Wir möchten Dr. Carolyn Preis für wissenschaftliche Diskussionen und aufschlussreiche Anregungen bedanken. Diese Arbeit wird durch NIH AI066095 (: Bibo Li PI) unterstützt.

<ol>
	<li>Munoz-Jordan, J. L., Cross, G. A., de Lange, T., Griffith, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=t-loops+at+trypanosome+telomeres.">t-loops at trypanosome telomeres.</a> EMBO J. 20, 579-588 (2001).</li><li>Li, B., Espinal, A., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+telomeres+are+protected+by+a+homologue+of+mammalian+TRF2.">Trypanosome telomeres are protected by a homologue of mammalian TRF2.</a> Mol Cell Biol. 25, 5011-5021 (2005).</li><li>Wellinger, R. J., Wolf, A. J., Zakian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+telomeres+acquire+single-strand+TG1-3+tails+late+in+S+phase.">Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase.</a> Cell. 72, 51-60 (1993).</li><li>McElligott, R., Wellinger, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+terminal+DNA+structure+of+mammalian+chromosomes.">The terminal DNA structure of mammalian chromosomes.</a> EMBO J. 16, 3705-3714 (1997).</li><li>Harrington, L. A., Greider, 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=Telomerase+primer+specificity+and+chromosome+healing.">Telomerase primer specificity and chromosome healing.</a> Nature. 353, 451-454 (1991).</li><li>Jacob, N. K., Skopp, R., Price, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-overhang+dynamics+at+Tetrahymena+telomeres.">G-overhang dynamics at Tetrahymena telomeres.</a> Embo J. 20, 4299-4308 (2001).</li><li>Barry, J. D., McCulloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+trypanosomes:+enhanced+phenotypic+variation+in+a+eukaryotic+parasite.">Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite.</a> Adv Parasitol. 49, 1-70 (2001).</li><li>Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAP1+is+essential+for+silencing+telomeric+variant+surface+glycoprotein+genes+in+Trypanosoma+brucei.">RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei.</a> Cell. 137, 99-109 (2009).</li></ol>

Trypanosoma brucei verursacht Schlafkrankheit beim Menschen. Diese Krankheit, die unbehandelt unweigerlich tödlich. T. brucei Zellen in Säugetierwirten unterziehen antigene Variation regelmäßig, so dass das Immunsystem des Wirts Attacke 7 auszuweichen. Daher ist es sehr schwierig, T. beseitigen brucei Zellen einmal eine Infektion festgestellt wird. Wir haben kürzlich gezeigt, dass die Telomere eine wichtige Rolle spielen bei der Regulierung der Expression von T. br...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SeaKem LE Agarose</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>50004</td>
<td>&nbsp;</td>
<tr>
<td>Agarose Type VII (low melting agarose)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4018</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K, recombinant, PCR grade</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>03 115801001</td>
<td>&nbsp;</td>
<tr>
<td>Exo I</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0293</td>
<td>&nbsp;</td>
<tr>
<td>Exo T</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0265</td>
<td>&nbsp;</td>
<tr>
<td>T7 exonuclease</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0263</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0203</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick nucleotide removal kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28304</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

examination of the telomere g-overhang structure in trypanosoma brucei

Die Telomer-G-Überhang Struktur hat sich in vielen Eukaryonten, einschließlich Hefe, Wirbeltieren und Trypanosoma brucei identifiziert worden. Es dient als Substrat für die Telomerase für die de novo Telomere DNA-Synthese und ist daher für Telomererhaltung wichtig. T. brucei ist ein einzelliger Parasit, dass die Schlafkrankheit bei Menschen und Nagana bei Rindern verursacht. Einmal infiziert Säugerwirt, T. brucei Zelle regelmäßig wechselt seinen Oberflächen-Antigen auf das Immunsystem des Wirts Angriff auszuweichen. Wir haben kürzlich gezeigt, dass die T. brucei Telomer-Struktur spielt eine wesentliche Rolle bei der Regulation der Oberflächen-Antigen Genexpression, die kritisch für T. ist brucei Pathogenese. Allerdings T. brucei Telomer-Struktur noch nicht ausführlich untersucht worden aufgrund der Begrenzung von Methoden zur Analyse dieser speziellen Struktur. Wir haben nun erfolgreich die einheimische In-Gel-Hybridisierung und Ligation-vermittelte Primer-Extension-Methoden zur Untersuchung der Telomer-G-Überhang-Struktur und eine Adaptorligation Methode zur Bestimmung der Telomere terminalen Nukleotids in T. angenommen brucei Zellen. Hier werden wir die Protokolle im Detail zu beschreiben und vergleichen ihre verschiedenen Vor-und Nachteile.

Watch this Scientific Journal Video about Examination of the Telomere G-overhang Structure in Trypanosoma brucei at JoVE.com

Untersuchung der Telomer-G-Überhang-Struktur in Trypanosoma brucei | Immunologie und Infektion | Video

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomere sind unerlässlich für das Chromosom Stabilität und die Telomer-G-Überhang Struktur ist essentiell für Telomerase-vermittelte Telomererhaltung. Wir haben vor kurzem zwei Verfahren zum Nachweis der Telomer-G-Überhang Struktur in verabschiedet<em&gt; Trypanosoma brucei</em&gt; Sind die einheimischen In-Gel-Hybridisierung und Ligation-vermittelte Primer-Extension, die beschrieben werden.

Die Untersuchung der Telomere G-Überhang Struktur in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

examination-of-the-telomere-g-overhang-structure-in-trypanosoma-brucei

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Genetics

DNA Structure and Function

SCIENTISTS IN ACTION

14.2K Aufrufe.  Cleveland State University. Telomere sind unerlässlich für das Chromosom Stabilität und die Telomer-G-Überhang Struktur ist essentiell für Telomerase-vermittelte Telomererhaltung. Wir haben vor kurzem zwei Verfahren zum Nachweis der Telomer-G-Überhang Struktur in verabschiedet Trypanosoma brucei Sind die einheimischen In-Gel-Hybridisierung und Ligation-vermittelte Primer-Extension, die beschrieben werden.

Serie: Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Forschung

Immunologie und Infektion

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Hochdurchsatz-Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Nachweis von<em&gt; Trypanosoma brucei</em&gt; Variante Oberflächen-Glykoprotein Switching von Magnetic Activated Cell Sorting und Durchflusszytometrie

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

Die Prüfung der Host-Phänotypen in<em&gt; Gambusia affinis</em&gt; Nach der Behandlung mit Antibiotika

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Nachweis von extravaskulären Trypanosoma Parasiten durch Feine Nadelaspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Ein Mausmodell der chronischen Leberfibrose zur Untersuchung der Gallengangsatresie

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Entwicklung eines pHluorin2-basierten Sensors für den glykosomalen pH-Wert bei Trypanosoma brucei

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

Autor im Rampenlicht: Fortschritte bei der glykosomalen pH-Überwachung bei Trypanosoma brucei mit dem pHluorin2-Biosensor

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

Messung dynamischer glykosomaler pH-Änderungen bei lebenden Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

Quantifizierung der Pyroptose in mit dem Virus der infektiösen Schleimbeutelkrankheit infizierten DF-1-Zellen

Examination of Pyroptosis by Flow Cytometry

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P &#60; 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

Autor im Rampenlicht: Durchflusszytometrische Bestimmung der Pyroptose in Vogelzellen

This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).

Untersuchung der Pyroptose mittels Durchflusszytometrie

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...