scholarly journals Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Alexandra N. Ii ◽  
Shih-Chao Lin ◽  
Benjamin Lepene ◽  
Weidong Zhou ◽  
Kylene Kehn-Hall ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Yersinia Pestis ◽  
Western Blotting ◽  
Human Blood ◽  
Whole Blood ◽  
Acid Yield ◽  
Blood Samples ◽  
High Affinity ◽  
Magnetic Hydrogel

Abstract Background Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. Results CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. Conclusion Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods. Graphic Abstract

A Modified Indirect Method for Determining Erythrocyte Sodium and Potassium Concentrations

1973 ◽  
Vol 19 (5) ◽  
pp. 506-510 ◽  
Author(s):  
Larry L Small ◽  
Dwight B Coulter
Keyword(s):  
Cell Volume ◽  
Human Blood ◽  
Whole Blood ◽  
Indirect Method ◽  
Indirect Methods ◽  
Blood Samples ◽  
Automated Method ◽  
Human Blood Samples ◽  
Erythrocyte Sodium ◽  
Sodium And Potassium

Abstract An automated method was adapted to measure the concentration of Na and K in plasma, nonhemolyzed whole blood, and hemolyzed whole blood, and thus allow the calculation of erythrocyte electrolyte concentrations by a modified indirect method. The Na concentrations of nonhemolyzed whole blood and plasma were used to calculate the percent cell volume (hematocrit) of a blood sample. The percent cell volume and concentrations of Na and K in nonhemolyzed whole blood were used to modify the indirect method of calculating erythrocyte Na and K concentrations in canine, porcine, and human blood samples. Significant differences were found between the two indirect methods (indirect and modified indirect) used to calculate erythrocyte Na and K concentrations of human blood samples.

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3471-3471
Author(s):  
Jennifer Whangbo ◽  
Marshall Thomas ◽  
Geoffrey McCrossan ◽  
Aaron Deutsch ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nuclear Localization ◽  
Serine Proteases ◽  
Rna Binding ◽  
Nuclear Accumulation ◽  
Target Cells ◽  
Nucleic Acid Binding ◽  
High Affinity ◽  
Nucleic Acid Binding Proteins

Abstract When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates. To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Stabilization of gene expression in whole blood: high quality RNA with room temperature handling of human blood samples.

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Vasco Liberal ◽  
Angela Stassinopoulos ◽  
Scott Whitney ◽  
Steven Wilkinson ◽  
Winnie Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Blood ◽  
Whole Blood ◽  
Room Temperature ◽  
High Quality ◽  
Blood Samples ◽  
Human Blood Samples

scholarly journals Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

2015 ◽  
Vol 2015 ◽  
pp. 1-31 ◽  
Author(s):  
Ka Lok Hong ◽  
Letha J. Sooter
Keyword(s):  
Nucleic Acid ◽  
Molecular Recognition ◽  
Nucleic Acids ◽  
Antibody Fragments ◽  
Small Peptides ◽  
Basic Principles ◽  
Single Stranded Dna ◽  
High Affinity ◽  
Ssdna Aptamer ◽  
Recognition Elements

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed.

Abstract WP287: Deterioration in Recombinant Tissue Plasminogen Activator After Repeated Freezing and Thawing Cycles for Thromboelastography

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Sean Calo ◽  
Anja-Kathrin Jaehne ◽  
Kelly A Keenan ◽  
Jun Xu ◽  
Baruch Tawil ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Blood ◽  
Whole Blood ◽  
Recombinant Tissue Plasminogen Activator ◽  
Healthy Human ◽  
Clot Strength ◽  
Blood Samples ◽  
Freeze Dried ◽  
Tissue Plasminogen

