Name: air-sampled filter analysis for endotoxins and dna content
Uploaded: 2016-03-07T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Air-sampled Filter Analysis for Endotoxins and DNA Content at JoVE.com

The authors thank Dr. Yoav Barak from the Chemistry Faculty, Weizmann Institute, for support and advice. This study was supported by the Israel Science Foundation (grant # 913/12), and by the Minerva Foundation with funding from the Federal German Ministry for Education and Research.

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The authors have nothing to disclose.

Diese Arbeit beschreibt Extraktion und Nachweismethoden für beide Endotoxinen und DNA, die in Aerosolproben auf Filtern gesammelt zu quantifizieren. Die Methoden erfordern eine genaue Routinen und kann leicht so lange durchgeführt werden, wie der Experimentator auf wenige wesentliche und wichtige Punkte hält sich hier diskutiert. Für die Endotoxin-Nachweisschritt zu beachten, dass das Lysat Lösung ziemlich viskos und neigt dazu, Blasen beim Pipettieren zu erzeugen. Es ist schwierig, dic...

Air Probenahme auf Filter ist ein grundlegendes Instrument in atmosphärischen Aerosolen Forschung. 1 Die abgetasteten Filter der Ausgangspunkt für verschiedene chemische, physikalische und biologische Charakterisierungen der gesammelten Umgebungspartikel sind. 11.02 Der Vorteil dieses Ansatzes besteht darin , dass verschiedene Analysen sein kann off-line auf der gleichen Probe durchgeführt. Kompilieren der Daten aus den verschiedenen Analysen der Forscher ermöglicht bei der Lösung komplexer Fragestellungen in den atmosphärischen Wissenschaften ein gutes Verständnis für die Eigenschaften der gesammelten Partikel und Hilfsmittel zu erhalten. 12,13 Zum Beispiel Meeres- und Binnenluftproben , die während der gleichen genommen Zeitraum kann in Bezug auf das abgetastete Partikel Toxizität und biologische Zusammensetzung verglichen. 14 Für diese Studie Lipopolysaccharide (LPS), Komponenten auf gram-negativen Bakterienzellwände, auch als Endotoxine bekannt sind , wurden von den Filtern on-shore abgetastete extrahiert und ein Binnenstelle und wurden unter Verwendung bewertet dieLimulusamöbocytenlysat (LAL-Test). Verwendung der quantitative Polymerase-Kettenreaktion (q-PCR) parallel wird eine genomische Auswertung des Bakteriengehalt (total Bakterien, gramnegative und Cyanobakterien) wurde an derselben Probe durchgeführt. Der LAL - Test basiert auf der Messung der Trübung nach der Zugabe eines wässrigen Extrakts aus Amöbozyten von dem Pfeilschwanzkrebs Limulus polyphemus gebildet, zu einer wässrigen Lösung , die Endotoxine enthält. Je höher die Endotoxin - Konzentration in der Probe entwickelt die schneller Trübung. 15 die q-PCR - Analyse auf einem Fluoreszenzsignal basiert , als ein spezifisches DNA - Fragment emittiert wird verstärkt. 16 durch Echtzeit - Überwachung des Signals während der exponentiellen Phase der PCR Reaktion und Kalibrierung mit einer Standardkurve, die anfängliche DNA-Menge quantifiziert werden kann. Die Kombination dieser beiden Analysen zusammen mit anderen, wie anderswo detailliert, 14 eine gute Schätzung des Gehalts an Endotoxin liefern kann und dasMenge der Quell Bakterien in den Proben. Der Zweck der hier vorgestellten Verfahren ist es, eine hochgenaue und zuverlässige Analyse des Endotoxin und DNA-Gehalt von Bio-Aerosole aus den Filtern extrahiert zu erhalten. Während Verfahren die physikalischen und anorganischen chemischen Eigenschaften von Aerosolen zur Abtastung bekannt sind und in jüngster Zeit sind Verfahren entwickelt worden , dessen organischer Substanz Komponente zu untersuchen, 17 dort hat auf die biologische Komponente von Aerosolen wenig Forschung. 18 Der Grund für die aktuelle Methode ist , diese Lücke zu schließen, indem zum Extrahieren im Detail ein robustes Verfahren präsentiert, die Analyse und die biologische Fraktion in der Luft Aerosole zu identifizieren. 14 Die Methode , die hier detailliert wird erwartet , dass weit verbreitete Verwendung in der biologischen Innen- und Außenaerosolforschungsprojekte mit Filteranalyse zu finden. 20-24

