Direct oral anticoagulants (DOACs) represent a new generation of drugs that have been increasingly used in the prevention and treatment of thromboembolic states. According to the mechanism of ...anticoagulant action, DOACs are divided into two groups: direct inhibitors of thrombin (dabigatran) and direct inhibitors of activated factor X (FXa) (rivaroxaban, apixaban, edoxaban, betrixaban). Compared to the vitamin K antagonists, DOACs are superior in terms of onset of action, pharmacokinetic and pharmacodynamics properties and fixed daily dose without the need for routine coagulation monitoring. Despite these advantages, there are clinical conditions in which laboratory measurement of DOACs should be performed. Although DOACs have an impact on screening haemostasis assays (prothrombin time, PT; activated partial thromboplastin time, aPTT; and thrombin time, TT), these tests are not appropriate for quantifying drug levels. Therefore, specific quantitative methods (LC-MS/MS as a gold standard method for all DOACs, coagulometric and chromogenic assays for dabigatran, and chromogenic anti-Xa assays with drug-specific calibrators for inhibitors of FXa) should only be used for determination of DOACs concentration. The aim of this review is to present all aspects of laboratory assessment of DOACs, including pre-analytical, analytical and post-analytical factors in the overall testing process with a special accent on the available specific quantitative methods for measurement of DOACs in circulation.
All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic ...antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.
Nanomedicine is a booming medical field that utilises nanoparticles (NPs) for the development of medicines, medical devices, and diagnostic tools. The behaviour of NPs in vivo may be quite complex ...due to their interactions with biological molecules. These interactions in biological fluids result in NPs being enveloped by dynamic protein coronas, which serve as an interface between NPs and their environment (blood, cell, tissue). How will the corona interact with this environment will depend on the biological, chemical, and physical properties of NPs, the properties of the proteins that make the corona, as well as the biological environment. This review summarises the main characteristics of protein corona and describes its dynamic nature. It also presents the most common analytical methods to study the corona, including examples of protein corona composition for the most common NPs used in biomedicine. This knowledge is necessary to design NPs that will create a corona with a desired efficiency and safety in clinical use.
It has been shown that one recreational SCUBA (rSCUBA) diving session is sufficient to cause changes in plasma level of cardiovascular (CV) and muscular biomarkers. To explore whether repetitive ...rSCUBA diving triggers an adaptive response of the CV, muscular, and immune system, we measured the cardiac damage (NT‐proBNP, hs‐TnI, and CK‐MB), muscle damage (myoglobin (Mb), galectin‐3, CK, and LDH), vascular endothelial activation (ET‐1 and VEGF), and inflammatory (leukocyte count (Lkc), CRP, and IL‐6) biomarkers. A longitudinal intervention study included divers (N = 14) who conducted one dive per week over 5 weeks at the depth of 20–30 m for 30 min after a non‐dive period of 5 months. The blood samples were collected before and after the first, third, and fifth dives and specific biomarkers were measured in plasma or serum by the standard laboratory methods. The concentrations of the majority of measured biomarkers increased after every single dive; the exception was ET‐1 concentration that decreased. The cumulative effect of five dives has been reflected in diminishing changes in hs‐TnI, Mb, galectin‐3, ET‐1, VEGF, and IL‐6 levels, and more pronounced increases in NT‐proBNP and hs‐CRP levels. The median values of all measured biomarkers in all time points, except Mb, remained within the corresponding reference range. Repeatedly performed rSCUBA diving activates an adaptive response of the CV, muscular, and immune system that is reflected in changes in the specific biomarker concentration.
Under challenging environmental conditions, stress forces organisms to adapt to the rapidly changing surroundings. This is the first study on the cumulative effect of rSCUBA diving on the blood concentration of the majority of the selected biomarkers. Repeatedly performed recreational SCUBA diving triggered activation of an adaptive response of the cardiovascular, muscular, and immune systems that were reflected in changes in the specific biomarkers. No clinically relevant impairment of any observed systems was seen, but all measured biomarkers showed different dynamics of change with repeatedly performed rSCUBA diving. Studies on the cumulative effect of recreational SCUBA diving should be carefully designed, because the frequency of repeated dives seems to be crucial for triggering the adaptive response that could be recorded by the blood levels of the specific biomarkers.
