scholarly journals Platelet Phenotyping and Function Testing in Thrombocytopenia

2021 ◽  
Vol 10 (5) ◽  
pp. 1114
Author(s):  
Kerstin Jurk ◽  
Yavar Shiravand
Keyword(s):  
Function Analysis ◽  
Point Of Care ◽  
Bleeding Complications ◽  
Diagnostic Significance ◽  
Blood Samples ◽  
Skilled Personnel ◽  
Life Threatening ◽  
And Function ◽  
Function Testing

Patients who suffer from inherited or acquired thrombocytopenia can be also affected by platelet function defects, which potentially increase the risk of severe and life-threatening bleeding complications. A plethora of tests and assays for platelet phenotyping and function analysis are available, which are, in part, feasible in clinical practice due to adequate point-of-care qualities. However, most of them are time-consuming, require experienced and skilled personnel for platelet handling and processing, and are therefore well-established only in specialized laboratories. This review summarizes major indications, methods/assays for platelet phenotyping, and in vitro function testing in blood samples with reduced platelet count in relation to their clinical practicability. In addition, the diagnostic significance, difficulties, and challenges of selected tests to evaluate the hemostatic capacity and specific defects of platelets with reduced number are addressed.

scholarly journals Scenarios of Genes-to-Terpenoids Network Led to the Identification of a Novel α/β-Farnesene/β-Ocimene Synthase in Camellia sinensis

2020 ◽  
Vol 21 (2) ◽  
pp. 655
Author(s):  
Jieyang Jin ◽  
Shangrui Zhang ◽  
Mingyue Zhao ◽  
Tingting Jing ◽  
Na Zhang ◽  
...  
Keyword(s):  
Function Analysis ◽  
Pearson Correlation ◽  
Integrated Approach ◽  
In Planta ◽  
Terpene Synthases ◽  
Gene Transcripts ◽  
Pearson Correlation Analysis ◽  
Tea Plants ◽  
And Function

Terpenoids play vital roles in tea aroma quality and plants defense performance determination, whereas the scenarios of genes to metabolites of terpenes pathway remain uninvestigated in tea plants. Here, we report the use of an integrated approach combining metabolites, target gene transcripts and function analyses to reveal a gene-to-terpene network in tea plants. Forty-one terpenes including 26 monoterpenes, 14 sesquiterpenes and one triterpene were detected and 82 terpenes related genes were identified from five tissues of tea plants. Pearson correlation analysis resulted in genes to metabolites network. One terpene synthases whose expression positively correlated with farnesene were selected and its function was confirmed involved in the biosynthesis of α-farnesene, β-ocimene and β-farnesene, a very important and conserved alarm pheromone in response to aphids by both in vitro enzymatic assay in planta function analysis. In summary, we provided the first reliable gene-to-terpene network for novel genes discovery.

Anti-Xa Potentiating Effect Of Low Molecular Weight Heparin

1981 ◽  
Author(s):  
W Junker ◽  
J Harenberg ◽  
F Fussi ◽  
K Mattes ◽  
R Zimmermann ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Intestinal Mucosa ◽  
Low Molecular Weight Heparin ◽  
Bleeding Complications ◽  
Low Molecular Weight ◽  
Chromogenic Substrate ◽  
Clotting Time ◽  
Blood Samples ◽  
Increased Risk

