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.

<ol>
	<li>Lodge, J. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of Air Sampling and Analysis. , (1988).</li><li>Costa, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+carbonaceous+aerosols+in+Emilia-Romagna+(Northern+Italy)+based+on+two+fall/winter+field+campaigns.">Characteristics of carbonaceous aerosols in Emilia-Romagna (Northern Italy) based on two fall/winter field campaigns.</a> Atmos. Res. , (2015).</li><li>Brent, L. 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Environ. 97, 552-555 (2014).</li><li>Hospodsky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+airborne+fungal+and+bacterial+concentrations+and+emission+rates+in+six+occupied+children's+classrooms.">Characterizing airborne fungal and bacterial concentrations and emission rates in six occupied children's classrooms.</a> Indoor air. , (2014).</li><li>Bottos, E., Woo, A., Zawar-Reza, P., Pointing, S., Cary, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+Bacterial+Populations+Above+Desert+Soils+of+the+McMurdo+Dry+Valleys,+Antarctica.">Airborne Bacterial Populations Above Desert Soils of the McMurdo Dry Valleys, Antarctica.</a> Microb. Ecol. 67, 120-128 (2014).</li><li>Ovadnevaite, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicron+NE+Atlantic+marine+aerosol+chemical+composition+and+abundance:+Seasonal+trends+and+air+mass+categorization.">Submicron NE Atlantic marine aerosol chemical composition and abundance: Seasonal trends and air mass categorization.</a> J. Geophys. Res. Atmos. 119, (2014).</li><li>Lodge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ES&amp;T+Books:+Methods+of+Air+Sampling+and+Analysis,+3rd+ed.">ES&amp;T Books: Methods of Air Sampling and Analysis, 3rd ed.</a> Environ. Sci. Technol. 23, 938 (1989).</li><li>Pramod, K., Baron, P. A., Willeke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Measurement: Principles, Techniques, and Applications. , (2011).</li><li>Vincent, J. 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> Aerosol Sampling: Science, Standards, Instrumentation and Applications. , (2007).</li><li>Duquenne, P., Marchand, G., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Endotoxins+in+Bioaerosols+at+Workplace:+A+Critical+Review+of+Literature+and+a+Standardization+Issue.">Measurement of Endotoxins in Bioaerosols at Workplace: A Critical Review of Literature and a Standardization Issue.</a> Ann. Occup. Hyg. 57, 137-172 (2013).</li><li>Okuda, T., Schauer, J. J., Shafer, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brock Biology of Microorganisms. , (2011).</li><li>Watson, J. G., Chow, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Measurement: Principles and Techniques. , 591-613 (2011).</li><li>Mueller-Anneling, L., Avol, E., Peters, J. M., Thorne, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+endotoxin+concentrations+in+PM10+from+Southern+California.">Ambient endotoxin concentrations in PM10 from Southern California.</a> Environ. 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The authors have nothing to disclose.

This work describes extraction and detection methods for quantifying both endotoxins and DNA present in aerosol samples collected on filters. The methods require accurate routines and can be performed easily as long as the experimentalist adheres to a few essential and important points discussed here.
For the endotoxin detection step, note that the lysate solution is quite viscous and tends to produce bubbles upon pipetting. It is difficult to remove thee bubbles, and they lead to changes in t...

Air sampling on filters is a basic tool in atmospheric aerosols research.1 The sampled filters are the starting point for various chemical, physical, and biological characterizations of the collected ambient particles.2-11 The advantage of this approach is that various analyses can be performed off-line on the same sample. Compiling the data from all the different analyses enables the researcher to obtain a good understanding of the characteristics of the collected particles and aids in solving complex questions in the atmospheric sciences.12,13 For example, marine and inland air-samples taken during the same period can be compared with respect to the sampled particle toxicity and biological composition.14 For this study, lipopolysaccharides (LPS), components on gram-negative bacterial cell-walls, also known as endotoxins, were extracted from filters sampled on-shore and at an inland site, and were evaluated using the limulus amebocyte lysate (LAL) test. In parallel, a genomic evaluation of the bacterial content (total bacteria, gram negative, and cyanobacteria) was performed on the same sample using the quantitative polymerase chain reaction (q-PCR). The LAL test is based on measurements of turbidity formed following the addition of an aqueous extract of amebocytes from the horseshoe crab, Limulus polyphemus, to an aqueous solution containing the endotoxins. The higher the endotoxin concentration in the sample, the faster turbidity develops.15 The q-PCR analysis is based on a fluorescence signal emitted as a specific DNA fragment is amplified.16 By real time monitoring of the signal during the exponential phase of the PCR reaction and calibrating with a standard curve, the initial DNA amount can be quantified. The combination of these two analyses together with others, as detailed elsewhere,14 can provide a good estimation of the levels of endotoxin and the amount of the source bacteria in the samples.
The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. While methods for sampling the physical and inorganic chemical characteristics of aerosols are well known and, more recently, methods have been developed to investigate its organic matter component,17 there has been scant research on the biological component of aerosols.18 The rationale for the current method is to address this gap by presenting in detail a robust method for extracting, analyzing, and identifying the biological fraction of airborne aerosols.14
The method detailed here is expected to find wide-spread use in biological indoor and outdoor aerosol research projects involving filter analysis.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

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

It is common to study atmospheric aerosols using &#34;off-line&#34; analyses of sampled filters (see Figure 2).32 Chemical analyses of the sampled matter include organic (e.g. protein, hydrocarbon molecules, saccharides) and inorganic (e.g. metals, salts) content. Biological analyses include viable and non-viable microorganism content, species identification using a DNA approach or microscopy, as well as DNA-based quantification.
<p class=...

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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.

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

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Universidad San Sebastian

Research

JoVE Journal

Environment

9.6K Views.  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.

Video: 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.

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

Chemistry

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.

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

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.

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.

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

Bioengineering

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.

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

Biology

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.

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.

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

Immunology and Infection

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

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.

Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

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.

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

Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

Source: Neun, B. W. et al., <a href="https://www.jove.com/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019).
This video describes a gel-clot assay to determine the presence of bacterial endotoxins in nano-formulations using Limulus Amoebocyte Lysate derived from the blood cells of horseshoe crabs. The assay helps test for endotoxin contamination of pharmaceutical products, including nano-based formulations.

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019).
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Biological Techniques

Assay Techniques

Biochemical assays

An Assay for TLR-Dependent NF-&kcy;B/AP-1 Transcription Factor Signaling in Macrophages

Source: McKiel, L. A., et al. <a href="https://app.jove.com/v/60317">A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to examine the toll-like receptor(TLR)-dependent activity of the transcription factors nuclear factor-kappa-B (NF-&#954;B) and activator protein-1 (AP-1) in macrophages. The TLRs trigger a pro-inflammatory signaling cascade upon recognizing damage-associated molecular patterns (DAMPs), activating NF-&#954;B and AP-1. The engineered reporter macrophages produce secreted embryonic alkaline phosphatase (SEAP) enzyme upon NF-&#954;B and AP-1 activation, which is detected via a chromogenic reaction.

1. Media and Reagent Preparation

	Prepare fibroblast media. Combine 450 mL of Dulbecco&#39;s modified Eagle medium (DMEM), 50 mL of fetal bovine serum ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

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