Blood compatibility evaluations of two-dimensional Ti3C2Txnanosheets.

Two-dimensional nanomaterial Ti3C2Txis a novel biomaterial used for medical apparatus. For its application, biosafety serves as a prerequisite for their usein vivo. So far, no research has systematically reported how Ti3C2Txinteracts with various components in the blood. In this work, we evaluated the hemocompatibility of Ti3C2Txnanosheets which we prepared by HF etching. Effects of the concentration and size of Ti3C2Txon the morphology and hemolysis rate of human red blood cells (RBCs), the structure and conformation of plasma proteins, the complement activation, as well asin vitroblood coagulation were studied. In general, Ti3C2Txtakes on good blood compatibility, but in the case of high concentration (>30 µg ml-1) and 'small size' (about 100 nm), it led to the rupture of RBCs membrane and a higher rate of hemolysis. Meanwhile, platelets and complement were inclined to be activated with the increased concentration, accompanying the changed configuration of plasma proteins dependent on concentration. Surprisingly, the presence of Ti3C2Txdid not significantly disrupt the coagulation.In vitrocell culture, the results prove that when the Ti3C2Txconcentration is as high as 60 µg ml-1and still has good biological safety. By establishing a fuzzy mathematical model, it was proved that the hemocompatibility of Ti3C2Txis more concentration-dependent than size-dependent, and the hemolysis rate is the most sensitive to the size and concentration of the Ti3C2Tx. These findings provide insight into the potential use of Ti3C2Txas biofriendly nanocontainers for biomaterialsin vivo.

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Protective effects of chitosan and chitosan oligosaccharide against oxidative damage in liver tissue of rats with fluorine poisoning.

Fluorine toxicity has negative effects on soft tissue besides skeletal and dental tissues. In the present study, we have investigated the protective effect of chitosan (CS) and chitosan oligosaccharide (COS) on liver tissue of fluorine-intoxicated rats taking the antioxidant characteristics of chitosan and its derivatives into consideration. In this study, 42 male Wistar albino rats were randomly selected to determine the control and experimental fluorosis groups. Our study lasted for 12 weeks. As a consequence of the study, MDA significantly increased in the liver tissue of NaF group while some antioxidant values significantly decreased. It was detected that serum AST and LDH levels increased significantly while ALB and TP values significantly decreased in NaF group. The degenerations were identified in the liver histopathology of all fluoride-treated groups. We have concluded according to the results that chitosan oligosaccharide can be more effective compared with chitosan.

In vitro and in vivo blood compatibility of concentrated polymer brushes.

Concentrated polymer brushes (CPBs) and semi-dilute polymer brushes (SDPBs) of poly(2-hydroxyethyl methacrylate), poly(2-hydroxyethyl acrylate), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA) and poly(2-methoxyetyl acrylate) were prepared on silica particles and silicon wafers by surface-initiated atom transfer radical polymerization (SI-ATRP). In order to evaluate in vitro blood compatibility, plasma protein adsorption on the brushes was quantified with a BCA protein assay, and the adsorbed proteins on the brushes were identified using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). All four CPBs displayed much less protein adsorption than their corresponding SDPBs. Interestingly, the number and type of identified proteins differed on the brushes. Platelet adhesion was then examined on the brushes, whereby CPBs suppressed platelet adhesion to a greater extent than the corresponding SDPBs, although platelet activation was observed on all surfaces. As a result, the CPBs of PPEGMA prevented platelet adhesion the most. After screening the polymers by in vitro evaluation, CPBs of PPEGMA were then grafted on a catheter by SI-ATRP. The catheter with the CPBs was implanted into the jugular vein of a rabbit. The in vivo assessment after three weeks of implantation confirmed that the CPBs caused little coagulation or inflammation, whereas the pristine catheter exhibited inflammation and encapsulation.

The effect of pathogen inactivation on cryoprecipitate: a functional and quantitative evaluation.