Recently special attention has been drawn to bleeding complications of commercial heparins in patients with increased risk for haemorrhages. Alternative heparin preparations with high antithrombotic and low haemostaseological properties have been developed. We now report on a new low molecular weight (LMW) heaprin (mean MW 5000, 85 USP/mg), which has been obtained by depolymerisation of a heparin from pig intestinal mucosa (mean MW 15000, 154 USP/mg).In vitro the anti-Xa-activity (chromogenic substrate S2222) was 15% higher for the LMW heparin in a range of 0.01-2.0 USP/ml plasma. No difference was seen on the anti-IIa-activity (thrombin clotting time)and the aPTT for both heparins in the same range. Both Heparins were injected s.c. in a dose of 100, 50 and 25 USP/kg bodyweight into each of six volunteers randomly at weekly intervalIs. The pharmacodynamic effects were controlled for 6-10 hrs by 8-12 blood samples in relation to the dose applied. Increasing dosis the effects of each heparin increased in all test systems. The anti- Xa-activity of LMW heparin was somewhat higher at 100 and 25 USP/kg. At 50 USP/kg the effect of LMW heaprin was in the same range as 100 USP/kg of the original preparation (MW 15000). The factor Ila activity and aPTT were not influenced differently by the two heparins at each dose.The data indicate, that the LMW heparin presented here may have a more pronounced antithrombotic property by a specific anti-Xa-activity than the compaired commercial heparin. This effect is most pronounced at doses, which have only small haemostaseological effects.

scholarly journals Aptamers that bind filovirus GP : a new frontier for detection and neutralization of Ebola and Marburg virus

2020 ◽  
Author(s):  
◽  
Kwaku Dwumah Tawiah
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Marburg Virus ◽  
Whole Cells ◽  
Affinity Enrichment ◽  
Disease Diagnostics ◽  
Individual Sequences ◽  
The Individual ◽  
And Function

Aptamers are single chained, nucleic acid-based affinity probes that bind to their targets with strong affinity and specificity. They are made through an in vitro combinatorial selection method, wherein large libraries of nucleic acids with randomized sequences are subjected to an iterative process of affinity enrichment, partitioning, and amplification. Evolved libraries are sequenced, and the individual sequences are screened and characterized for their structure and function. Aptamers have been developed to target many molecules, including small molecules, purified proteins, whole cells, bacteria, and viruses. They have been developed for therapeutics and as research and diagnostic probes. Aptamers that have an affinity for virus surfaces are excellent probes for developing low-cost biosensors and potentially antiviral therapeutics. In this work, I present the development of aptamers that have an affinity for filovirus surfaces. I first describe the development of an improved method for purifying highly lytic vesicular stomatitis virus-based filovirus GP displayed surrogate viruses. Filoviruses are highly pathogenic and thus require highly secured containment facilities for their studies. The use of attenuated surrogates facilitates filovirus research at biosafety level 2 facilities. This work outlines the steps required to propagate and generate pure virus particles to be used as selection targets. I then describe the development of aptamer probes that differentially recognize GPs from MARV and EBOV. This work represents the first step in the development of aptamer based-low-cost point of care devices for filovirus disease diagnostics. Finally, this work describes the use of a hybrid selection approach that combines two different selection platforms to generate aptamers that bind to EBOV surfaces.

scholarly journals Non-Canonical G-quadruplexes cause the hCEB1 minisatellite instability in Saccharomyces cerevisiae

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aurèle Piazza ◽  
Xiaojie Cui ◽  
Michael Adrian ◽  
Frédéric Samazan ◽  
Brahim Heddi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structure Function ◽  
Genomic Instability ◽  
Function Analysis ◽  
Structural Features ◽  
Detectable Effect ◽  
Definition Of ◽  
And Function

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids in vitro, but the sequence and structural features dictating their formation and function in vivo remains uncertain. Here we report a structure-function analysis of the complex hCEB1 G4-forming sequence. We isolated four G4 conformations in vitro, all of which bear unusual structural features: Form 1 bears a V-shaped loop and a snapback guanine; Form 2 contains a terminal G-triad; Form 3 bears a zero-nucleotide loop; and Form 4 is a zero-nucleotide loop monomer or an interlocked dimer. In vivo, Form 1 and Form 2 differently account for 2/3rd of the genomic instability of hCEB1 in two G4-stabilizing conditions. Form 3 and an unidentified form contribute to the remaining instability, while Form 4 has no detectable effect. This work underscores the structural polymorphisms originated from a single highly G-rich sequence and demonstrates the existence of non-canonical G4s in cells, thus broadening the definition of G4-forming sequences.

scholarly journals Structure-guided disruption of pseudopilus tip inhibits Type II secretion in Pseudomonas aeruginosa

