Linking aberrant glycosylation of plasma glycoproteins with progression of myelodysplastic syndromes: a study based on plasmonic biosensor and lectin array.

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

Oxidative Stress as a Reliable Biomarker of Carotid Plaque Instability: A Pilot Study.

Background: Predicting stroke risk in patients with carotid artery stenosis (CS) remains challenging. Circulating biomarkers seem to provide improvements with respect to risk stratification. Methods: Study patients who underwent carotid endarterectomy were categorized into four groups according to symptomatology and compared as follows: symptomatic with asymptomatic patients; and asymptomatic patients including amaurosis fugax (AF) (asymptomatic + AF group) with patients with a transient ischemic attack (TIA) or brain stroke (BS) (hemispheric brain stroke group). Carotid specimens were histologically analyzed and classified based on the American Heart Classification (AHA) standard. As a marker of OS, the plasma levels of malondialdehyde (MDA) were measured. Comparisons of MDA plasma levels between groups were analyzed. Results: In total, 35 patients were included in the study. There were 22 (63%) patients in the asymptomatic group and 13 (37%) in the symptomatic group. Atheromatous plaque (p = 0.03) and old hemorrhage (p = 0.05), fibrous plaque (p = 0.04), myxoid changes (p = 0.02), plaques without hemorrhage (p = 0.04), significant neovascularization (p = 0.04) and AHA classification (p = 0.006) had significant correlations with clinical presentation. There were 26 (74%) patients in the asymptomatic group and 9 (26%) in the hemispheric brain stroke group. Atheromatous plaque (p = 0.02), old hemorrhage (p = 0.05) and plaques without neovascularization (p = 0.02), fibrous plaque (p = 0.03), plaques without hemorrhage (p = 0.02) and AHA classification (p = 0.01) had significant correlations with clinical presentation. There was no significant difference between symptomatic and asymptomatic groups with respect to MDA plasma levels (p = 0.232). A significant difference was observed when MDA plasma levels were compared to asymptomatic + AF and the hemispheric stroke group (p = 0.002). Conclusions: MDA plasma level correlates with the risk of hemispheric stroke (TIA or BS) and is a reliable marker of plaque vulnerability in carotid artery stenosis.

Fibrinogenolysis in Venom-Induced Consumption Coagulopathy after Viperidae Snakebites: A Pilot Study.

Envenomations that are caused by Viperidae snakebites are mostly accompanied by venom-induced consumption coagulopathy (VICC) with defibrination. The clinical course of VICC is well described; however, reports about its detailed effects in the hemocoagulation systems of patients are sparse. In this pilot study, we prospectively analyzed the changes in plasma fibrinogen that were caused by the envenomation of six patients by five non-European Viperidae snakes. Western blot analysis was employed and fibrinogen fragments were visualized with the use of specific anti-human fibrinogen antibodies. All of the studied subjects experienced hypo- or afibrinogenemia. The western blot analysis demonstrated fibrinogenolysis of the fibrinogen chains in all of the cases. Fibrinogenolysis was considered to be a predominant cause of defibrination in Crotalus, Echis, and Macrovipera envenomation; while, in the cases of VICC that were caused by Atheris and Calloselasma envenomation, the splitting of the fibrinogen chains was present less significantly.

Long-Term Effects on the Lipidome of Acute Coronary Syndrome Patients.

Lipids modified by oxidative stress are key players in atherosclerosis progression. Superimposed thrombosis with subsequent closure of the coronary artery leads to the clinical manifestation of acute coronary syndrome (ACS). While several studies focusing on alterations in lipid metabolism in the acute phase have been conducted, no information is available on patients' lipidome alterations over longer time periods. In the current follow-up study, we analyzed plasma samples obtained from 17 patients three years after their ACS event (group AC). Originally, these patients were sampled 3-5 days after an index event (group B). Lipidome stability over time was studied by untargeted lipidomics using high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). Multi-dimensional statistics used for data processing indicated that plasmalogen lipids were the most prominent lipids separating the above patient groups and that they increased in the follow-up AC group. A similar trend was observed for lysophosphatidylethanolamine (LPE) and phosphatidylethanolamine (PE). The opposite trend was observed for two fatty acyls of hydroxy fatty acid (FAHFAs) lipids and free stearic acid. In addition, a decrease in the "classic" oxitadive stress marker, malondialdehyde (MDA), occurred during the follow-up period. Our findings present unique information about long-term lipidome changes in patients after ACS.

Fibrin Clot Formation under Oxidative Stress Conditions.