BACKGROUND: As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. MATERIALS AND METHODS: Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. RESULTS: Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. DISCUSSION: Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.

Triphala Ameliorates Nephropathy via Inhibition of TGF-ß1 and Oxidative Stress in Diabetic Rats.

INTRODUCTION: Advanced glycation end products, oxidative stress, and TGF-ß expression play a crucial role in pathophysiology of diabetic nephropathy. Inhibition of oxidative stress and TGF-ß expression by natural traditional medicines may give an economic and safe alternative treatment option. Triphala churna, a traditional medicine, has been proved to have potent antioxidant activity, and individual components of it have shown significant antidiabetic activity. Hence, the present study was designed to study the effect of Triphala churna in diabetic nephropathy in rats. METHODS: Diabetes was induced in rats by administration of streptozotocin (55 mg/kg i.p.). Four weeks after induction of diabetes, the animals were treated with Triphala churna at the doses of 250, 500, and 1,000 mg/kg for next 4 weeks. Various biochemical and urine parameters such as glucose, creatinine, blood urea nitrogen (BUN), total protein, and albumin were assessed at the end of study. Creatinine clearance, BUN clearance, and glomerular filtration rate were determined. Oxidative stress parameters such as malondialdehyde, catalase, reduced glutathione, and superoxide dismutase were determined in kidney tissues. TGF-ß1 expression was measured with ELISA, immunohistochemistry, and western blot techniques. Histopathology study was carried out with haemotoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining to determine histological changes. RESULTS: Treatment with Triphala churna significantly improved urine parameters. Triphala churna treatment also improved plasma proteins, albumin, creatinine, and BUN levels. The oxidative stress was reduced in the kidney with the treatment of Triphala churna. Histopathological studies revealed that Triphala churna reduced kidney damage. Immunohistochemistry, ELISA, and western blotting study revealed that treatment with Triphala decreased the expression of TGF-ß in kidney tissues. CONCLUSION: From the results, it can be concluded that Triphala churna has a significant nephroprotective effect because of its capability of inhibiting oxidative stress and TGF-ß in diabetes.

Mechanisms of Sodium-Glucose Cotransporter 2 Inhibition: Insights From Large-Scale Proteomics.

OBJECTIVE: To assess the effects of empagliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, on broad biological systems through proteomics. RESEARCH DESIGN AND METHODS: Aptamer-based proteomics was used to quantify 3,713 proteins in 144 paired plasma samples obtained from 72 participants across the spectrum of glucose tolerance before and after 4 weeks of empagliflozin 25 mg/day. The biology of the plasma proteins significantly changed by empagliflozin (at false discovery rate-corrected P < 0.05) was discerned through Ingenuity Pathway Analysis. RESULTS: Empagliflozin significantly affected levels of 43 proteins, 6 related to cardiomyocyte function (fatty acid-binding protein 3 and 4 [FABPA], neurotrophic receptor tyrosine kinase, renin, thrombospondin 4, and leptin receptor), 5 to iron handling (ferritin heavy chain 1, transferrin receptor protein 1, neogenin, growth differentiation factor 2 [GDF2], and ß2-microglobulin), and 1 to sphingosine/ceramide metabolism (neutral ceramidase), a known pathway of cardiovascular disease. Among the protein changes achieving the strongest statistical significance, insulin-like binding factor protein-1 (IGFBP-1), transgelin-2, FABPA, GDF15, and sulphydryl oxidase 2 precursor were increased, while ferritin, thrombospondin 3, and Rearranged during Transfection (RET) were decreased by empagliflozin administration. CONCLUSIONS: SGLT2 inhibition is associated, directly or indirectly, with multiple biological effects, including changes in markers of cardiomyocyte contraction/relaxation, iron handling, and other metabolic and renal targets. The most significant differences were detected in protein species (GDF15, ferritin, IGFBP-1, and FABP) potentially related to the clinical and metabolic changes that were actually measured in the same patients. These novel results may inform further studies using targeted proteomics and a prospective design.

