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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</ol>

The authors have nothing to disclose.

עבודה זו מתארת ​​שיטות חילוץ וזיהוי לכימות הוא אנדוטוקסינים ובהווה DNA בדגימות אירוסול שנאספו על מסננים. השיטות דורשות שגרות מדויקות וניתן לבצעו בקלות כל עוד בניסויים דבק כמה נקודות חיוניות וחשיבות דנו כאן. עבור שלב זיהוי רעלן ה...

דגימת אוויר על מסננים הוא כלי בסיסי במחקר אירוסולים אטמוספריים. 1 המסננים שנדגמו הם נקודת המוצא כימית שונה, פיזי, ומאפיינים ביולוגיים של חלקיקי הסביבה שנאספו. 2-11 היתרון של גישה זו הוא כי ניתוחים שונים יכולים להיות ביצע off-line על מדגם זהה. ליקוט נתונים מכל הניתוחים השונים מאפשר לחוקר לקבל הבנה טובה של המאפיינים של חלקיקים שנאספו ועזרים בפתרון שאלות מורכבות בתחום מדעי האטמוספרה. 12,13 לדוגמה, ימיית יבשת אוויר-דגימות שנלקחו במהלך אותה תקופת ניתן להשוות ביחס רעילות החלקיקים שנדגמו והרכב ביולוגי. 14 לצורך המחקר, lipopolysaccharides (LPS), רכיבים על קירות תא החיידק גרם שליליים, הידוע גם בשם אנדוטוקסינים, חולצו מן מסננים שנדגמו על-shore ב אתר יבש, וכן הוערך באמצעותLimulus lysate amebocyte (LAL) הבדיקה. במקביל, ערכה הגנומי של תוכן החיידקים (חיידקים הכולל, גרמו שלילית, כחוליות) בוצעו על המדגם הזהה באמצעות תגובת שרשרת פולימרז כמוני (q-PCR). מבחן LAL מבוסס על מדידות של עכירות נוצרו בעקבות התוספת של תמצית מימית של amebocytes מן סרטן הפרסה, פוליפמוס Limulus, כדי בתמיסה מימית המכילה את אנדוטוקסינים. ריכוז רעלן הפנימי הגבוה ב המדגם, העכירות מהר מפתחת. 15 ניתוח q-PCR מבוסס על אותות קרינה נפלטים כמו קטע DNA ספציפי מוגבר. 16 על ידי ניטור בזמן אמת של האות בשלב המעריכים של PCR ניתן לכמת תגובת כיול עם עקומת סטנדרט, כמות ה- DNA הראשונית. השילוב של שני אלה מנתח יחד עם אחרים, כמפורט במקומות אחרים, 14 יכול לספק הערכה טובה של רמות של רעלן פנימי ואתכמות חיידקי המקור בדגימות. מטרת השיטה המוצגת כאן היא להשיג ניתוח מדויק והאמין ביותר של תוכן רעלן הפנימי ו- DNA של ביו-אירוסולים מופקים מסננים. בעוד שיטות הדגימו את המאפיינים הכימיים הפיסיים אנאורגניים אירוסולים ידועות, ולאחרונה, שיטות פותחו כדי לחקור מרכיב החומר האורגני שלה, 17 חלו מחקר דל על המרכיב הביולוגי של אירוסולים. 18 רציונל הזרם שיטה היא לתת מענה לפער זה על ידי ההצגה בפירוט שיטה חזקה לחילוץ, ניתוח וזיהוי החלק הביולוגי של אירוסולים מוטסים. 14 השיטה המפורטת כאן צפויה למצוא שימוש רחב התפשט בפרויקטים מחקריים אירוסול מקורה ביולוגיים החיצוניים המעורבים ניתוח מסנן. 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

מחקר אירוסול חיצוני כלל משתמש חומר חלקיקי נדגמים מסננים. הליך זה מאפשר אפיונים שונים של חלקיקים שנאספו להתבצע במקביל. מטרת השיטה המוצגת כאן היא להשיג ניתוח מדויק והאמין ביותר של תוכן רעלן הפנימי ו- DNA של ביו-אירוסולים מופקים מסננים. הפקת מולקולות אורגניות משקל מולקולרי גבוה, כגון lipopolysaccharides, ממסנני שנדגמו כרוך רועד המדגם בתווך על בסיס מים pyrogen חינם. הניתוח שלאחר מכן מבוסס על תגובה האנזימטית כי ניתן לאתר באמצעות מדידת turbidimetric. כתוצאת התוכן האורגני הגבוה על מסנני שנדגמו, החילוץ של דנ&quot;א הדגימות מתבצע באמצעות ערכת חילוץ DNA מסחרית אשר תוכננה במקור עבור קרקעות שונות כדי לשפר את תשואת DNA. איתור וכימות של מינים ספציפיים של חיידקים באמצעות Reacti שרשרת פולימראז כמותייםעל (q-PCR) ניתוח מתואר ולעומת משיטות אחרות.

<html> זה נפוץ ללמוד אירוסולים באטמוספירה באמצעות &quot;off-line&quot; מנתח של מסננים שנדגמו (ראה איור 2). 32 בדיקות כימיות של שנדגמו משנה כוללים אורגני (חלבון למשל, מולקולות פחמימנים, סכרידים) ואי-אורגניים (מתכות למשל, מלחים) תוכן . ניתוחים...

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-שנדגמו ניתוח סינון עבור אנדוטוקסינים ותוכן DNA

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 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.

בידוד ואפיון כימי ליפידים מחיידקים גראם שליליים

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

Chemistry

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.

הלחנה וניתוח הפצה Bioaerosols בתנאים סביבתיים שונים

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.

זיהוי של אנדוטוקסין ניסוחים-Nano באמצעות מבחני Lysate (LAL) Amoebocyte Limulus

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

Bioengineering

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

ייצור של E. coli-הביע עצמית הרכבה חלקיקי חלבון עבור חיסונים הדורשים Trimeric אפירופה מצגת

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

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.

תא עיתונאי מקרופאג שיטה לבחון קולטן שיחת כמו-תיווך NF-kB/AP-1 איתות על שכבות חלבון Adsorbed על משטחים פולימריים

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

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

שיטת פעילות אנדוטוקסין לאיתור דם כולו אנדוטוקמיה בחולים חולי אנוש

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Medicine

טכניקה יעילה להערכת AMR סביבתי על ידי העשרת DNA בהספק MW נמוך

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 Combined 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.

מיצוי משופר של DNA במשקל מולקולרי נמוך משפכים להערכה מקיפה של עמידות מיקרוביאלית 

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

Biology

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.

בדיקת ג'ל-קריש לימולוס אמבוציטים ליזט: שיטה לזיהוי אנדוטוקסין חיידקי בפורמולציות ננו-חלקיקים

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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