign--an accurate and fast multiple sequence alignment algorithm.</a> BMC Bioinformatics. 6, 298 (2005).</li><li>Katoh, K., Kuma, K., Toh, H., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT+version+5:+Improvement+in+accuracy+of+multiple+sequence+alignment.">MAFFT version 5: Improvement in accuracy of multiple sequence alignment.</a> Nucleic Acids Res. 33 (2), 511-518 (2005).</li><li>Katoh, K., Misawa, K., Kuma, K., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT:+A+novel+method+for+rapid+multiple+sequence+alignment+based+on+fast+fourier+transform.">MAFFT: A novel method for rapid multiple sequence alignment based on fast fourier transform.</a> Nucleic Acids Res. 30 (14), 3059-3066 (2002).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+Multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: Multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Notredame, C., Higgins, D. G., Heringa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-coffee:+A+novel+method+for+fast+and+accurate+multiple+sequence+alignment.">T-coffee: A novel method for fast and accurate multiple sequence alignment.</a> J. Mol. Biol. 302 (1), 205-217 (2000).</li><li>Do, C. B., Mahabhashyam, M. S., Brudno, M., Batzoglou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProbCons:+Probabilistic+consistency-based+multiple+sequence+alignment.">ProbCons: Probabilistic consistency-based multiple sequence alignment.</a> Genome Res. 15 (2), 330-340 (2005).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProtTest+3:+Fast+selection+of+best-fit+models+of+protein+evolution.">ProtTest 3: Fast selection of best-fit models of protein evolution.</a> Bioinformatics. 27 (8), 1164-1165 (2011).</li><li>Page, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeView:+An+application+to+display+phylogenetic+trees+on+personal+computers.">TreeView: An application to display phylogenetic trees on personal computers.</a> Comput. Appl. Biosci. 12 (4), 357-358 (1996).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=jModelTest+2:+More+models,+new+heuristics+and+parallel+computing.">jModelTest 2: More models, new heuristics and parallel computing.</a> Nat. Methods. 9 (8), 772 (2012).</li><li>Chevenet, F., Brun, C., Banuls, A. L., Jacq, B., Christen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeDyn:+Towards+dynamic+graphics+and+annotations+for+analyses+of+trees.">TreeDyn: Towards dynamic graphics and annotations for analyses of trees.</a> BMC Bioinformatics. 7, 439 (2006).</li></ol>

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La nostra speranza per questo articolo è che servirà come punto di partenza per guidare ricercatori o studenti che sono nuovi per filogenesi. Progetti di sequenziamento del genoma sono diventati meno costosi nel corso degli ultimi anni e di conseguenza la domanda degli utenti per questa tecnologia è in aumento, e ora la produzione di grandi quantità di dati di sequenza è comune in piccoli laboratori. Questi dataset spesso forniscono ai ricercatori insiemi di geni che richiedono un quadro filogenetico per iniziare a...

Al fine di comprendere come due (o più) delle specie si è evoluta, è prima necessario ottenere sequenza o dati morfologici di ogni campione; questi dati rappresentano quantità che possiamo utilizzare per misurare il loro rapporto attraverso lo spazio evolutivo. Proprio come quando si misura la distanza lineare, avendo più dati disponibili (ad esempio miglia, pollici, micron) saranno equivalere a una misurazione più accurata. Ergo, la precisione con cui un ricercatore può dedurre la distanza evolutiva è fortemente influenzato dal volume di dati informativi disponibili per misurare i rapporti. Inoltre, poiché diversi campioni si evolvono a ritmi diversi e da diversi meccanismi, il metodo che usiamo per misurare la relazione tra due taxa influenza direttamente anche la precisione delle misurazioni evolutivi. Pertanto, poiché le relazioni evolutive non osservati direttamente ma invece sono estrapolate dalla sequenza o dati morfologici, il problema di inferire evolutivorelazioni diventa una delle statistiche. Phylogenetics è la branca della biologia in questione con l&#39;applicazione di modelli statistici a modelli di evoluzione al fine di ricostruire in modo ottimale la storia evolutiva tra i taxa. Questa ricostruzione tra taxa viene indicato come filogenesi della specie. Per contribuire a colmare il divario di conoscenze tra biologi molecolari e biologi evolutivi che descriviamo qui un passo per passo cantiere per inferire filogenesi da un insieme di sequenze. In primo luogo, abbiamo dettaglio i passaggi necessari per l&#39;interrogazione del database utilizzando la base Local Alignment Search Tool (BLAST 1) algoritmo attraverso l&#39;interfaccia web based e anche utilizzando eseguibili locali, questo è spesso il primo passo per ottenere un elenco di sequenze simili a un non meglio identificato interrogazione, anche se alcuni ricercatori potrebbero anche essere interessati a raccogliere dati per un singolo gruppo tramite interfacce web come Phylota (http://www.phylota.net/). BLAST è un algoritmo per comparing aminoacidica primaria o dati di sequenza nucleotidica con un database di sequenze per la ricerca di &quot;hits&quot; che ricordano la sequenza query. Il programma BLAST è stato progettato da Stephen Altschul et al. presso il National Institutes of Health (NIH) 1. Il server BLAST è costituito da un certo numero di programmi diversi, e qui è un elenco di alcuni dei programmi BLAST più comuni: i) Nucleotide-nucleotide BLAST (BLASTN): Questo programma richiede un ingresso sequenza di DNA e restituisce le sequenze di DNA più simili dal database del DNA che l&#39;utente specifica (ad esempio, per un organismo specifico). ii) proteina-proteina BLAST (BLASTP): Qui l&#39;utente immette una sequenza proteica e il programma ritorna sequenze proteiche più simili dal database proteina che l&#39;utente specifica. iii) BLAST iterativo Position-Specific (PSI-BLAST) (blastpgp): L&#39;input utente è un prin sequenza che restituisce un insieme di proteine ​​strettamente correlate, e da questo insieme di dati viene generato un profilo conservati. Successiva una nuova interrogazione viene generata utilizzando solo questi &quot;motivi&quot; conservati che viene utilizzato per interrogare un database di proteine ​​e questo restituisce un ampio gruppo di proteine ​​da cui una nuova serie di &quot;motivi&quot; conservati sono estratti e poi utilizzato per interrogare un database proteina fino ancora più grande insieme di proteine ​​sono retuned e viene generato un altro profilo e il processo ripetuto. Includendo proteine ​​correlate nella query in ogni passo questo programma permette all&#39;utente di identificare sequenze che sono più divergenti. iv) Nucleotide 6-frame traduzione in proteine ​​(BLASTX): qui l&#39;utente fornisce un ingresso sequenza nucleotidica che viene convertito nei sei fotogrammi concettuali prodotti di traduzione (cioè entrambi i filamenti) nei confronti di un database di sequenza della proteina.. v) Nucleotide 6-frame traduzione nucleotideTraduzione 6-frame (tblastx): Questo programma richiede un ingresso sequenza nucleotidica del DNA e traduce l&#39;ingresso in tutti i sei fotogrammi prodotti di traduzione concettuali che si confronta contro le traduzioni sei fotogrammi di un database sequenza nucleotidica. vi) Protein-nucleotide Traduzione 6-frame (TBLASTN): Questo programma utilizza un ingresso sequenza proteica da confrontare con tutte e sei le fasi di lettura di un database sequenza nucleotidica. Avanti, descriviamo i programmi comunemente utilizzati per generare un allineamento di sequenze multiple (MSA) da un dataset sequenza, e questo è seguito da una guida d&#39;uso di programmi che determinano i modelli di best-fit di evoluzione per un dataset sequenza. Ricostruzione filogenetica è un problema statistico, e per questo, metodi filogenetici necessario incorporare un quadro statistico. Questo quadro statistico diventa un modello evolutivo che incorpora il cambiamento sequenza all&#39;interno del dataset. Questo mo evolutivodel comprende un insieme di ipotesi circa il processo di nucleotide o ammino-acidi sostituzioni, e il modello più adatto per un particolare insieme di dati può essere selezionata tramite test statistico. L&#39;adattamento ai dati di diversi modelli possono essere confrontati con i test del rapporto di verosimiglianza (LRTS) o criteri di informazione per selezionare il modello più adatto all&#39;interno di un insieme di quelli possibili. Due criteri informativi comuni sono l&#39;informazione criterio di Akaike (AIC) 2 e il criterio di informazione bayesiano (BIC) 3. Una volta che un allineamento ottimale è generato, ci sono molti metodi diversi per creare una filogenesi dai dati allineati. Ci sono numerosi metodi di inferenza relazioni evolutive, in generale, possono essere suddivisi in due categorie: metodi basati sulla distanza e metodi basati su sequenza. Metodi Distance-based calcolare le distanze a coppie di sequenze, e quindi utilizzare queste distanze per ottenere l&#39;albero. Metodi basati sequenza-usano l&#39;allineamento di sequenza diretta, e di solito ricerche nel tspazio ree utilizzando un criterio di ottimalità. Noi delineare due metodi basati su sequenza per ricostruire le relazioni filogenetiche: sono PhyML 4 che implementa il quadro di massima verosimiglianza, e MrBayes 5 che utilizza bayesiano Markov Chain Monte Carlo inferenza. Probabilità e metodi bayesiani forniscono un quadro statistico per la ricostruzione filogenetica. Fornendo informazioni utente a comunemente usati strumenti di albero di costruzione, si introduce il lettore ai dati necessari richiesti per dedurre relazioni filogenetiche.

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			<td height="21" style="height:21px;">BLAST webpage&#160;</td>
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			<td><a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a></td>
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			<td height="21" style="height:21px;">BLAST executables&#160;</td>
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			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/">ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Preformatted BLAST databases</td>
			<td></td>
			<td></td>
			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/db/">ftp://ftp.ncbi.nlm.nih.gov/blast/db/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustal</td>
			<td></td>
			<td></td>
			<td><a href="http://www.clustal.org/">http://www.clustal.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kalign</td>
			<td></td>
			<td></td>
			<td><a href="http://msa.sbc.su.se/cgi-bin/msa.cgi">http://msa.sbc.su.se/cgi-bin/msa.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAFFT</td>
			<td></td>
			<td></td>
			<td><a href="http://mafft.cbrc.jp/alignment/software/">http://mafft.cbrc.jp/alignment/software/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MUSCLE</td>
			<td></td>
			<td></td>
			<td><a href="http://www.drive5.com/muscle/">http://www.drive5.com/muscle/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-Coffee</td>
			<td></td>
			<td></td>
			<td><a href="http://www.tcoffee.org/Projects/tcoffee/">http://www.tcoffee.org/Projects/tcoffee/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROBCONS</td>
			<td></td>
			<td></td>
			<td><a href="http://toolkit.tuebingen.mpg.de/probcons">http://toolkit.tuebingen.mpg.de/probcons&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Se-Al&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://tree.bio.ed.ac.uk/software/seal/">http://tree.bio.ed.ac.uk/software/seal/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSEdit&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.bsedit.org/">http://www.bsedit.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JalView</td>
			<td></td>
			<td></td>
			<td><a href="http://www.jalview.org/">http://www.jalview.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeaView</td>
			<td></td>
			<td></td>
			<td><a href="http://pbil.univ-lyon1.fr/software/seaview.html">http://pbil.univ-lyon1.fr/software/seaview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProtTest&#160;</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/prottest3/">https://code.google.com/p/prottest3/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Java Runtime&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.java.com/en/download/chrome.jsp">http://www.java.com/en/download/chrome.jsp</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Readseq</td>
			<td></td>
			<td></td>
			<td><a href="http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi">http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">jModelTest</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/jmodeltest2/">https://code.google.com/p/jmodeltest2/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PhyML</td>
			<td></td>
			<td></td>
			<td><a href="https://code.google.com/p/phyml/">https://code.google.com/p/phyml/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MrBayes</td>
			<td></td>
			<td></td>
			<td><a href="http://mrbayes.sourceforge.net/download.php">http://mrbayes.sourceforge.net/download.php</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeView</td>
			<td></td>
			<td></td>
			<td><a href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html">http://taxonomy.zoology.gla.ac.uk/rod/treeview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeDyn</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="http://www.treedyn.org/">http://www.treedyn.org/</a></td>
		</tr>
	</tbody>
</table>

a practical guide to phylogenetics for nonexperts

Molti ricercatori, attraverso foci incredibilmente varia, stanno applicando filogenesi alla loro domanda di ricerca (s). Tuttavia, molti ricercatori sono nuovi a questo tema e quindi presenta problemi inerenti. Qui compiliamo un&#39;introduzione pratica alla filogenesi per non esperti. Abbiamo delineare in maniera step-by-step, un gasdotto per la generazione di filogenesi affidabili da insiemi di dati di sequenza del gene. Iniziamo con una interfaccia user-guida per somiglianza strumenti di ricerca attraverso interfacce online così come eseguibili locali. Avanti, esploriamo i programmi per la generazione di allineamenti multipli di sequenze seguite da protocolli per l&#39;utilizzo di software per determinare i modelli di best-fit dell&#39;evoluzione. Abbiamo poi delineare protocolli per ricostruire le relazioni filogenetiche tramite massima verosimiglianza e criteri bayesiani e, infine, descrive gli strumenti per la visualizzazione di alberi filogenetici. Anche se questo non è affatto una descrizione esaustiva di approcci filogenetici, fornisce al lettore pratico informat di partenzaion su applicazioni software chiave comunemente utilizzati da phylogeneticists. La visione di questo articolo sarebbe che potrebbe servire come strumento di formazione pratica per i ricercatori intraprendono studi filogenetici ed anche servire come risorsa educativa che potrebbe essere inserito in una classe o di insegnamento-lab.

Trovare analogie a una query consente ai ricercatori di attribuiscono un potenziale identità nuove sequenze e anche deducono le relazioni tra le sequenze. Il tipo di input di file per BLAST 1 è FASTA sequenza di testo formattato o GenBank numero di accesso. Sequenza FASTA formattato inizia con una riga di descrizione indicata da un segno &quot;&gt;&quot; (Figura 2). La descrizione deve seguire immediatamente dopo il segno &quot;&gt;&quot;, la sequenza (ad es. Nucleotidi o amminoaci...

Watch this Scientific Journal Video about A Practical Guide to Phylogenetics for Nonexperts at JoVE.com

A Practical Guide to Phylogenetics for Nonexperts

Qui si descrive un oleodotto step-by-step per la generazione di filogenesi affidabili da nucleotidiche o aminoacidiche set di dati di sequenza. Questa guida si propone di servire i ricercatori o studenti nuovi ad analisi filogenetica.

Una guida pratica per Phylogenetics per i non esperti

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

a-practical-guide-to-phylogenetics-for-nonexperts

Research

JoVE Journal

Biology

35.2K Views.  The George Washington University.  Qui si descrive un oleodotto step-by-step per la generazione di filogenesi affidabili da nucleotidiche o aminoacidiche set di dati di sequenza. Questa guida si propone di servire i ricercatori o studenti nuovi ad analisi filogenetica. 

Video: A Practical Guide to Phylogenetics for Nonexperts

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Il ramo Ladder Walking attività: un sistema di punteggio e la sua applicazione pratica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Ricerca

Biologia

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Un approccio pratico alla mappatura genetica Destino inducibile: una guida visiva a Mark e Pista Cellule In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Un metodo pratico e innovativo per estrarre il DNA genomico da kit di raccolta del sangue per Plasma Proteina Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

Flusso di lavoro senza macchie V3 per un controllo pratico, conveniente e affidabile del carico totale delle proteine nell'blotting occidentale

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Una guida al moderno Quantitative Fluorescent Western Blotting con strategie di risoluzione dei problemi

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the avoidance of airway obstruction, prevention of gastric tympany, and to allow ventilatory support. Due to the difficulty of intubation, alternative methods such as the use of laryngeal mask airways or laryngeal tubes have been explored. However, these methods do not result in direct access to the trachea and thus may present a risk for development of complications. In addition, lack of direct intubation of the trachea can result in personnel exposure to waste anesthetic gases. Numerous methods for endotracheal intubation have been described, including blind placement, use of a fiberoptic laryngoscope or endoscope, and cricoid placement. Despite these numerous publications, many still struggle to achieve success. Here we provide a detailed description of an intubation technique that can be taught with minimal training with a short time to proficiency. Briefly, after administration of injectable anesthesia and proper positioning of the rabbit, a polypropylene catheter is placed into the trachea by direct visualization using a laryngoscope. The catheter is then used as a guide to direct the endotracheal tube into the trachea. This method allows for intubation without the need for expensive equipment and can be performed by a single individual without the need for an assistant. In conclusion, this technique can be easily taught and performed at very little cost in any clinical or research setting.

Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting.

Intubazione endotracheale di conigli utilizzando un catetere guida in polipropilene

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

Uso pratico di interferenza del RNA: consegna orale del RNA Double-stranded in elementi portanti di liposomi per scarafaggi

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Guida all'elaborazione di sezioni, coring e immagini per l'approvvigionamento e l'analisi di campioni ossei corticali ad alta produttività per synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Una guida passo-passo all'elettroantennography delle zanzare

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

Sviluppo di un modello murino ovariectomizzato con livelli elevati di FSH

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Autore in primo piano: Indagine sulla relazione tra FSH e cambiamenti fisiopatologici nelle donne in perimenopausa - Approfondimenti da un modello murino 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

Methods Article

Una guida pratica per Phylogenetics per i non esperti