Concentration of dipotassium ethylenediaminetetraacetic acid but not lithium heparin affects total protein determination in equine synovial fluid.

BACKGROUND: Refractometric determination of total protein (TP) in synovial fluid (SF) is commonly used for diagnosis and monitoring of synovial sepsis in horses. Previous studies have shown that elevated concentrations of certain anticoagulants may overestimate refractometric determination of TP concentration. OBJECTIVES: The aim of the study was to evaluate the effect of different concentrations of dipotassium EDTA (K2EDTA) and lithium heparin (LH) on TP determination by using a hand-held refractometer in equine synovial fluid. STUDY DESIGN: Cross-section observational study. METHODS: Thirty samples of synovial fluid obtained from 22 horses with different synovial conditions were collected. Synovial fluid samples were separated into different aliquots and placed in commercially available collection tubes containing K2EDTA or LH at four different concentrations (1.76, 3.52, 7.04 and 17.6 mg/ml for K2EDTA; 16, 32, 64 and 160 IU/ml for LH) . Refractometric TP determination was performed on untreated and K2EDTA and LH aliquots with a hand-held refractometer and by spectophotometric Biuret method as the gold standard. RESULTS: Refractometric TP determination was overestimated in SF samples containing 10 times the recommended K2EDTA concentrations. Lower concentrations of K2EDTA and LH concentrations did not affect refractometric TP determinations. MAIN LIMITATIONS: Limited number of samples mostly obtained from large synovial structures. CONCLUSION: To avoid incorrect TP determination, the use of LH containing collection tubes may be an appropriate alternative when the SF volume available is not enough to fill the K2EDTA collection tube.

Purification and Assays of Tachylectin-5.

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 µg/mL for tachylectin-5A and 0.077-0.27 µg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 µg/mL for tachylectin-5A and 0.05-0.11 µg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 µg/mL for tachylectin-5A and 15.1-26.8 µg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Graphene Surfaces Interaction with Proteins, Bacteria, Mammalian Cells, and Blood Constituents: The Impact of Graphene Platelet Oxidation and Thickness.

Graphene-based materials (GBMs) have been increasingly explored for biomedical applications. However, interaction between GBMs-integrating surfaces and bacteria, mammalian cells, and blood components, that is, the major biological systems in our body, is still poorly understood. In this study, we systematically explore the features of GBMs that most strongly impact the interactions of GBMs films with plasma proteins and biological systems. Films produced by vacuum filtration of GBMs with different oxidation degree and thickness depict different surface features: graphene oxide (GO) and few-layer GO (FLGO) films are more oxidized, smoother, and hydrophilic, while reduced GO (rGO) and few-layer graphene (FLG) are less or nonoxidized, rougher, and more hydrophobic. All films promote glutathione oxidation, although in a lower extent by rGO, indicating their potential to induce oxidative stress in biological systems. Human plasma proteins, which mediate most of the biological interactions, adsorb less to oxidized films than to rGO and FLG. Similarly, clinically relevant bacteria, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, adhere less to GO and FLGO films, while rGO and FLG favor bacterial adhesion and viability. Surface features caused by the oxidation degree and thickness of the GBMs powders within the films have less influence toward human foreskin fibroblasts; all materials allow cell adhesion, proliferation and viability up to 14 days, despite less on rGO surfaces. Blood cells adhere to all films, with higher numbers in less or nonoxidized surfaces, despite none having caused hemolysis (<5%). Unlike thickness, oxidation degree of GBMs platelets strongly impact surface morphology/topography/chemistry of the films, consequently affecting protein adsorption and thus bacteria, fibroblasts and blood cells response. Overall, this study provides useful guidelines regarding the choice of the GBMs to use in the development of surfaces for an envisioned application. Oxidized materials appear as the most promising for biomedical applications that require low bacterial adhesion without being cytotoxic to mammalian cells.