<table><tbody><tr><td>&lt;strong&gt;Filter sampling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HiVol 3000 - High Volume Air Sampler</td><td>Ecotech</td><td></td><td></td></tr><tr><td>Quartz Microfiber Filters</td><td>Whatman</td><td>1851-865</td><td>203 mm x 254 mm</td></tr><tr><td>ELF - Laboratory Chamber Furnaces</td><td>Carbolite</td><td>ELF 11series</td><td></td></tr><tr><td>Aluminum foil</td><td>Opal</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Endotoxin&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>16368&amp;nbsp;</td><td>Laboratory Reagent, 96%</td></tr><tr><td>Airstream Class II Biological Safety Cabinet AC-4E1</td><td>ESCO</td><td>10011712</td><td></td></tr><tr><td>Pyrotell -T</td><td>Associates of Cape Cod, Inc.</td><td>T0051</td><td></td></tr><tr><td>Control Standard Endotoxin</td><td>Associates of Cape Cod, Inc.</td><td>E0005-1</td><td>&lt;em&gt;Escherichia coli&lt;/em&gt;&amp;nbsp;O113:H10, 0.5 &amp;micro;g/vial 1 Pack</td></tr><tr><td>LAL Reagent Water</td><td>Associates of Cape Cod, Inc.</td><td>W0051</td><td></td></tr><tr><td>10 ml sterile syringe with Luer-Lok Tip</td><td>Becton-Dickinson &amp;amp; Co.</td><td>309605</td><td></td></tr><tr><td>BD Precisionglide syringe needle</td><td>Becton-Dickinson &amp;amp; Co.</td><td>305129</td><td>Sterile&amp;nbsp;</td></tr><tr><td>Parafilm-M sealing tape</td><td>Parafilm</td><td>P7543</td><td>Sigma catalog number</td></tr><tr><td>Microtubes</td><td>Axigen</td><td>MCT-200-C</td><td>2 ml, pyrogen free</td></tr><tr><td>1.12 cm diameter Cork Borer</td><td>Boekel Scientific</td><td>1601 BD Series - Steel</td><td>Part of a cork borer set containing borers with various diameters.&amp;nbsp;</td></tr><tr><td>50 mm Petri Dish</td><td>Miniplast Ein-Shemer</td><td>72050-01</td><td>Aseptic</td></tr><tr><td>Vortex Genie 2&amp;nbsp;</td><td>Scientific Industries, inc.</td><td>SI-0297</td><td></td></tr><tr><td>Microcentrifuge 5415 D</td><td>Eppendorf</td><td>22621408</td><td></td></tr><tr><td>TC MicroWell 96 F SI w/lid</td><td>Nunc</td><td>167008</td><td>Flat bottom wells (with lid (individually wrapped)), sterile, pyrogen free</td></tr><tr><td>Synergy HT Multi-Detection Microplate Reader</td><td>Biotek</td><td>7091000</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DNA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA away</td><td>Sigma Aldrich</td><td>7010</td><td></td></tr><tr><td>Standard DNA of the microbial species of interest</td><td>ATCC or other culture collection</td><td></td><td>Either the appropriate microbial strain for DNA extraction or the extracted DNA</td></tr><tr><td>Neubauer-improved</td><td>Marienfeld</td><td>640030</td><td>hemocytometer</td></tr><tr><td>TE buffer, Low EDTA</td><td>Life Technologies</td><td>12090-015</td><td>10 mM Tris-HCl (pH 8.0)&amp;nbsp;0.1 mM EDTA&amp;nbsp;</td></tr><tr><td>Nuclease-free PCR-grade water&amp;nbsp;</td><td>Sigma Aldrich</td><td>3315959001</td><td></td></tr><tr><td>PCR primers</td><td>Sigma Aldrich</td><td></td><td>Targets the microbial species of interest</td></tr><tr><td>Dual-Labeled Probes</td><td>Sigma Aldrich</td><td></td><td>Targets the microbial species of interest</td></tr><tr><td>Screw cap tubes</td><td>Axigen</td><td>ST-200-SS</td><td>2 ml&amp;nbsp;</td></tr><tr><td>PowerSoil DNA extraction kit&amp;nbsp;</td><td>Mo Bio Laboratories</td><td>12888-100</td><td></td></tr><tr><td>Glass beads, acid-washed 425-600 &amp;micro;m</td><td>Sigma Aldrich</td><td>G8772-100G</td><td></td></tr><tr><td>Glass beads, acid-washed &amp;lt;106 &amp;micro;m</td><td>Sigma Aldrich</td><td>G4649-100G</td><td></td></tr><tr><td>PowerSoil Solution C1</td><td>Mo Bio Laboratories</td><td>12888-100-1</td><td>Cell lysis buffer , Power soil Kit</td></tr><tr><td>Magic Touch ice bucket</td><td>Bel-Art</td><td>18848-4001</td><td></td></tr><tr><td>Mini-Beadbeater-16</td><td>BioSpec</td><td>607EUR</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Applied Biosystems</td><td>4376600</td><td></td></tr><tr><td>Fast SYBR Green Master Mix</td><td>Applied Biosystems</td><td>4385612</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Applied Biosystems</td><td>4370048</td><td></td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 ml</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>MicroAmp Splash-Free 96-Well Base</td><td>Applied Biosystems</td><td>4312063</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied Biosystems</td><td>4311971</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td>5811 000.010</td><td>Rotor A-4-62 with MTP buckets&amp;nbsp;</td></tr></tbody></table>

