In Vitro Assay for Single-cell Characterization of Impaired Deformability in Red Blood Cells under Recurrent Episodes of Hypoxia

Lab on a Chip ◽  
2021 ◽  
Author(s):  
YUHAO QIANG ◽  
Jia Liu ◽  
Ming Dao ◽  
E Du
Keyword(s):  
Shear Stress ◽  
Red Blood Cells ◽  
Single Cell ◽  
Blood Cells ◽  
Blood Circulation ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Cyclic Shear

Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that...

scholarly journals A new in vitro assay for carbon dioxide excretion by trout red blood cells: effects of catecholamines

1991 ◽  
Vol 157 (1) ◽  
pp. 349-366 ◽  
Author(s):  
C. M. Wood ◽  
S. F. Perry
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Time Course ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Acid Base Status ◽  
Excretion Rates ◽  
Co2 Excretion

A new in vitro assay was developed and critically characterized to measure the rate of CO2 excretion by trout red blood cells (RBCs) from HCO3- in their natural plasma under normal in vivo conditions of acid-base status. The assay is based on the addition of [14C]bicarbonate to the whole blood and collection of the resultant 14CO2 in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2 excretion rates are representative of those occurring in vivo. Rates are linear over the 3 min time course of the assay, related to haematocrit in a non-linear fashion, elevated by the addition of carbonic anhydrase, reduced by blockade with acetazolamide, and sensitive to variations of equilibration PCO2. Large variations in plasma [HCO3-] have only a small effect on CO2 excretion rates when the blood is chronically equilibrated at these levels. Acute elevations in [HCO3-], however, create a non-equilibrium situation, resulting in large increases in CO2 excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO2 excretion. The response is transient, reaching a peak 5–8 min after addition of adrenaline and disappearing by 30–60 min. The magnitude of the adrenergic inhibition is correlated with the magnitude of the RBC pHi regulatory response, expressed as the RBC transmembrane pH difference (pHe-pHi). These results support the ‘CO2 retention theory’ explaining observed increases in blood PCO2 in vivo after exhaustive exercise and catecholamine infusions in fish.

Cinnamomum zeylanicum extract on the radiolabelling of blood constituents and the morphometry of red blood cells: In vitro assay

2008 ◽  
Vol 66 (2) ◽  
pp. 139-146 ◽  
Author(s):  
M.O. Benarroz ◽  
A.S. Fonseca ◽  
G.S. Rocha ◽  
J.N.G. Frydman ◽  
V.C. Rocha ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
In Vitro Assay ◽  
Cinnamomum Zeylanicum ◽  
Vitro Assay ◽  
Blood Constituents

scholarly journals Plasmodium falciparum: Assessment of Selectivity of Action of Chloroquine, Alchornea cordifolia, Ficus polita, and Other Drugs by a Tetrazolium-Based Colorimetric Assay

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Nana Kofi Ayisi ◽  
Regina Appiah-Opong ◽  
Ben Gyan ◽  
Kwasi Bugyei ◽  
Fred Ekuban
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Incubation Time ◽  
Blood Cells ◽  
Colorimetric Assay ◽  
In Vitro Assay ◽  
Effective Concentration ◽  
Vitro Assay ◽  
Number Of Cells

A tetrazolium-based colorimetric selective assay (MTT-based CSA) was developed to assess the selectivity of antimalarial drugs. This in vitro assay, unlike all others, measures the ability of drugs to indirectly protect red blood cells (RBCs) from Plasmodium-falciparum-induced destruction. Optimum incubation time and number of cells needed were 5 days and 23×106 RBCs per well, respectively. A parasitemia range of 0.375% to 3% was found to be suitable for this assay. The MTT-based CSA determined anti-P. falciparum strain DD2 activity of chloroquine at a higher 50% effective concentration (EC50) value (21.0 μg/mL) than the isotopic microtest (10.0 μg/mL). Artesunate and oxytetracycline achieved 90% effect against DD2 with minimal or no toxicity to RBCs. Against chloroquine sensitive strain 3D7, chloroquine and Alchornea cordifolia had EC50 values of 0.025 μg/mL and 4.9 μg/mL respectively, and selective index (SI) values of >2,000 and >69.4 μg/mL, respectively.

scholarly journals In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (7) ◽  
pp. 2033-2041 ◽  
Author(s):  
Meriyem Aktas ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
In Vitro Assay ◽  
Enzyme Properties ◽  
Vitro Assay ◽  
Plant Infection ◽  
In Vitro Characterization ◽  
End Products ◽  
And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

Eulerian-Eulerian Multiphase Modeling of Blood Cells Segregation in Flow Through Microtubes

2017 ◽  
Author(s):  
Yertay Mendygarin ◽  
Luis R. Rojas-Solórzano ◽  
Nurassyl Kussaiyn ◽  
Rakhim Supiyev ◽  
Mansur Zhussupbekov
Keyword(s):  
Shear Stress ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Heart Diseases ◽  
Accurate Determination ◽  
Shear Stresses ◽  
High Shear ◽  
Blood Damage ◽  
Solid Phases

Cardiovascular Diseases, the common name for various Heart Diseases, are responsible for nearly 17.3 million deaths annually and remain the leading global cause of death in the world. It is estimated that this number will grow to more than 23.6 million by 2030, with almost 80% of all cases taking place in low and middle income countries. Surgical treatment of these diseases involves the use of blood-wetted devices, whose relatively recent development has given rise to numerous possibilities for design improvements. However, blood can be damaged when flowing through these devices due to the lack of biocompatibility of surrounding walls, thermal and osmotic effects and most prominently, due to the excessive exposure of blood cells to shear stress for prolonged periods of time. This extended exposure may lead to a rupture of membrane of red blood cells, resulting in a release of hemoglobin into the blood plasma, in a process called hemolysis. Moreover, exposure of platelets to high shear stresses can increase the likelihood of thrombosis. Therefore, regions of high shear stress and residence time of blood cells must be considered thoroughly during the design of blood-contacting devices. Though laboratory tests are vital for design improvements, in-vitro experiments have proven to be costly, time-intensive and ethically controversial. On the other hand, simulating blood behavior using Computational Fluid Dynamics (CFD) is considered to be an inexpensive and promising tool to help predicting blood damage in complex flows. Nevertheless, current state-of-the-art CFD models of blood flow to predict hemolysis are still far from being fully reliable and accurate for design purposes. Previous work have demonstrated that prediction of hemolysis can be dramatically improved when using a multiphase (i.e., phases are plasma, red blood cells and platelets) model of the blood instead of assuming the blood as a homogeneous mixture. Nonetheless, the accurate determination of how the cells segregate becomes the critical issue in reaching a truthful prediction of blood damage. Therefore, the attempt of this study is to develop and validate a numerical model based on Granular Kinetic Theory (GKT) for solid phases (i.e., cells treated as particles) that provides an improved prediction of blood cells segregation within the flow in a microtube. Simulations were based on finite volume method using Eulerian-Eulerian modeling for treatment of three-phase (liquid-red blood cells and platelets) flow including the GKT to deal with viscous properties of the solid phases. GKT proved to be a good model to predict particle concentration and pressure drop by taking into account the contribution of collisional, kinetic and frictional effects in the stress tensor of the segregated solid phases. Preliminary results show that the improved segregated model leads to a better prediction of spatial distribution of blood cells. Simulations were performed using ANSYS FLUENT platform.

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