During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients-with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the in vitro-formed fibrin network. Fibers from patients with AIS were significantly thicker in comparison with control and ACS fibers. The number of fibrin fibers in patients with AIS was significantly lower in comparison with ACS and control groups. Thus, oxidative stress-mediated changes in fibrin clot formation, structure and dissolution may affect the effectiveness of thrombolytic therapy.

Lipidomic Analysis to Assess Oxidative Stress in Acute Coronary Syndrome and Acute Stroke Patients.

Alterations in lipid metabolism mediated by oxidative stress play a key role in the process of atherosclerosis and superimposed thrombosis; these can lead to acute coronary syndrome (ACS) and acute ischemic stroke (AIS). Multiple studies have shown that the formation of atheromatous lesions is initiated by oxidation of low-density lipoproteins incorporated into the intima of the vessel wall. Here, we studied lipids in plasma samples from three cohorts: 61 patients with ACS (group A), 49 patients with AIS (group D), and 82 controls (group K). Untargeted lipidomics based on high-performance liquid chromatography coupled to mass spectrometry (UHPLC-HRMS) was employed to obtain comprehensive information on whether relationships exist between these patient categories based on lipid patterns. In addition, malondialdehyde (MDA) as a standard marker of oxidative stress was monitored. The most characteristic lipids in group K were fatty acyls of hydroxyfatty acids (FAHFAs). As expected, MDA concentrations were the lowest in group K. Our findings can better explain ongoing pathologies, both acute and chronic, with the potential for future diagnosis and treatment.

Low Plasma Citrate Levels and Specific Transcriptional Signatures Associated with Quiescence of CD34+ Progenitors Predict Azacitidine Therapy Failure in MDS/AML Patients.

To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients' HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.

Psychophysiological responses to treadwall and indoor wall climbing in adult female climbers.

The purpose of the study was to compare the psychophysiological response of climbers of a range of abilities (lower grade to advanced) when ascending identical climbing routes on a climbing wall and a rotating treadwall. Twenty-two female climbers (31.2 ± 9.4 years; 60.5 ± 6.5 kg; 168.6 ± 5.7 cm) completed two identical 18 m climbing trials (graded 4 on the French Sport scale) separated by 1 week, one on the treadwall (climbing low to the ground) and the other on the indoor wall (climbing in height). Indirect calorimetry, venous blood samples and video-analysis were used to assess energy cost, hormonal response and time-load characteristics. Energy costs were higher during indoor wall climbing comparing to those on the treadwall by 16% (P < 0.001, [Formula: see text] = 0.48). No interaction of climbing ability and climbing condition were found. However, there was an interaction for climbing ability and post-climbing catecholamine concentration (P < 0.01, [Formula: see text] = 0.28). Advanced climbers' catecholamine response increased by 238% and 166% with respect to pre-climb values on the treadwall and indoor wall, respectively; while lower grade climbers pre-climb concentrations were elevated by 281% and 376% on the treadwall and indoor wall, respectively. The video analysis showed no differences in any time-motion variables between treadwall and indoor wall climbing. The study demonstrated a greater metabolic response for indoor wall climbing, however, the exact mechanisms are not yet fully understood.

The ω-3 Polyunsaturated Fatty Acids and Oxidative Stress in Long-Term Parenteral Nutrition Dependent Adult Patients: Functional Lipidomics Approach.

Omega-3 polyunsaturated fatty acids (ω-3PUFAs) are introduced into parenteral nutrition (PN) as hepatoprotective but may be susceptible to the lipid peroxidation while olive oil (OO) is declared more peroxidation resistant. We aimed to estimate how the lipid composition of PN mixture affects plasma and erythrocyte lipidome and the propensity of oxidative stress. A cross-sectional comparative study was performed in a cohort of adult patients who were long-term parenterally administered ω-3 PUFAs without (FO/-, n = 9) or with (FO/OO, n = 13) olive oil and healthy age- and sex-matched controls, (n = 30). Lipoperoxidation assessed as plasma and erythrocyte malondialdehyde content was increased in both FO/- and FO/OO groups but protein oxidative stress (protein carbonyls in plasma) and low redox status (GSH/GSSG in erythrocytes) was detected only in the FO/- subcohort. The lipidome of all subjects receiving ω-3 PUFAs was enriched with lipid species containing ω-3 PUFAs (FO/-˃FO/OO). Common characteristic of all PN-dependent patients was high content of fatty acyl-esters of hydroxy-fatty acids (FAHFAs) in plasma while acylcarnitines and ceramides were enriched in erythrocytes. Plasma and erythrocyte concentrations of plasmanyls and plasmalogens (endogenous antioxidants) were decreased in both patient groups with a significantly more pronounced effect in FO/-. We confirmed the protective effect of OO in PN mixtures containing ω-3 PUFAs.