air-sampled filter analysis for endotoxins and dna content

Außenaerosolforschung verwendet allgemein Partikel auf Filter abgetastet. Dieses Verfahren ermöglicht es, verschiedene Charakterisierungen der gesammelten Partikel in parallel durchgeführt werden. Der Zweck der hier vorgestellten Verfahren ist es, eine hochgenaue und zuverlässige Analyse des Endotoxin und DNA-Gehalt von Bio-Aerosole aus den Filtern extrahiert zu erhalten. Die Gewinnung von hochmolekularen organischen Moleküle, wie Lipopolysacchariden, aus abgetasteten Filter beinhaltet Schütteln der Probe in einer pyrogenfreien Medium auf Wasserbasis. Die nachfolgende Analyse basiert auf einer Enzymreaktion basiert, die unter Verwendung einer turbidimetrischen Messung erfasst werden können. Als Ergebnis des hohen organischen Anteils auf den abgetasteten Filter wird die Extraktion der DNA aus den Proben durchgeführt, eine kommerzielle DNA-Extraktions-Kits verwenden, die ursprünglich für Böden entworfen und modifiziert, um die DNA-Ausbeute verbessern. Der Nachweis und die Quantifizierung von spezifischen mikrobiellen Spezies unter Verwendung quantitative polymerase chain Reactiauf (q-PCR) Analyse mit anderen verfügbaren Methoden beschrieben und verglichen.

Es ist üblich , atmosphärischer Aerosole zu studieren &quot;off-line&quot; , analysiert der abgetasteten Filter verwenden (siehe Abbildung 2). 32 Chemische Analysen des abgetasteten Materie umfassen organische (zB Protein, Kohlenwasserstoffmoleküle, Saccharide) und anorganischen (zB Metalle, Salze) Gehalt . Biologische Analysen umfassen lebensfähigen und nicht lebensfähige Mikroorganismen Gehalt, Artbestimmung einer DNA-Ansatz oder Mikro...

Watch this Scientific Journal Video about Air-sampled Filter Analysis for Endotoxins and DNA Content at JoVE.com

Air-sampled Filter Analysis for Endotoxins and DNA Content

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Air-abgetastete Filteranalyse für Endotoxine und DNA-Gehalt

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

air-sampled-filter-analysis-for-endotoxins-and-dna-content

Research

JoVE Journal

Environment

9.6K Aufrufe.  Weizmann Institute of Science. Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Serie: Air-sampled Filter Analysis for Endotoxins and DNA Content

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Isolierung und chemische Charakterisierung von Lipid A von gramnegativen Bakterien

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Forschung

Chemie

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Die Verwendung einer Filterpatrone für die Filtration von Wasserproben und Extraktion von Umwelt-DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Umwelt

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Zusammensetzung und Verteilungsanalyse Bioaerosole unter verschiedenen Umweltbedingungen

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Biotechnik

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Biologie

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Ein Makrophagen-Reporter-Zell-Assay zur Untersuchung der toll-like Receptor-vermittelten NF-kB/AP-1-Signalisierung auf adsorbierten Proteinschichten auf polymeren Oberflächen

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

Quantifizierung von drei DNA-Mäsionen durch Massenspektrometrie und Bewertung ihrer Niveaus in den Themen von Mäusen, die der zämatten Feinstaub ausgesetzt sind

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Immunologie und Infektion

Effiziente Technik zur Bewertung von antimikrobiellen Resistenzen in der Umwelt durch Anreicherung von DNA mit niedrigem MW

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Autor im Rampenlicht: Verständnis und Nachweis von antimikrobiellen Resistenzen in der Umwelt durch Kombination kulturbasierter Techniken und Genomik

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

Verbesserte Extraktion von niedermolekularer DNA aus Abwasser zur umfassenden Bewertung von Antibiotikaresistenzen 

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

Air-abgetastete Filteranalyse für Endotoxine und DNA-Gehalt

Zusammenfassung

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Die Verwendung einer Filterpatrone für die Filtration von Wasserproben und Extraktion von Umwelt-DNA

Zusammensetzung und Verteilungsanalyse Bioaerosole unter verschiedenen Umweltbedingungen

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Ein Makrophagen-Reporter-Zell-Assay zur Untersuchung der toll-like Receptor-vermittelten NF-kB/AP-1-Signalisierung auf adsorbierten Proteinschichten auf polymeren Oberflächen

Quantifizierung von drei DNA-Mäsionen durch Massenspektrometrie und Bewertung ihrer Niveaus in den Themen von Mäusen, die der zämatten Feinstaub ausgesetzt sind

Verbesserte Extraktion von niedermolekularer DNA aus Abwasser zur umfassenden Bewertung von Antibiotikaresistenzen

Verbesserte Extraktion von niedermolekularer DNA aus Abwasser zur umfassenden Bewertung von Antibiotikaresistenzen

Verbesserte Extraktion von niedermolekularer DNA aus Abwasser zur umfassenden Bewertung von Antibiotikaresistenzen