2018 ◽  
Author(s):  
Zongchao Jia ◽  
Yichen Zhang ◽  
Frederick Faucher ◽  
Wenwen Zhang ◽  
Shu Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Function Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Core Complex ◽  
Wide Range ◽  
Interaction Interface ◽  
And Function

Pseudomonas aeruginosa utilizes the Type II secretion system (T2SS) to translocate a wide range of large, structured protein virulence factors through the periplasm to the extracellular environment for infection. In the T2SS, five pseudopilins assemble into the pseudopilus that acts as a piston to extrude exoproteins out of cells. Through structure determination of the pseudopilin complexes of XcpVWX and XcpVW and function analysis, we have confirmed that two minor pseudopilins, XcpV and XcpW, constitute a core complex indispensable to the pseudopilus tip. The absence of either XcpV or -W resulted in the non-functional T2SS. Our small-angle X-ray scattering experiment for the first time revealed the architecture of the entire pseudopilus tip and established the working model. Based on the interaction interface of complexes, we have developed inhibitory peptides. The structure-based peptides not only disrupted of the XcpVW core complex and the entire pseudopilus tip in vitro but also inhibited the T2SS in vivo. More importantly, these peptides effectively reduced the virulence of P. aeruginosa towards Caenorhabditis elegans.

scholarly journals Measurement of Circulating Forms of Prostate-specific Antigen in Whole Blood Immediately after Venipuncture: Implications for Point-of-Care Testing

2001 ◽  
Vol 47 (4) ◽  
pp. 703-711 ◽  
Author(s):  
Timo Piironen ◽  
Martti Nurmi ◽  
Kerttu Irjala ◽  
Olli Heinonen ◽  
Hans Lilja ◽  
...  
Keyword(s):  
Physical Exercise ◽  
Prostate Specific Antigen ◽  
Whole Blood ◽  
Point Of Care ◽  
Prostate Gland ◽  
Specific Antigen ◽  
Point Of Care Testing ◽  
Blood Samples ◽  
Whole Blood Samples

Abstract Background: The purpose of this study was to validate the use of whole-blood samples in the determination of circulating forms of prostate-specific antigen (PSA). Methods: Blood samples of hospitalized prostate cancer and benign prostatic hyperplasia patients were collected and processed to generate whole-blood and serum samples. Three different rapid two-site immunoassays were developed to measure the concentrations of total PSA (PSA-T), free PSA (PSA-F), and PSA-α1-antichymotrypsin complex (PSA-ACT) to detect in vitro changes in whole-blood samples immediately after venipuncture. The possible influence of muscle movement on the release of PSA from prostate gland was studied in healthy men by measuring the rapid in vitro whole-blood kinetics of PSA forms before and after 15 min of physical exercise on a stationary bicycle. Results: Rapid PSA-T, PSA-F, and PSA-ACT assays were designed using a 10-min sample incubation. No significant changes were detected in the concentrations of PSA-T, PSA-F, and PSA-ACT from the earliest time point of 12–16 min compared with measurements performed up to 4 h after venipuncture. Physical exercise did not influence the concentrations of the circulating forms of PSA. Hematocrit-corrected whole-blood values of PSA-T and PSA-F forms were comparable to the respective serum values. Calculation of the percentage of PSA-F (PSA F/T ratio × 100) was similar irrespective of the sample format used, i.e., whole blood or serum. Conclusions: We found that immunodetectable PSA forms are likely at steady state immediately after venipuncture, thus enabling the use of anticoagulated whole-blood samples in near-patient settings for point-of-care testing, whereas determinations of PSA (e.g., PSA-T, PSA-F, or PSA-ACT) performed within the time frame of the office visit would provide results equivalent to conventional analyses performed in serum.

scholarly journals Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

2020 ◽  
Author(s):  
Michal Wieczorek ◽  
Shih-Chieh Ti ◽  
Linas Urnavicius ◽  
Kelly R. Molloy ◽  
Amol Aher ◽  
...  
Keyword(s):  
Self Assembly ◽  
Function Analysis ◽  
The Self ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Partial Cone ◽  
Ring Complex ◽  
Microtubule Networks ◽  
And Function

AbstractThe formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MDa assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge”. The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP), a ∼35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex, γ-TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that γ-TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native γ-TuRCs. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCmini-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the γ-TuRC.

scholarly journals A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaojian Han ◽  
Yingming Wang ◽  
Shenglong Li ◽  
Chao Hu ◽  
Tingting Li ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Inhibitory Concentration ◽  
Function Analysis ◽  
Screening Method ◽  
Emerging Infectious Diseases ◽  
Receptor Binding Domain ◽  
Blood Samples ◽  
High Affinity ◽  
And Function ◽  
Efficient Screening

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

scholarly journals Comparison of three common whole blood platelet function tests for in vitro P2Y12 induced platelet inhibition

2019 ◽  
Vol 50 (1) ◽  
pp. 135-143 ◽  
Author(s):  
Joao D. Dias ◽  
Torben Pottgiesser ◽  
Jan Hartmann ◽  
Daniel Duerschmied ◽  
Christoph Bode ◽  
...  
Keyword(s):  
Platelet Function ◽  
Whole Blood ◽  
Linear Models ◽  
Point Of Care ◽  
Platelet Function Tests ◽  
Blood Samples ◽  
P2y12 Inhibitors ◽  
Function Tests ◽  
Increased Risk

Abstract In the context of interventional cardiology, platelet function testing may identify patients treated with P2Y12-inhibitors at an increased risk of mortality, thrombosis and bleeding. Several whole blood point-of-care platelet function analyzers are available; however, inter-device differences have not been examined systematically. To compare three platelet function tests under standardized in vitro conditions. Healthy volunteer (n = 10) blood samples were spiked with increasing concentrations of ticagrelor (0–7500 ng/mL) and/or ASA (0–3280 ng/mL), measured on three platelet function analyzers (TEG®6s, Multiplate®, and VerifyNow®) and respective Effective Concentration (EC) levels EC10, EC50 and EC90 were calculated. Repeatability was assessed in a separate group of pooled blood samples (n = 10) spiked with ticagrelor at EC10, EC50 and EC90. ASA had no impact on ADP-activated channels for all three devices. TEG®6s was able to distinguish (p ≤ 0.05) between all ticagrelor EC zones; VerifyNow® and Multiplate® were able to distinguish between three and two zones, respectively. Multiplate® showed the largest window between EC10 and EC90 (19–9153 ng/mL), followed by TEG®6s (144–2589 ng/mL), and VerifyNow® (191–1100 ng/mL). Drug effect models distribution of disagreements were identified for TEG®6s (5.0%), VerifyNow® (8.3%), and Multiplate® (13.3%). TEG®6s showed the smallest average coefficient of variation between EC conditions (5.1%), followed by Multiplate® (14.1%), and VerifyNow® (17.7%). Linear models could be generated between TEG®6s and Multiplate®, but not VerifyNow®. Significant differences were found between whole blood point-of-care platelet function analyzers and the clinical impact of these differences needs to be further investigated.

Neutrophil isolation from feline blood using discontinuous Percoll dilutions

2018 ◽  
Vol 46 (06) ◽  
pp. 399-402 ◽  
Author(s):  
Ayse Cakmak ◽  
Kader Yildiz ◽  
Neslihan Sursal
Keyword(s):  
Intact Cell ◽  
Venous Blood ◽  
Neutrophil Activation ◽  
Limiting Factor ◽  
Blood Samples ◽  
Percoll Gradients ◽  
Life Threatening ◽  
The Mean ◽  
Adult Cats

Summary Objective: Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. Material and methods: Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63 %, 54 % and 45 %). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72–63 % Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. Results: The mean PMN number was 22 x 105 per ml (minimum – maximum: 20–26 x 105/ml). Neutrophil homogeneity was > 95 % in the cell suspensions. The viability of isolated neutrophils was > 98 %. The technique did not result in neutrophil activation. Conclusion and clinical relevance: Discontinuous Percoll gradients (72 %, 63 %, 54 % and 45 %) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.

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