DOAC Dipstick Testing Can Reliably Exclude the Presence of Clinically Relevant DOAC Concentrations in Circulation.

In certain clinical situations, it is necessary to determine whether clinically relevant plasma levels of direct oral anticoagulants (DOACs) are present. We examined whether qualitative testing of DOACs in urine samples can exclude DOAC plasma concentrations of ≥30 ng/mL. This prospective single-center cohort study included consecutive patients treated with an oral direct factor Xa inhibitor (DXI) (apixaban, n = 31, rivaroxaban, n = 53) and direct thrombin inhibitor (DTI) (dabigatran, n = 44). We aimed to define the negative predictive value (NPV) and other statistical parameters of detecting DXIs and DTIs by DOAC Dipstick at plasma concentrations of ≥30 ng/mL. We also determined the best-fit threshold plasma levels using chromogenic substrate assays by logistic regression analysis. Between July 2020 and July 2021, 128 eligible patients (mean age 66 years, 55 females) were included into the study. The NPVs and sensitivities for DXI and DTI of DOAC Dipstick were 100% at ≥30 ng/mL plasma, for specificities 6 and 21% and for positive predictive values 62 and 72%, respectively. All diagnostic statistical tests improved to values between 86 and 100% at best-fitting plasma thresholds of ≥14 ng/mL for DXI and ≥19 ng/mL for DTI. Visual analysis using the DOAC Dipstick was 100% in agreement with that of the optoelectronic DOASENSE Reader for all the three DOACs.DOAC Dipstick testing can reliably exclude the presence of DOACs in urine samples at best-fitting thresholds of >14 and >19 ng/mL in plasma. The performance of the DOAC Dipstick at detecting lower DOAC concentrations in plasma requires confirmation.

Direct oral anticoagulants (DOACs): From the laboratory point of view.

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.

Galectin-3 and Cardiovascular Biomarkers Reflect Adaptation Response to Scuba Diving.

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.

How protein coronas determine the fate of engineered nanoparticles in biological environment.

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.

Estimation of human age using N-glycan profiles from bloodstains.

Protein glycosylation is the most common epiproteomic modification involved in numerous physiological and pathological processes. Previous studies reported strong associations between human plasma N-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 N-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)(2)+ 17.07 × GP6 - 5.30 × (GP10)(2) - 16.56 × GP16 + 20.07 × GP20 - 7.54 × (GP20)(2) + 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 N-glycan profile could be a new tool in forensics, offering an approximate human age estimation from dried bloodstains found at a crime scene.

Galectin-3 expression in response to LPS, immunomodulatory drugs and exogenously added galectin-3 in monocyte-like THP-1 cells.

Galectin-3, a structurally unique beta-galactoside-binding lectin, through the specific protein-protein and protein-carbohydrate interactions participates in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation. Its expression and secretion by until now an unknown mechanism are modulated by diverse molecules and are dependent on different physiological and pathophysiological conditions. By autocrine and paracrine actions, galectin-3 modulates many immune reactions and affects various immune cells, particularly those of monocyte-macrophage lineage. This is why galectin-3 has recently become an attractive therapeutic target. However, molecular mechanisms of its actions as well as regulatory mechanism of its expression and activation are still largely unknown. In this study, we show that lipopolysaccharide (LPS) provokes upregulation of galectin-3 expression on both gene and protein level in monocyte-like THP-1 cells, which can be inhibited by dexamethasone, but not with non-steroidal anti-inflammatory drugs aspirin and indomethacin. Resting and LPS-challenged monocyte-like THP-1 cells do not have detectable amount of surface-bound galectin-3, but are able to bind exogenously added galectin-3 with the same capacity. Although galectin-3 is generally considered to be a pro-inflammatory molecule, here we show that the exogenously added galectin-3 does not affect interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α production in resting and LPS-activated monocyte-like THP-1 cells nor influences its own gene expression level in those cells.

Insights into complexity of congenital disorders of glycosylation.

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.