Clinical application of rivaroxaban and apixaban does not require therapeutic monitoring. Commercial anti-activated factor X (anti-FXa) inhibition methods for all anti-FXa drugs are based on the same ...principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. We aimed to evaluate relationship between anti-FXa methods calibrated with low molecular weight heparin (LMWH) and with drug specific calibrators, and to determine whether commercial LMWH anti-FXa assay can be used to exclude the presence of clinically relevant concentrations of rivaroxaban and apixaban.
Low molecular weight heparin calibrated reagent (Siemens Healthineers, Marburg, Germany) was used for anti-FXa activity measurement. Innovance heparin (Siemens Healthineers, Marburg, Germany) calibrated with rivaroxaban and apixaban calibrators (Hyphen BioMed, Neuville-sur-Oise, France) was used for quantitative determination of FXa inhibitors.
Analysis showed good agreement between LMWH calibrated and rivaroxaban calibrated activity (κ = 0.76) and very good agreement with apixaban calibrated anti-Xa activity (κ = 0.82), respectively. Low molecular weight heparin anti-FXa activity cut-off values of 0.05 IU/mL and 0.1 IU/mL are suitable for excluding the presence of clinically relevant concentrations (< 30 ng/mL) of rivaroxaban and apixaban, respectively. Concentrations above 300 ng/mL exceeded upper measurement range for LMWH anti-FXa assay and cannot be determined by this method.
Low molecular weight heparin anti-FXa assay can be used in emergency clinical conditions for ruling out the presence of clinically relevant concentrations of rivaroxaban and apixaban. However, use of LMWH anti-FXa assay is not appropriate for their quantitative determination as an interchangeable method.
To understand better the adaptation response of the cardiovascular system (CVS) to self-contained underwater breathing apparatus (SCUBA) diving, Galectin-3 (Gal-3) and specific CVS biomarkers were ...measured in plasma of 16 male recreational divers before and after (30 min, 3 and 6 h) diving (total time of 30 min at 30 m depth) undertaken a after long non-dive period. The one-time SCUBA dive caused a significant increase in Gal-3, N-terminal prohormone of brain natriuretic peptide (NT-proBNP), high-sensitivity troponin-I (hs-TnI), and myoglobin immediately after diving. Whereas Gal-3 and myoglobin dropped down to the basal levels during the recovery period, NT-proBNP and hs-TnI concentration continued to increase. An immediate increase of vascular endothelial growth factor, detected immediately after diving, was followed by a significant decrease and return to the basal level, 3 and 6 h after diving, respectively. After a significant initial decrease, endothelin-1 increased during the recovery period, but did not return to the basal level. The observed changes in these biomarkers reflect comprehensive, but transient adaptation of CVS and muscular system to the specific environmental conditions during the SCUBA dive. Whether the recurrent activation of these adaption mechanisms due to repetitive dives has positive or negative effects on CVS remains to be elucidated.
Recreational SCUBA (rSCUBA) diving has become a highly popular and widespread sport. Yet, information on molecular events underlying (patho)physiological events that follow exposure to the specific ...environmental conditions (hyperbaric conditions, coldness, immersion, and elevated breathing pressure), in which rSCUBA diving is performed, remain largely unknown. Our previous study suggested that repeated rSCUBA diving triggers an adaptive response of cardiovascular and immune system. To elucidate further molecular events underlying cardiac and immune system adaptation and to exclude possible adverse effects we measured blood levels of specific cardiac and inflammation markers.
This longitudinal intervention study included fourteen recreational divers who performed five dives, one per week, on the depth 20-30 m that lasted 30 min, after the non-dive period of 5 months. Blood samples were taken immediately before and after the first, third, and fifth dives. Copeptin, immunoglobulins A, G and M, complement components C3 and C4, and differential blood count parameters, including neutrophil-to-lymphocyte ratio (NLR) were determined using standard laboratory methods. Cell-free DNA was measured by qPCR analysis and N-glycans released from IgG and total plasma proteins (TPP), were analyzed by hydrophilic interaction ultra-performance liquid chromatography.
Copeptin level increased after the first dive but decreased after the third and fifth dive. Increases in immunoglobulins level after every dive and during whole studied period were observed, but no changes in C3, C4, and cfDNA level were detected. NLR increased only after the first dive. IgG and TPP N-glycosylation alterations toward anti-inflammatory status over whole studied period were manifested as an increase in monogalyctosylated and core-fucosylated IgG N-glycans and decrease in agalactosylated TPP N-glycans.
rSCUBA diving practiced on a regular basis promotes anti-inflammatory status thus contributing cardioprotection and conferring multiple health benefits.
