A Cell-based Model of Hemostasis

2001 ◽  
Vol 85 (06) ◽  
pp. 958-965 ◽  
Author(s):  
Dougald Monroe ◽  
Maureane Hoffman
Keyword(s):  
Large Scale ◽  
Coagulation Factors ◽  
Cellular Receptors ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Coagulation Proteins ◽  
A Cell ◽  
And Control ◽  
Prevailing View

SummaryBased on our work and that of many other workers, we have developed a model of coagulation in vivo. Many workers have demonstrated mechanisms by which cells can influence the coagulation process. Nonetheless, the prevailing view of hemostasis remains that the protein coagulation factors direct and control the process with cells serving primarily to provide a phosphatidylserine containing surface on which the procoagulant complexes are assembled. By contrast, we propose a model in which coagulation is regulated by properties of cell surfaces. This model emphasizes the importance of specific cellular receptors for the coagulation proteins. Thus, cells with similar phosphatidylserine content can play very different roles in hemostasis depending on their complement of surface receptors. We propose that coagulation occurs not as a “cascade”, but in three overlapping stages: 1) initiation, which occurs on a tissue factor bearing cell; 2) amplification, in which platelets and cofactors are activated to set the stage for large scale thrombin generation; and 3) propagation, in which large amounts of thrombin are generated on the platelet surface. This cell based model explains some aspects of hemostasis that a protein-centric model does not.

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2531-2531
Author(s):  
Pani A. Apostolidis ◽  
Stephan Lindsey ◽  
William M. Miller ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Reticulated Platelets ◽  
High Ploidy ◽  
And Control ◽  
Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Integrative glycoproteomics reveals protein N-glycosylation aberrations and glycoproteomic network alterations in Alzheimer’s disease

2020 ◽  
Vol 6 (40) ◽  
pp. eabc5802
Author(s):  
Qi Zhang ◽  
Cheng Ma ◽  
Lih-Shen Chin ◽  
Lian Li
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Human Brain ◽  
Large Scale ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
System Level ◽  
Disease Signatures ◽  
And Control

Protein N-glycosylation plays critical roles in controlling brain function, but little is known about human brain N-glycoproteome and its alterations in Alzheimer’s disease (AD). Here, we report the first, large-scale, site-specific N-glycoproteome profiling study of human AD and control brains using mass spectrometry–based quantitative N-glycoproteomics. The study provided a system-level view of human brain N-glycoproteins and in vivo N-glycosylation sites and identified disease signatures of altered N-glycopeptides, N-glycoproteins, and N-glycosylation site occupancy in AD. Glycoproteomics-driven network analysis showed 13 modules of co-regulated N-glycopeptides/glycoproteins, 6 of which are associated with AD phenotypes. Our analyses revealed multiple dysregulated N-glycosylation–affected processes and pathways in AD brain, including extracellular matrix dysfunction, neuroinflammation, synaptic dysfunction, cell adhesion alteration, lysosomal dysfunction, endocytic trafficking dysregulation, endoplasmic reticulum dysfunction, and cell signaling dysregulation. Our findings highlight the involvement of N-glycosylation aberrations in AD pathogenesis and provide new molecular and system-level insights for understanding and treating AD.

scholarly journals Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation

2019 ◽  
Vol 3 (7) ◽  
pp. 1154-1166 ◽  
Author(s):  
Alyssa J. Moroi ◽  
Nicole M. Zwifelhofer ◽  
Matthew J. Riese ◽  
Debra K. Newman ◽  
Peter J. Newman
Keyword(s):  
Platelet Activation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Negative Regulator ◽  
Platelet Surface ◽  
Rotational Thromboelastometry ◽  
Surface Receptors ◽  
Glycoprotein Vi ◽  
Granule Secretion

Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride–induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.

Coagulation-Related Effect of Bortezomib Treatment in Patients with Relapsed or Refractory Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2733-2733
Author(s):  
Maurizio Zangari ◽  
Jose Guerrero ◽  
Federica Cavallo ◽  
Michael J. Burns ◽  
Keshava Prasad ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Factor V Leiden ◽  
Coagulation Factors ◽  
Factor V ◽  
Post Dose ◽  
Platelet Surface ◽  
Induced Aggregation

