Prevalence of readily detected amyloid blood clots in ‘unclotted’ Type 2 Diabetes Mellitus and COVID-19 plasma: a preliminary report

Background Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. Methods Platelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals. Results In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. Conclusions This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P < 0.0001) and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.

Prevalence of readily detected amyloid blood clots in ‘unclotted’ Type 2 Diabetes Mellitus and COVID-19 plasma: A preliminary report

Background: Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. Methods: Platelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals.Results: In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. Conclusions: This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P<0.0001) and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.

Prevalence of readily detected amyloid blood clots in ‘unclotted’ Type 2 Diabetes Mellitus and COVID-19 plasma

Background Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. Methods We show here that microclots can be detected in the native plasma of COVID-19, as well as T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. Conclusions This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P < 0.0001), and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.

Blood Clot Phenotyping by Rheometry: Platelets and Fibrinogen Chemistry Affect Stress-Softening and -Stiffening at Large Oscillation Amplitude

(1) Background: Together with treatment protocols, viscoelastic tests are widely used for patient care. Measuring at broader ranges of deformation than currently done will add information on a clot’s mechanical phenotype because fibrin networks follow different stretching regimes, and blood flow compels clots into a dynamic non-linear response. (2) Methods: To characterize the influence of platelets on the network level, a stress amplitude sweep test (LAOStress) was applied to clots from native plasma with five platelet concentrations. Five species were used to validate the protocol (human, cow, pig, rat, horse). By Lissajous plots the oscillation cycle for each stress level was analyzed. (3) Results: Cyclic stress loading generates a characteristic strain response that scales with the platelet quantity at low stress, and that is independent from the platelet count at high shear stress. This general behavior is valid in the animal models except cow. Here, the specific fibrinogen chemistry induces a stiffer network and a variant high stress response. (4) Conclusions: The protocol provides several thresholds to connect the softening and stiffening behavior of clots with the applied shear stress. This points to the reversible part of deformation, and thus opens a new route to describe a blood clot’s phenotype.

Prevalence of amyloid blood clots in COVID-19 plasma

The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated whether the individual was infected or not would be highly desirable. Coagulaopathies are a common accompaniment to COVID-19, especially micro-clots within the lungs. We show here that microclots can be detected in the native plasma of COVID-19 patient, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. This provides a rapid and convenient test (P<0.0001), and suggests that the early detection and prevention of such clotting could have an important role in therapy.

Research of the activity and viability of spermatozoa at different concentrations and proportions of diluents after incubation

The results of studies of the viability of spermatozoa with different incubation time at different concentrations and using different diluents are highlighted in the article. (Un) concentrated spermatozoa were diluented: 1) with their native plasma; 2) medium 199; 3) a mixture of equal volumes of plasma and medium 199. The experiment was designed to generate experimental samples with spermatozoa concentrations prepared according to the method, namely: 0.2; 0.1; 0.05; 0.025 billion / ml. The sperm was evaluated after 2, 4, 6 and 8 hours. The perspective of such a study is significant and makes it possible to research various aspects of the subject in a wide range. In this regard, a series of experiments were conducted in this area. The data obtained are statistically processed and allow us to highlight the results that relate to each stage of the study. In particular, in this article it was found out some regularities between the viability of sperm, the type of diluent and the rate of rarefaction, as evidenced by the data presented in the tables. As a result of sperm incubation, the viability of spermatozoa remains at least the highest trend when sperm are diluted to a concentration of 0.1 billion / ml, regardless of the type of diluent used. To maintain the viability of sperm using this concentration of medium 199 is not better than its native plasma, and its mixture with an equal volume of plasma through any length of time incubation of such sperm. Most often it is at this concentration of sperm that their viability is characterized by the lowest coefficient of variation, regardless of the type of diluent used, which may indicate the greatest stability of the result under these conditions. The viability of spermatozoa with a concentration of 0.1 billion / ml is statistically significantly reduced only after 6 or even 8 hours of incubation. If the sperm are incubated for only 2 hours, regardless of the type of diluent used, the sperm concentrations tested do not affect the viability of the sperm. Key words: boar, spermatozoa, sperm plasma, concentration, incubation, medium 199, activity, viability, rarefaction.

Peptide-based NTA(Ni)-nanodiscs for studying membrane enhanced FGFR1 kinase activities

Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.

Current and Emerging Reconstituted HDL-apoA-I and HDL-apoE Approaches to Treat Atherosclerosis

Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1−/− × Apoe−/− mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.

Multicenter Evaluation of Two Next-Generation HIV-1 Quantitation Assays, Aptima Quant Dx and Cobas 6800, in Comparison to the RealTime HIV-1 Reference Assay

ABSTRACT High accuracy and precision at the lower end of quantification are crucial requirements of a modern HIV viral load (VL) assay, since some clinically relevant thresholds are located at 50 and 200 copies/ml. In this study, we compared the performance of two new fully automated HIV-1 VL assays, Aptima HIV-1 Quant Dx and Cobas HIV-1 (Cobas 6800), with the established RealTime m2000 assay. Assay precision and accuracy were evaluated in a retrospective evaluation out of excess plasma material from four HIV-1+ individuals (subtypes B, C, CRF01_AE, and CRF02_AG). Native plasma samples were diluted to nominal concentrations at 50 and 200 copies/ml (according to the RealTime m2000 assay). All dilutions were tested in triplicate in five independent runs over 5 days and in three labs per system. Assay concordance was determined using 1,011 surplus clinical routine samples, as well as selected retrospective longitudinal samples from 7 patients on treatment. The three assays yielded highly concordant results for individual clinical samples (R2 > 0.98; average difference, ≤0.2 log copies/ml) and retrospective longitudinal samples from patients on treatment. The Aptima and RealTime assays showed similar high precision, meeting the 5σ criterion for the majority of samples across all labs and subtypes. The Cobas assay was less precise, missing the 5σ criterion for the majority of samples at low concentrations. In this analysis, results from the Cobas assay appeared less reliable near the clinically relevant cutoff and should be interpreted with more caution in this context. Due to high precision, full automation, and high concordance with the RealTime assay, the Aptima assay represents a good alternative in routine VL monitoring.