Protein glycosylation is the most common epiproteomic modification involved in numerous physiological and pathological processes. Previous studies reported strong associations between human plasma
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...-glycans and age, prompting us to evaluate the potential application of this biological phenomenon in the field of forensics. Blood from 526 blood donors from different parts of Croatia was collected on bloodstain cards during the period 2004–2007 and stored at 4°C for 6–9 years. Glycosylation profiles of the bloodstains were analysed using hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) and divided into 38 glycan groups (GP1-GP38). A statistically significant correlation between
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-glycan profiles of bloodstains and chronological age was found and a statistical model that can be used for the age prediction was designed (Age = 75.59 – 5.15 × (GP4)
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+ 17.07 × GP6 – 5.30 × (GP10)
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– 16.56 × GP16 + 20.07 × GP20 – 7.54 × (GP20)
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+ 16.47 × GP22). This model explains 47.78 % of the variation in age, with a prediction error of 9.07 years. Our findings demonstrate that analysing the
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-glycan profile could be a new tool in forensics, offering an approximate human age estimation from dried bloodstains found at a crime scene.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests have been suggested as an additional diagnostic tool in highly suspected cases with a negative molecular test and ...determination of seroprevalence in population. We compared the diagnostic performance of eight commercial serological assays for IgA, IgM, and IgG antibodies to the SARS-CoV-2 virus.
The comparison study was performed on a total of 76 serum samples: 30 SARS-CoV-2 polymerase chain reaction (PCR)-negative and 46 SARS-CoV-2 PCR-positive patients with asymptomatic to severe disease and symptoms duration from 3-30 days. The study included: three rapid lateral flow immunochromatographic assays (LFIC), two enzyme-linked immunosorbent assays (ELISA), and three chemiluminescence immunoassays (CLIA).
Agreement between IgM assays were minimal to moderate (kappa 0.26 to 0.63) and for IgG moderate to excellent (kappa 0.72 to 0.92). Sensitivities improved with > 10 days of symptoms and were: 30% to 89% for IgM; 89% to 100% for IgG; 96% for IgA; 100% for IgA/IgM combination; 96% for total antibodies. Overall specificities were: 90% to 100% for IgM; 85% to 100% for IgG; 90% for IgA; 70% for IgA/IgM combination; 100% for total antibodies. Diagnostic accuracy for IgG ELISA and CIA assays were excellent (AUC ≥ 0.90), without significant difference. IgA showed significantly better diagnostic accuracy than IgM (P < 0.001).
There is high variability between IgM assays independently of the assay format, while IgG assays showed moderate to perfect agreement. The appropriate time for testing is crucial for the proper immunity investigation.
Biochemical and biological properties of glycoconjugates are strongly determined by the specific structure of its glycan parts. Glycosylation, the covalent attachment of sugars to proteins and ...lipids, is very complex and highly-coordinated process involving > 250 gene products. Deficiency of glycosylation enzymes or transporters results in impaired glycosylation, and consequently pathological modulation of many physiological processes. Inborn defects of glycosylation enzymes, caused by the specific mutations, lead to the development of rare, but severe diseases - congenital disorders of glycosylation (CDGs). Up today, there are more than 45 known CDGs. Their clinical manifestations range from very mild to extremely severe (even lethal) and unfortunately, only three of them can be effectively treated nowadays. CDG symptoms highly vary, though some are common for several CDG types but also for other unrelated diseases, especially neurological ones, leaving the possibility that many CDGs cases are under- or misdiagnosed. Glycan analysis of serum transferrin (by isoelectric focusing or more sophisticated methods, such as HPLC (high-performance liquid chromatography) or MALDI (matrix-assisted laser desorption/ionization)) or serum N-glycans (by MS), enzyme activity assays and DNA sequence analysis are the most frequently used methods for CDG screening and identification, since no specific tests are available yet. In this review we summarize the current knowledge on the clinical, biochemical and genetic characteristic of distinct CDGs, as well as existing diagnostic and therapeutic procedures, aiming to contribute to the awareness on the existence of these rare diseases and encourage the efforts to elucidate its genetic background, improve diagnostics and develop new strategies for their treatment.
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