Abstract Proteasomes inhibitors have been recently identified as potential agents, which can influence processes leading to thrombosis. We have previously reported a reduced incidence of deep vein thrombosis (DVT) in 69 patients enrolled in Total Therapy III and treated with (B), dexamethasone, thalidomide, cisplatin and doxorubicin with prophylactic anticoagulation. Patients with relapse MM, not previously exposed to B with normal baseline coagulation parameters (PT, PTT fibrinogen and platelet count) were eligible for the study. Except for the two PCR-based genotyping assays Factor V Leiden and Prothrombin Gene, which were assessed only at enrollment, in table one are listed coagulation factors serially assessed at baseline and within 1 hour after the first dose of B on day 1 and 4 of the first treatment cycle. Platelet Aggregation, with epinephrine, collagen, arachidonic acid, ADP and P- selectin by flow cytometry, were obtained at same time points. Results: Median values of coagulation factors pre and post B infusion are shown in table 1. Platelet count on platelet rich plasma (PRP) decreased after each B treatment (204.1±69.2 × 103 platelets/μL versus 160.8±50.3 × 103 platelets/μL). Platelet aggregation in PRP induced by 10 μM ADP was reduced by 20% and 29% in post-B on day one and four with p-values of 0.033 and 0.009 rispectively. Ristocetin induced platelet agglutination and epinephrine-induced aggregation were both negatively affected by B administration, p=0.0077 and p=0.034. Platelet aggregations elicited by collagen and arachidonic were not affected by B P-selectin expression on platelet surface was overall decrease with each agonist after B administration however no statistical differences were observed. In conclusion this pilot study has shown that even a short in vivo B exposure can significantly impaired platelet number and function and could explain clinical observations. Table 1 Test Day 1 Pre-dose Median Day 1 Post-dose Median Day 4 Pre-dose Median Day 4 Post-dose Median PT (sec) 13.4 13.85 13.55 14.4 PTT (sec) 17.35 26.2 26.95 25.5 Fibrinogen (mg/dL) 385 360 386 341.5 Protein C (70%–160%) 114 102 120 115 Protein S (65–140%) 89 85 92 92.5 Antithrombin II (190–127%) 98 97.5 100.5 102 APC Ration 2.345 2.33 2.3 2.22 T.T. (<20s) 18.2 17.9 17.7 19.45 D-Dimer (ug/mL) 0.56 0.5 0.5 0.59 Homocysteine (umol/L) 8.77 8.99 8.69 8.13

The Development of Novel Hemostatic Bypassing Molecules.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-20-SCI-20
Author(s):  
Rodney M. Camire ◽  
Matthew Bunce ◽  
Lacramioara Ivanciu ◽  
Raffaella Toso ◽  
Harre D. Downey ◽  
...  
Keyword(s):  
Active Site ◽  
Half Life ◽  
Research Funding ◽  
Critical Role ◽  
Coagulation Factors ◽  
In Vivo Studies ◽  
Intrinsic Pathway ◽  
Platelet Surface ◽  
Injury Model

Abstract Abstract SCI-20 Blood coagulation factors VIII and IX are part of the intrinsic pathway and play a critical role in maintaining normal hemostasis by activating factor X. Deficiency of either of these proteins caused by mutations in the genes encoding FVIII or FIX leads to hemophilia A or B (HA and HB), respectively. In each disease, there is impairment of the intrinsic pathway with inadequate thrombin (IIa) generation and defective hemostasis. Unfortunately, ∼20-30% of patients with FVIII deficiency and ∼3-5% of patients with FIX deficiency develop inhibitory antibodies to infused factor replacement products. This has prompted the research community to develop so-called “bypass strategies” which use other coagulation factors to provide hemostasis in these patients. In principle, infusion of FXa should bypass deficiencies in the intrinsic pathway; however it is generally thought that it has limited utility since the infused FXa could cause excessive activation of coagulation and/or more importantly, FXa is rapidly inactivated by plasma inhibitors resulting in a very short half-life (<2 min). We recently characterized variants of FXa (FXa-I16L and FXa-V17a) which have “zymogen-like” properties that could circumvent these associated problems (JBC 2008; 283: 18627). For example we have found that i) these proteins have an incompletely formed active site, making them resistant to plasma protease inhibitors; ii) in the absence of FVa, the FXa variants are, in general, refractory to active site functions and thus do not activate FVII, FV, FVIII, and prothrombin very well; and iii) the variants are thermodynamically rescued by FVa; thus at the site of injury on the activated platelet surface, where FVa is present, prothrombinase rapidly forms generating a burst of thrombin. We have begun to exploit these unique properties and evaluate whether these FXa variants could be effective and safe in bypassing the hemophilic phenotype both in vitro and in vivo. Clotting and IIa generation assays in human HA, HB and inhibitor plasma revealed that FXa-I16L could completely restore IIa generation in a FVa-dependent fashion. Furthermore, the zymogen-like conformation protects FXa-I16L in human plasma as it has a prolonged half-life (∼2 hr) versus wt-FXa (<2 min). In vivo studies using HB mice revealed that administration of FXa-I16L via tail vein almost completely corrected the prolonged aPTT. The aPTT was shortened for more than 2 hours and returned close to the starting value after 24 hr. In these experiments, infusion of the protein was well tolerated as platelet levels were unaffected over the course of the experiment with little or no change in the values for TAT, D-dimer, and fibrinogen. Next we tested whether the improvement of the clotting times was associated with in vivo hemostatic performance. Using three separate injury models (tail clip assay, FeCl2 carotid artery injury model, and cremaster muscle laser injury model) infusion FXa-I16L provided effective hemostasis. This was in contrast to the infusion of wild-type FXa which was ineffective. Taken together our data show that FXa-I16L is highly effective in correcting the hemostatic defect in human hemophilic plasma. Furthermore it improves the hemophilic phenotype in HB mice following a series of hemostatic challenges and can restore thrombus formation upon injury at both micro and macrocirculation levels. Thus zymogen-like variants of FXa have properties that indicate their ability to serve as superior therapeutic procoagulants for bypassing deficiencies upstream of the common pathway. Disclosures Camire: Wyeth: Patents & Royalties, Research Funding. Bunce:Wyeth: Research Funding. Ivanciu:Wyeth: Research Funding. Downey:Wyeth: Research Funding.

scholarly journals Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

2022 ◽  
Author(s):  
Benjamin J. Orlando ◽  
Pawel K. Dominik ◽  
Sourav Roy ◽  
Chinemerem Ogbu ◽  
Satchal K. Erramilli ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Protein Complexes ◽  
Gastrointestinal Diseases ◽  
Phage Display Library ◽  
Surface Receptors ◽  
Terminal Domain ◽  
A Cell ◽  
And Function ◽  
Development Structure

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating 𝛽-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via 𝛽-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound 𝛽-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

scholarly journals Membrane-Tethered Ligands: Tools for Cell-Autonomous Pharmacological Manipulation of Biological Circuits

Physiology ◽  
2013 ◽  
Vol 28 (3) ◽  
pp. 164-171 ◽  
Author(s):  
Charles Choi ◽  
Michael N. Nitabach
Keyword(s):  
Cell Surface ◽  
Biological Significance ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Membrane Tethering ◽  
A Cell ◽  
Cellular Circuits ◽  
Communication Pathway ◽  
Tethered Ligands

Detection of secreted signaling molecules by cognate cell surface receptors is a major intercellular communication pathway in cellular circuits that control biological processes. Understanding the biological significance of these connections would allow us to understand how cellular circuits operate as a whole. Membrane-tethered ligands are recombinant transgenes with structural modules that allow them to act on cell-surface receptors and ion channel subtypes with pharmacological specificity in a cell-autonomous manner. Membrane-tethered ligands have been successful in the specific manipulation of ion channels as well as G-protein-coupled receptors, and, in combination with cell-specific promoters, such manipulations have been restricted to genetically defined subpopulations within cellular circuits in vivo to induce specific phenotypes controlled by those circuits. These studies establish the membrane-tethering approach as a generally applicable method for dissecting neural and physiological circuits.

Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 138-138 ◽  
Author(s):  
Gerard Jansen ◽  
Emma C. Josefsson ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Shape Change ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Surface Receptors ◽  
Platelet Survival ◽  
Platelet Receptor

Abstract Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.

Phenethyl-4-ANPP: A Marginally Active Byproduct Suggesting a Switch in Illicit Fentanyl Synthesis Routes

2021 ◽  
Author(s):  
Marthe M Vandeputte ◽  
Alex J Krotulski ◽  
Fabian Hulpia ◽  
Serge Van Calenbergh ◽  
Christophe P Stove
Keyword(s):  
Valuable Insight ◽  
Unknown Compound ◽  
The Usa ◽  
A Cell ◽  
Synthesis Routes ◽  
And Control ◽  
First Time ◽  
Single Reaction

Abstract Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply.

Sensitive Bioassay for Detection of Biologically Active Ricin in Food

2012 ◽  
Vol 75 (5) ◽  
pp. 951-954 ◽  
Author(s):  
REUVEN RASOOLY ◽  
XIAOHUA HE
Keyword(s):  
Large Scale ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Biologically Active ◽  
Green Fluorescent Protein Gene ◽  
Mouse Bioassay ◽  
Human Embryonic Kidney Cells ◽  
Green Fluorescent ◽  
And Control

The potential use of ricin as an agent of biological warfare highlights the need to develop fast and effective methods to detect biologically active ricin. The current “gold standard” for ricin detection is an in vivo mouse bioassay; however, this method is not practical to test on a large number of samples and raises ethical concerns with regard to the use of experimental animals. In this work, we generated adenoviral vectors that express the green fluorescent protein gene and used the relative fluorescence units intensity inhibition by transduced cells for quantitative measurement of biologically active ricin. The detection limit of the assay was 200 pg/ml, which is over 500,000 times greater than the adult human lethal oral dose. The inhibition of fluorescence intensity between ricin treatment and control was higher in 72-h posttransduction Vero cells than 24-h human embryonic kidney cells. Therefore, to detect biologically active ricin in food matrices that might influence the assay, we used 72-h posttransduction Vero cells. This simple assay could be used for large-scale screening to detect biologically active ricin in food without added substrates or use of cell fixation methods.

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