Tryptophan Metabolism, Inflammation, and Oxidative Stress in Patients with Neurovascular Disease.

Atherosclerosis is a leading cause of major vascular events, myocardial infarction, and ischemic stroke. Tryptophan (TRP) catabolism was recognized as an important player in inflammation and immune response having together with oxidative stress (OS) significant effects on each phase of atherosclerosis. The aim of the study is to analyze the relationship of plasma levels of TRP metabolites, inflammation, and OS in patients with neurovascular diseases (acute ischemic stroke (AIS), significant carotid artery stenosis (SCAS)) and in healthy controls. Blood samples were collected from 43 patients (25 with SCAS, 18 with AIS) and from 25 healthy controls. The concentrations of twelve TRP metabolites, riboflavin, neopterin (NEO, marker of inflammation), and malondialdehyde (MDA, marker of OS) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations of seven TRP metabolites (TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), anthranilic acid (AA), melatonin (MEL), tryptamine (TA)), NEO, and MDA were significantly different in the studied groups. Significantly lower concentrations of TRP, KYN, 3-HAA, MEL, TA, and higher MDA concentrations were found in AIS compared to SCAS patients. MDA concentration was higher in both AIS and SCAS group (p < 0.001, p = 0.004, respectively) compared to controls, NEO concentration was enhanced (p < 0.003) in AIS. MDA did not directly correlate with TRP metabolites in the study groups, except for 1) a negative correlation with kynurenine acid and 2) the activity of kynurenine aminotransferase in AIS patients (r = -0.552, p = 0.018; r = -0.504, p = 0.033, respectively). In summary, TRP metabolism is clearly more deregulated in AIS compared to SCAS patients; the effect of TRP metabolites on OS should be further elucidated.

The effect of ω-3 polyunsaturated fatty acids on the liver lipidome, proteome and bile acid profile: parenteral versus enteral administration.

Parenteral nutrition (PN) is often associated with the deterioration of liver functions (PNALD). Omega-3 polyunsaturated fatty acids (PUFA) were reported to alleviate PNALD but the underlying mechanisms have not been fully unraveled yet. Using omics´ approach, we determined serum and liver lipidome, liver proteome, and liver bile acid profile as well as markers of inflammation and oxidative stress in rats administered either ω-6 PUFA based lipid emulsion (Intralipid) or ω-6/ω-3 PUFA blend (Intralipid/Omegaven) via the enteral or parenteral route. In general, we found that enteral administration of both lipid emulsions has less impact on the liver than the parenteral route. Compared with parenterally administered Intralipid, PN administration of ω-3 PUFA was associated with 1. increased content of eicosapentaenoic (EPA)- and docosahexaenoic (DHA) acids-containing lipid species; 2. higher abundance of CYP4A isoenzymes capable of bioactive lipid synthesis and the increased content of their potential products (oxidized EPA and DHA); 3. downregulation of enzymes involved CYP450 drug metabolism what may represent an adaptive mechanism counteracting the potential negative effects (enhanced ROS production) of PUFA metabolism; 4. normalized anti-oxidative capacity and 5. physiological BAs spectrum. All these findings may contribute to the explanation of ω-3 PUFA protective effects in the context of PN.

Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.

A New Approach for the Diagnosis of Myelodysplastic Syndrome Subtypes Based on Protein Interaction Analysis.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies with a high risk of transformation to acute myeloid leukemia (AML). MDS are associated with posttranslational modifications of proteins and variations in the protein expression levels. In this work, we present a novel interactomic diagnostic method based on both protein array and surface plasmon resonance biosensor technology, which enables monitoring of protein-protein interactions in a label-free manner. In contrast to conventional methods based on the detection of individual biomarkers, our presented method relies on measuring interactions between arrays of selected proteins and patient plasma. We apply this method to plasma samples obtained from MDS and AML patients, as well as healthy donors, and demonstrate that even a small protein array comprising six selected proteins allows the method to discriminate among different MDS subtypes and healthy donors.

Enhanced plasma protein carbonylation in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of pre-leukemic disorders, characterized by ineffective hematopoiesis and the abnormal blood cell development of one or more lineages. Oxidative stress, as an important factor in the carcinogenesis of onco-hematological diseases, is also one of the known factors involved in the pathogenesis of MDS. An increase of reactive oxygen species (ROS) may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. Protein carbonylation caused by ROS is defined as an irreversible post-translational oxidative modification of amino acid side chains, and could play an important role in signaling processes. The detection of protein carbonyl groups is a specific useful marker of oxidative stress. In this study, we examined 32 patients divided into three different subtypes of MDS according to the World Health Organization (WHO) classification criteria as refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts-1,2 (RAEB-1,2). We found significant differences in protein carbonylation between the group of all MDS patients and healthy controls (P=0.0078). Furthermore, carbonylated protein levels were significantly elevated in RARS patients compared to healthy donors (P=0.0013) and to RCMD patients (P=0.0277). We also found a significant difference in the total iron binding capacity (TIBC) between individual subgroups of MDS patients (P=0.0263). Moreover, TIBC was decreased in RARS patients compared to RCMD patients (P=0.0203). TIBC moderately negatively correlated with carbonyl levels (r=-0.5978, P=0.0054) in the MDS patients as a whole. Additionally we observed changes in the carbonylated proteins of RARS patients in comparison with healthy controls and their negative controls. Using tandem mass spectrometry (LC-MS/MS) we identified 27 uniquely carbonylated proteins of RARS patients, which were generated by ROS and could influence the pathophysiology of low-risk MDS. These data indicate that increased protein carbonylation is related with RARS as low-risk MDS subgroup. We suggest that this type of post-translational modification in MDS disease is not "only" a consequence of oxidative stress, but also plays an active role in the pathophysiology and iron metabolism within the RARS subgroup of MDS. Measurement of plasma carbonyl levels and the isolation of carbonylated plasma proteins, followed by their identification, could serve as a potential diagnostic and prognostic tool in MDS.

The effect of potential fall distance on hormonal response in rock climbing.

The aim of this study was to examine the effect of alterations in potential lead fall distance on the hormonal responses of rock climbers. Nine advanced female climbers completed two routes while clipping all (PRO-all) or half (PRO-½) of the fixed points of protection. Venous blood samples were analysed for total catecholamines, noradrenaline (norepinephrine), adrenaline (epinephrine), dopamine, lactate, cortisol and serotonin. Differences between the two conditions pre, immediately post and 15 min post climbing were assessed using a 2 × 3 repeated measures ANOVA. All hormones and blood lactate concentrations increased significantly (P < 0.05) immediately post climb, except for cortisol. Peak cortisol concentrations did not occur until 15 min post ascent. Further, significant interactions between climbing and clipping conditions were found for total catecholamines (890% of basal concentration in PRO-½ vs. 568% in PRO-all), noradrenaline (794% vs. 532%) and dopamine (500% vs. 210%). There were no significant interactions for adrenaline (1920% vs. 1045%), serotonin (150% vs. 127%) or lactate (329% vs. 279%). The study showed a greater catecholamine response with an increase in potential lead fall distance. The most pronounced increases seen in catecholamine concentration were reported for dopamine and noradrenaline.

Two novel mutations in the fibrinogen γ nodule.

INTRODUCTION: Congenital dysfibrinogenemia and hypofibrinogenemia are rare diseases characterized by inherited abnormality in the fibrinogen molecule, resulting in functional defects (dysfibrinogenemia) or low fibrinogen plasma levels (hypofibrinogenemia). MATERIALS AND METHODS: We have described two abnormal fibrinogens - fibrinogen Hranice (γ Phe204Val) and Praha IV (γ Ser313Gly). The carrier of the Hranice mutation was a 40-year-old female with low fibrinogen levels. The carrier of the Praha IV mutation was a 42-year-old man with a history of idiopathic thrombosis, low functional fibrinogen levels, and a prolonged thrombin time. RESULTS: Fibrin polymerization kinetics measurement was normal in both cases (after the addition of either thrombin or reptilase), as well as was fibrinolysis. Scanning electron microscopy and confocal microscopy revealed significantly wider fibers in both cases, when compared with fibers prepared from healthy control samples. Although both cases are situated in the γ-nodule, they manifested differently. While the γ Ser313Gly mutation manifested as dysfibrinogenemia with a thrombotic background, the γ Phe204Val mutation manifested as hypofibrinogenemia without clinical symptoms. The mutation sites of both fibrinogens are in highly conserved regions of the fibrinogen γ chains. γ Ser313 is situated in a class 16:18 ß hairpin and is involved in hydrogen bonding with γ Asp320. γ Phe204 is situated in an inverse γ turn and may be involved in π-π interactions. CONCLUSIONS: Both mutations cause conformational changes in fibrinogen, which lead either to impaired fibrinogen assembly (fibrinogen Hranice) or abnormal fibrinogen function (fibrinogen Praha IV).