Frequency Determination of α-1,3 Glucosyltransferase p.Y131H and p.F304S Polymorphisms in the Croatian Population Revealed Five Novel Single Nucleotide Polymorphisms in the hALG6 Gene.

The congenital disorder of glycosylation (CDG)-Ic (ALG6-CDG, CDG-Ic) is caused by mutations in the hALG6 gene that encodes the N-glycosylation pathway enzyme, α-1,3-glucosyltransferase (NP_037471.2). The aim of our study was to estimate the frequencies of ALG6-CDG related p.Y131H and p.F304S polymorphisms in the Croatian population. Genomic DNA was isolated from blood samples collected from 600 healthy individuals. Functional single-nucleotide polymorphisms rs35383149 and rs17856039 causing p.Y131H and p.F304S, respectively, were genotyped by the TaqMan method and direct sequencing. The frequency of p.F304S polymorphism in the studied cohort was shown to be similar to the frequencies found in other tested populations (27%), whereas the frequency of p.Y131H was found to be three times higher (6.7%). Five novel base substitutions in the hALG6 gene were also found: three in exon 5 (c.383T>C, c.390G>A, and c.429G>C) and two in a downstream intervening sequence (IVS5+17C/T and IVS5+34G/A).

Employment of single-strand conformation polymorphism analysis in screening for α-1,3 glucosyltransferase gene mutation A333V in Croatian population.

Congenital disorder of glycosylation type Ic (CDG-Ic) is caused by mutations in hALG6 gene encoding α-1,3 glucosyltransferase (NP_037471.2), an enzyme that catalyzes the addition of the first glucose residue to the growing lipid-linked oligosaccharide precursor in N-glycosylation process. The most frequent mutation in hALG6 gene causing CDG-Ic is c.998C>T that results in p.A333V substitution. Up-to-date, no CDG-Ic patient has been detected in Croatia. However, as a part of the comprehensive project undertaken with the aim to estimate the frequencies of the carriers for specific mutations and polymorphisms related to particular CDGs in Croatian population, we screened genomic DNA samples obtained from 600 healthy nonconsanguineous Croatian residents to determine the frequency of the A333V mutation. For that purpose, we established the conditions for polymerase chain reaction-based single-strand conformation polymorphism analysis that is suitable for primary screening and in population studies, especially when the initial sample volume is small or DNA quantity is limited. None of the analyzed samples carried this mutation, indicating that the frequency of the patients carrying this homozygous mutation in Croatian population would be <1 in 1.4×10(6).

Rare TA repeats in promoter TATA box of the UDP glucuronosyltranferase (UGT1A1) gene in Croatian subjects.

BACKGROUND: Gilbert's syndrome is a chronic or recurrent mild unconjugated hyperbilirubinemia caused by decreased activity of UDP glucuronosyltranferase (UGT1A1). The most common cause of Gilbert's syndrome in Caucasians is homozygous variant of the A(TA)7TAA promoter polymorphism. Alleles with five or eight TA repeats have also been described, but they are very rare in Caucasian populations. METHODS: Over a 6-year period (2001-2006), 1109 subjects with suspected Gilbert's syndrome were included in this study. Genotyping of (TA)6 and (TA)7 alleles was performed using high-resolution electrophoretic separation of amplified PCR products on Spreadex EL300 gels. In seven subjects, aberrant electrophoretic patterns were observed and additionally sequenced on an ABI Prism 310 Genetic Analyzer. RESULTS: Genotype distributions for 1102 subjects with (TA)6 or (TA)7 alleles were as follows: 54.10%, 26.33% and 18.94% for the (TA)7/(TA)7, (TA)6/(TA)7 and (TA)6/(TA)6, respectively. Sequencing of seven samples that could not be identified as one of these alleles identified four subjects with the (TA)5/(TA)7, two with the (TA)7/(TA)8 and one with the (TA)6/(TA)8 genotype. CONCLUSION: Genotyping of TA repeats in the promoter region of the UGT1A1 gene revealed the presence of rare alleles with five or eight TA repeats, with a very high frequency of the (TA)7 allele in subjects suspected of having Gilbert's syndrome.