Termit tam-bağırsak DNA ekstraksiyonu için Usul özeti: <ol><li> Chill termitler, buz üzerinde steril cımbız kullanarak gut kaldırmak ve tampon bağırsak örnekleri stabilize. </li><li> PVPP / SDS / fenol tampon numune homojenize. </li><li> Qiagen DNeasy sütunlarını kullanarak ham lizat DNA Özü ve arındırmak. </li></ol> Protokol: <ol><li> Buz, steril forseps kullanarak işçi kast termitler cesareti çıkarın. </li><li> 50 mcL buz 1x moleküler biyoloji sınıf TE tampon içeren steril, nükleaz içermeyen tüp hemen cesareti ve içeriğini transfer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). -20 ° C&#39;de örnekleri Dondurma, ya da doğrudan homojenizasyon ile devam edin. </li><li> Transfer bağırsak örnekleri ve tampon steril, nükleaz içermeyen 2 ml vidalı kapaklı tüp, 500 mg ile% 1 w / v polyvinylpolypyrrolidone (PVPP içeren steril zirkon / silis boncuk (0.1 mm) ve 700 ul 1x TE tampon önceden yüklenmiş .) </li><li> 50 ul% 20 Sodyum dodesil sülfat (SDS) ve 500 ul örnekleri fenol ekleyin. </li><li> 30 sn homojenizasyon üç kür ve buz üzerinde tüyler ürpertici 30 saniye ile en yüksek ayarı (boncuk yendi) karıştırılır. </li><li> 1 dakika için 8.000 x g Tortu çözünmez malzeme. </li><li> Ham lizat arıtma için açıklanan yöntemi kullanarak Qiagen DNeasy sütunları ile sulu (üst) tabakasının 300-ul alikotları arındırın. </li><li> Daha sonra kullanmak için -20 ° C&#39;de nükleik asit içeriği ve dondurma örnekleri sayısal olarak. </li></ol>

<ol>
	<li>Berthelet, M., Whyte, L. G., Greer, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Extraction+of+DNA+from+Soils+for+PCR+Analysis+using+Polyvinylpolypyrrolidone+Spin+Columns.">Rapid, Direct Extraction of DNA from Soils for PCR Analysis using Polyvinylpolypyrrolidone Spin Columns.</a> FEMS Microbiol. Lett. 138, 17-22 (1996).</li><li>Kudo, T., Ohkuma, M., Moriya, S., Noda, S., Ohtoko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Phylogenetic+Identification+of+the+Intestinal+Anaerobic+Microbial+Community+in+the+Hindgut+of+the+Termite,+Reticulitermes+Speratus,+without+Cultivation.">Molecular Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Hindgut of the Termite, Reticulitermes Speratus, without Cultivation.</a> Extremophiles. 2, 155-161 (1998).</li><li>Mor&#233;, M. I., Herrick, J. B., Silva, M. C., Ghiorse, W. C., Madsen, 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=Quantitative+Cell+Lysis+of+Indigenous+Microorganisms+and+Rapid+Extraction+of+Microbial+DNA+from+Sediment.">Quantitative Cell Lysis of Indigenous Microorganisms and Rapid Extraction of Microbial DNA from Sediment.</a> Appl. Environ. Microbiol. 60, 1572-1580 (1994).</li><li>Odelson, D. A., Breznak, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+Fatty+Acid+Production+by+the+Hindgut+Microbiota+of+Xylophagous+Termites.">Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites.</a> Appl. Environ. Microbiol. 45, 1602-1613 (1983).</li><li>Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Digital+PCR+Enables+Multigene+Analysis+of+individual+Environmental+Bacteria.">Microfluidic Digital PCR Enables Multigene Analysis of individual Environmental Bacteria.</a> Science. 314, 1464-1467 (2006).</li><li>Purdy, K. J., Embley, T. M., Takii, S., Newdell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+DNA+and+rRNA+from+Sediments+by+a+Novel+Hydroxyapatite+Spin-Column+Method.">Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method.</a> Appl. Environ. Microbiol. 62, 3905-3907 (1996).</li><li>Salmassi, T. M., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Genes+of+Tetrahydrofolate-Dependent+Metabolism+from+Cultivated+Spirochaetes+and+the+Gut+Community+of+the+Termite+Zootermopsis+Angusticollis.">Analysis of Genes of Tetrahydrofolate-Dependent Metabolism from Cultivated Spirochaetes and the Gut Community of the Termite Zootermopsis Angusticollis.</a> Microbiology. 149, 2529-2537 (2003).</li><li>Schmitt-Wagner, D., Friedrich, M. W., Wagner, B., Brune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+Diversity,+Abundance,+and+Axial+Distribution+of+Bacteria+in+the+Intestinal+Tract+of+Two+Soil-Feeding+Termites+(Cubitermes+spp).">Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp).</a> Appl. Environ. Microbiol. 69, 6007-6017 (2003).</li><li>Tsai, Y. L., Olson, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Method+for+Separation+of+Bacterial+DNA+from+Humic+Substances+in+Sediments+for+Polymerase+Chain+Reaction.">Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction.</a> Appl. Environ. Microbiol. 58, 2292-2295 (1992).</li></ol>

Bizim tecrübelerimize göre, Zootermopis nevadensis gibi ahşap besleme termit türlerinin mikrobiyal topluluklar çıkarılan DNA ekstraksiyonu ve saflaştırma bir turdan sonra, PCR şablon için yeterince saf. Ancak, çöp besleme ve toprak besleme türleri gibi bazı termitler, bağırsak içeriği hümik asit daha yüksek bir konsantrasyon ve bağırsak mikrobiyal DNA ek arıtma gerekebilir. Toplam 5 Z. nevadensis işçilerin cesareti DNA verimi 10-30 mikrogram aralığında. Önemli ölçüde daha küçük veya daha büyük bu türü...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PVPP/SDS/phenol</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>homogeneization buffer</td>
</tr>
<tr>
<td>DNeasy Tissue Kit</td>
<td>Kit</td>
<td>Qiagen</td>
<td>77607</td>
<td>Used according to the protocol for isolation of genomic DNA from crude lysates (Appendix H, product manual version: July 2003)</td>
</tr>
<tr>
<td>TE</td>
<td>Buffer</td>
<td>Sigma</td>
<td>T9285</td>
<td>1x buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) from 100x concentrate</td>
</tr>
<tr>
<td>zirconia/silica beads </td>
<td>Supplies</td>
<td>BioSpec Products</td>
<td>11079101z</td>
<td>0.1 mm</td>
</tr>
<tr>
<td>PVPP</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6755</td>
<td>1% w/v polyvinylpolypyrrolidone prepared from dry reagent as a 1x suspension in TE buffer </td>
</tr>
<tr>
<td>Zootermopsis nevadensis</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Termites</td>
</tr>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4390</td>
<td>Sodium dodecyl sulfate 20% soln. in water from dry reagent</td>
</tr>
<tr>
<td>Phenol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>77607</td>
<td>TE-saturated, ~73%</td>
</tr>
<tr>
<td>MiniBeadbeater-8</td>
<td>Tool</td>
<td>BioSpec Products</td>
<td>963</td>
<td>&nbsp;</td>
<tr>
<td>BSS</td>
<td>Buffered Salt Solution, pH 7.2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Formulation per liter: 2.5 g K2HPO4, 1.0 g KH2PO4, 1.6 g KCl, 1.4 g NaCl, 0.075 g CaCl, 1 g MgCl, and 10 mL of a 1M soln. of NaHCO3</td>
</tr>
<tr>
<td>AxioPlan-2</td>
<td>Microscope</td>
<td>Carl Zeiss, Inc. USA</td>
<td>&nbsp;</td>
<td>Outfitted with 40x objective, 1.6x optivar and 10x ocular lenses. Samples were viewed using phase contrast illumination</td>
</tr>		</tbody>
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extracting dna from the gut microbes of the termite (zootermopsis angusticollis) and visualizing gut microbes

Termitler sadece alıcı ahşap geçinmeye kapasitesine sahip olduğu bilinen en az sayıda hayvan arasında yer almaktadır. Termit bağırsak yolu, ağırlıklı olarak asetat lignoselüloz bozulması yardımcı, yoğun ve türler zengin bir mikrobiyal nüfusun bulunduğu, termit metabolizma alevlendiren kilit besin (Odelson &amp; Breznak, 1983). (&quot;Düşük&quot;) bazı termitler de ökaryotik protozoa, bakteri, metan arkelerin ve bu mikrobik popülasyonları içinde. Böylece, termitler mükemmel araştırma konuları mikrobiyal türler ve ev sahibi yararına yapmak birçok biyokimyasal fonksiyonları arasındaki etkileşimleri çalışmak için. Termit bağırsaklar gibi çeşitli biyokimyasal süreçlerde yer alan anahtar genlerin mikrobiyal popülasyonların tür bileşimi, moleküler teknikler kullanılarak (Schmit-Wagner ve ark, 2003; Salmassi &amp; Leadbetter, 2003 Kudo ve ark, 1998) incelenmiştir. Bu teknikler, termit bağırsak ortamından yüksek kalitede nükleik asitlerin ekstraksiyonu ve saflaştırma bağlıdır. Berthelet ve ark, 1996; Purdy ve ark, 1996; Salmassi &amp; Leadbetter Bu video açıklanan çıkarma tekniği çevre örnekleri (Mor ve ark, 1994 nükleik asitlerin ekstraksiyonu ve saflaştırılması için geliştirilen protokoller değiştirilmiş bir derleme 2003;. Ottesen ve ark 2006) ve DNA polimeraz zincir reaksiyonu (PCR) için şablon olarak kullanmak için uygun termit hindgut malzeme üretir.

Watch this Scientific Journal Video about Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes at JoVE.com

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

Bu video termit hindgut ikamet mikropların türünün DNA ayıklamak için teknik göstermektedir. Bağırsak mikrobiyal topluluk görüntülenmesi için yararlı bir ıslak montaj kaydırağı, hazırlık da gösterilmiştir ve türler açısından zengin bir bağırsak ortamı üzerinden bir tur verilir.

Termit Gut Mikroplar (Zootermopsis Angusticollis) DNA ayıklanıyor ve Gut Mikroplar görselleştirme

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

extracting-dna-from-gut-microbes-termite-zootermopsis-angusticollis

Research

JoVE Journal

Biology

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

15.4K Görüntüleme.  California Institute of Technology - Caltech. Bu video termit hindgut ikamet mikropların türünün DNA ayıklamak için teknik göstermektedir. Bağırsak mikrobiyal topluluk görüntülenmesi için yararlı bir ıslak montaj kaydırağı, hazırlık da gösterilmiştir ve türler açısından zengin bir bağırsak ortamı üzerinden bir tur verilir.

Video: Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Röportaj - Termit Hindgut Mikrobiyal Topluluk incelenmesi

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Biyoloji

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Symbiosis Katmanları - Termit Hindgut Mikrobiyal Topluluk görselleştirme

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni, Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

Protozoa Termit Gut Karşı Ligandla litik Peptitler Protozoacidal Aktivitesi Test In vitro (Protozoa Kültür) ve In vivo (Mikroenjeksiyon içine Termit Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Vahşi Tırtıllar gelen Hemocytes ayıklamak için basit bir protokol

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

Kültürlü ve Gut metabolik Aktif Bakterilerin Tanımlanması<em&gt; Spodoptera littoralis</em&gt; DNA Kararlı İzotop yoluyla kullanma Probing<sup&gt; 13</sup&gt; C = Glukoz

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Bakteriyosin Etkilerini Farenin Bağırsak Mikrobiyolojisi Üzerine Etkileyen Bir Yöntem

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Murine Bağırsak Organoidlerinde Reaktif Oksijen Türlerine Duyarlı Florojenik Prob Kullanılarak Oksidatif Stresin Analizi

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

Yetişkin Drosophila Bağırsasında Bağırsak Asitleşmesini İzleme

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

Yaprak Böceğinin Host-Gut Mikrobiyota Etkileşim Çalışmaları için Doku Kültürlü Fidanlarla Aksenik Böceklerin Hazırlanması ve Yetiştirilmesi

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...