Adrenergic alpha-2 receptor antagonists cease augmented oxidation of plasma proteins and anxiety of rats caused by chronic noise exposure.

BACKGROUND: Noise is one of the environmental factors, which is considered as a powerful stressor for the organism. Generally, the acoustic stress affects the behavior and physiological state of humans and animals. AIMS: The goal of this study is to investigate the relationship between chronic noise exposure and the effects of adrenergic alpha-2 receptor antagonists, beditin and mesedin, on the anxiety and oxidation of plasma proteins and fibrinogen in rats. METHODS: The experiments were carried out on non-linear albino male rats, divided into four groups (six animals in each): 1. Healthy controls 2. Exposed to noise of a level 91 dB(A), eight hours daily, during 7, 30 and 60 days; 3. Injected with 2 mg/kg of beditin (2-(2-amino-4-thiazolyl)-1,4-benzodioxane hydrochloride)); 4. Injected with 10 mg/kg mesedin (2-(2-methyl-amino-thiozolyl)-1,4-benzodioxane hydrochloride). For evaluating the cognitive impairment, the Any-maze test was applied. The level of carbonylation of proteins was assessed by reaction with 2,4-dinitrophenylhydrazine, spectrophotometrically. RESULTS: Chronic noise decreased locomotor activity and increased anxiety and oxidation of plasma protein and fibrinogen. Intensity of these changes were dependent on the duration of noise exposure. CONCLUSION: The Alpha 2 adrenoblockers alleviate oxidative modification of plasma proteins and reduce the cognitive impairment caused by chronic exposure to noise.

Irreversible oxidations of platelet proteins after riboflavin-UVB pathogen inactivation.

Pathogen inactivation technologies are known to alter in vitro phenotype and functional properties of platelets. Because pathogen inactivation generates reactive oxygen species, oxidative stress is considered as one of the plausible cause at the origin of the platelet storage lesion acceleration after treatment. To date proteomics has been used to document the protein variations to picture out the impact. Here, platelet concentrates were prepared from buffy-coats in Intersol additive solution, leukoreduced and pathogen inactivated using a riboflavin/UVB treatment. At day 2 of storage the platelet proteomes of control (untreated) and treated platelet concentrates were investigated against the site specific oxidation by liquid chromatography coupled to tandem mass spectrometry in a shotgun experiment. The shotgun approach detected 9350 peptides (and 2534 proteins) of which 1714 were oxidized. Eighteen peptides were found exclusively oxidized in treated platelets whereas 3 peptides were only found oxidized in control. The present data evidenced an interference with several proteins involved in platelet aggregation and platelet shape change (such as talin and vinculin).

Plasma metabolomics in tuberculosis patients with and without concurrent type 2 diabetes at diagnosis and during antibiotic treatment.

Tuberculosis (TB) and type 2 diabetes mellitus (DM), a major TB risk factor, are both accompanied by marked alterations in metabolic processes. Dissecting the specific metabolic changes induced by disease through metabolomics has shown potential to improve our understanding of relevant pathophysiological mechanisms of disease, which could lead to improved treatment. Targeted tandem liquid chromatography-mass spectrometry (LC-MS/MS) was used to compare amine and acylcarnitine levels in plasma samples of patients with TB or TB-DM from Indonesia at time of diagnosis and during antibiotic treatment. Partial least squares discrimination analysis (PLS-DA) showed good separation of patient groups. Amine levels were strongly altered in both disease groups compared to healthy controls, including low concentrations of citrulline and ornithine. Several amino acid ratios discriminated TB from controls (phenylalanine/histidine; citrulline/arginine; kynurenine/tryptophan), possibly reflecting changes in indoleamine-pyrrole 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) activity. Choline, glycine, serine, threonine and homoserine levels were lower in TB-DM compared to TB, and, in contrast to other analytes, did not normalize to healthy control levels during antibiotic treatment. Our results not only provide important validation of previous studies but also identify novel biomarkers, and significantly enhance our understanding of metabolic changes in human TB and TB-DM.

Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress.

INTRODUCTION: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. METHODS: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. RESULTS: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. CONCLUSION: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.

Potential target-related proteins in rabbit platelets treated with active monomers dehydrocorydaline and canadine from Rhizoma corydalis.

BACKGROUND: Dehydrocorydaline (DHC) and canadine (THB) are two active alkaloid compounds in Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) (Rhizoma Corydalis). DHC and THC were previously shown to exert anti-platelet aggregation effect dose-dependently, but their exact mechanisms had not yet been addressed. Therefore, it is essential to study the mechanisms of DHC and THB affecting on platelet's function. PURPOSE: To investigate the anti-platelet effects and corresponding signal cascades of DHC and THB on platelet aggregation. METHODS: Firstly, in vitro anti-platelet aggregation of DHC and THB induced by different agonists including thrombin (THR), adenosine diphosphate (ADP) and arachidonic acid (AA) were determined through turbidimetry method. Then the possible target-related platelet proteins after treated with DHC/THB were separated and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS analysis, respectively. Finally, the signal cascades network induced by DHC/THB were predicted through functional analysis of these proteins along with the determination of platelet DAG concentration. RESULTS: The platelet aggregation stimulated by THR, ADP and AA were inhibited by DHC and THB dose-dependently to a certain degree. Meanwhile, DHC and THB had the strongest effect on ADP- and THR-induced platelet aggregation respectively. In addition, treatment of these two compounds caused regulations of about sixty proteins in platelet, including cytoskeleton proteins, cell signaling proteins, proteins related to material energy metabolism, etc. CONCLUSIONS: Using proteomic analysis combined with platelet aggregation test and ELISA, this study was successful in exploring the possible mechanisms of DHC/THB on platelet aggregation. DHC might inhibit platelet aggregation by a mechanism involving the ADP receptors P2Y1 and P2Y12, and the effect of THB on platelet function may be related to its binding to THR receptor PAR1 for mediated Gi signaling pathway. These results provide fundamental information for the anti-thrombotic effect of RC.

Effects of hetastarch 130/0.4 on plasma osmolality, colloid osmotic pressure and total protein in horses anaesthetised for elective surgical procedures.

Effects of lactated Ringer's solution (LRS) and hetastarch 130/0.4 (HES) on colloid osmotic pressure (COP), plasma osmolality (OSM) and total protein (TP) were investigated in 18 inhalational-anaesthetised healthy horses. Horses received 4-6 ml/kg LRS (LRS; n=9) or HES (HES; n=9) from anaesthesia induction through 60 min, after which all were administered LRS. COP, TP and OSM were measured before premedication (baseline), postinduction and 30 (n=18), 60 (n=18), 90 (n=18) and 120 (n=12) minutes. Baseline COP, OSM and TP were not different between groups. TP decreased in both groups at all time points after induction. OSM increased from baseline in HES at 30, 60, 90 and 120 minutes. COP decreased at 30-120 minutes in LRS, and at 90 and 120 minutes in HES. Mean COP was higher in HES than LRS at 30 (18.8±0.5 vs 16.3±0.4 mmHg (P=0.001)), 60 (19.1±0.5 vs 15.9±0.4 mmHg (P<0.0001)) and 90 (17.4±0.5 vs 15.4±0.5 mmHg (P=0.005)) minutes. Sixty minutes of HES infusion increases OSM and transiently maintains COP compared with an equal volume of LRS in anaesthetised horses.

Proteome analysis of the potential serum biomarkers for chronic benzene poisoning.