Background and Purpose: Thromboelastography (TEG) is often used to measure coagulation dynamics in the setting of acute ischemic stroke and thrombolytic therapy. The stability of thrombolytics has not been investigated in TEG. We conducted an experimental series to test the effects of recombinant tissue plasminogen activator (rtPA) on fibrinolysis in normal blood samples using TEG. Methods: Freeze dried rtPA powder was reconstituted in normal saline containing 0.2% bovine serum albumin (100 mg/24 mL), divided into 1 mL aliquots, and diluted to enable using a relatively large volume for complete mixing with blood samples. Aliquots and dilutions were frozen at -20°C. The same rtPA dilution was thawed to ambient temperature before each use and refrozen until the next use over 4 testing days. Blood was drawn into 3.2% sodium citrated collection tubes. rtPA (100 μL) was added to 1 mL whole blood to achieve a 636 ng/mL rtPA TEG sample concentration. Control-whole blood and rtPA-whole blood TEG was performed for 3 h on 4 healthy human blood samples. Maximum clot amplitude (mm) and absolute clot strength (dynes/cm 2 ) were measured. Data (mean±SD) were analyzed by t-tests and significance inferred at p <0.05. Results: Clot amplitude increased with thawing and refreezing (28±3, p=0.004; 35±2 p=0.01; 50±3, p=0.02; and 55±3, p=0.30; for testing cycles 1, 2, 3 and 4, respectively) compared to untreated samples (63±4). Clot strength also increased over the 4 cycles (2±0.3, p=0.007; 3±0.2, p=0.02; 5±05, p=0.01 and 6±0.7, p=0.30) compared to untreated (9±1.4). Lysis initiation time was gradually longer over the 4 tests (red arrows, Figure 1) suggesting delayed fibrinolysis. Conclusions: One repeatedly thawed and refrozen rtPA stock showed a delay in fibrinolysis in healthy human blood, suggesting a loss of potency. Thus, rtPA should be aliquoted for 1-time use for experiments using TEG. Further investigation into rtPA potency deterioration with storage after reconstitution is warranted.

scholarly journals Rapid Antibiotic Susceptibility Testing of Tier-1 Agents Bacillus anthracis, Yersinia pestis, and Francisella tularensis Directly From Whole Blood Samples

2021 ◽  
Vol 12 ◽  
Author(s):  
Shahar Rotem ◽  
Ohad Shifman ◽  
Moshe Aftalion ◽  
David Gur ◽  
Tamar Aminov ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Yersinia Pestis ◽  
Francisella Tularensis ◽  
Whole Blood ◽  
Antibiotic Susceptibility ◽  
Blood Samples ◽  
Wide Range ◽  
Tier 1 ◽  
Whole Blood Samples ◽  
Susceptibility Tests

Rapid antibiotic susceptibility tests, performed directly on whole blood samples, will offer great clinical advantages. This issue is of considerable importance when it comes to bioterror pathogens where prompt antibiotic treatment should be offered to infected patients as well as prophylaxis to suspected exposed individuals. Herein, we describe a novel and rapid method, named MAPt, that is based on the direct application of a blood sample onto solid agar that has been embedded with different concentrations of the tested antibiotic. Following a short incubation, bacterial growth is monitored by qPCR. The method was applied on blood cultures and whole blood samples inoculated with the Tier-1 pathogens Bacillus anthracis, Yersinia pestis, and Francisella tularensis. The use of agar medium, which better supports the growth of bacteria at low concentrations, together with the use of qPCR, which provides sensitivity and specificity, allowed minimal inhibitory concentration (MIC) determination to a wide range of bacterial concentrations, ranging from ∼5 × 102 cfu/ml up to 108 cfu/ml. The omission of the enrichment procedure in blood culture and the isolation step, both required in standard antibiotic susceptibility tests (ASTs), allowed a dramatic reduction in time to answer, from a few days to a few hours. The total time required for MIC determination was ∼6 h for fast-growing bacteria, such as B. anthracis, and 12–16 h for slow-growing bacteria, represented by Y. pestis and F. tularensis. Accordingly, MAPt may offer health authorities means for public preparedness in the case of a bioterror attack as well as prompt clinical treatment options in common blood stream infections.

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