The aim of our study is to seek novel specific biomarkers which could provide clues to the mechanism of chronic benzene poisoning (CBP) and might also be used as specific markers for early detection and diagnosis. In this study, a comparative serological proteome analysis between normal controls and CBP patients at three different levels of poisoning were performed via a 2D-DIGE and MALDI-TOF-MS. As the result a total of 10 proteins were found significantly altered between the normal and the mild, moderate and severe poisoning. The identified differentially expressed proteins were classified according to their molecular functions, biological processes, and protein classes, and three important serum proteins among them, apolipoproteinA-1, alpha-1-antitrypsin and complement C3, were further confirmed by immune turbidimetric analysis for their significant up-regulation in the CBP patients. Our findings suggest that these differential proteins may help elucidate the mechanism of CBP and provide potential biomarkers for diagnosis.

Ex vivo binding of the immunosuppressant mycophenolic acid to dog and cat plasma proteins and the effect of co-incubated dexamethasone and prednisolone.

Mycophenolic acid (MPA) has been shown to be promising for the treatment of autoimmune diseases in dogs and cats. In humans, MPA is highly bound to plasma proteins (~97%). It has been recommended to monitor free drug plasma concentrations because the free MPA correlates with its immunosuppressive effect. However, it is unknown if MPA is highly bound to plasma proteins in dogs and cats. The objectives of this study were to determine the extent of plasma protein binding of MPA and evaluate the effect of prednisolone and dexamethasone on the extent of protein binding of MPA in dogs and cats. The extent of plasma protein binding of MPA was determined in plasma collected from clinically healthy adult cats (n = 13) and dogs (n = 14) by combining high-throughput dialysis and ultra-high-liquid chromatography. This study reveals that MPA is highly bound to plasma proteins (>90%) in dogs and cats, mean extent of binding of MPA at 15 µg/ml to plasma proteins being 96% (range, 95%-97%) and 92% (range, 90%-93%) for dogs and cats, respectively. In dog plasma, MPA is primarily bound to albumin. In vitro, prednisolone increased the unbound MPA in dogs (p < .01) but not in cats (p = .07) while dexamethasone had no effect on MPA plasma binding in either species (p > .05). Results of this study provide valuable information for designing future pharmacokinetic and pharmacodynamic studies and also therapeutic monitoring programs for dogs and cats.

Mouse serum exosomal proteomic signature in response to asbestos exposure.

Asbestos-induced diseases like fibrosis and mesothelioma are very aggressive, without any treatment options. These diseases are diagnosed only at the terminal stages due to lack of early stage biomarkers. The recent discovery of exosomes as circulating biomarkers led us to look for exosomal biomarkers of asbestos exposure in mouse blood. In our model, mice were exposed to asbestos as a single bolus dose by oropharyngeal aspiration. Fifty-six days later blood was collected, exosomes were isolated from plasma and characterized and subjected to proteomic analysis using Tandem Mass Tag labeling. We identified many proteins, some of which were more abundant in asbestos exposed mouse serum exosomes, and three selected proteins were validated by immunoblotting. Our study is the first to show that serum exosomal proteomic signatures can reveal some important proteins relevant to asbestos exposure that have the potential to be validated as candidate biomarkers. We hope to extrapolate the positive findings of this study to humans in future studies.

Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality.

BACKGROUND: Photodynamic treatment using methylene blue (MB) and visible light is in routine use for pathogen inactivation of human plasma in different countries. Ambient and product temperature conditions for human plasma during production may vary between production sites. The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. METHODS: Plasma units equilibrated to 5 ± 2°C, room temperature (22 ± 2°C) or 30 ± 2°C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. RESULTS: Reduced solubility of the MB pill was observed at 5 ± 2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30 ± 2°C. Inactivation of suid herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5 ± 2°C than at higher temperatures. MB/light treatment affected clotting times and the activity of almost all investigated plasma proteins. Factor VIII (-17·7 ± 8·3%, 22 ± 2°C) and fibrinogen (-14·4 ± 16·4%, 22 ± 2°C) showed the highest decreases in activity. Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. CONCLUSIONS: Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended.