Detection of HOCl-driven degradation of the pericardium scaffolds by label-free multiphoton fluorescence lifetime imaging.

Artificial biomaterials can significantly increase the rate of tissue regeneration. However, implantation of scaffolds leads not only to accelerated tissue healing but also to an immune response of the organism, which results in the degradation of the biomaterial. The synergy of the immune response and scaffold degradation processes largely determines the efficiency of tissue regeneration. Still, methods suitable for fast, accurate and non-invasive characterization of the degradation degree of biomaterial are highly demandable. Here we show the possibility of monitoring the degradation of decellularized bovine pericardium scaffolds under conditions mimicking the immune response and oxidation processes using multiphoton tomography combined with fluorescence lifetime imaging (MPT-FLIM). We found that the fluorescence lifetimes of genipin-induced cross-links in collagen and oxidation products of collagen are prominent markers of oxidative degradation of scaffolds. This was verified in model experiments, where the oxidation was induced with hypochlorous acid or by exposure to activated neutrophils. The fluorescence decay parameters also correlated with the changes of micromechanical properties of the scaffolds as assessed using atomic force microscopy (AFM). Our results suggest that FLIM can be used for quantitative assessments of the properties and degradation of the scaffolds essential for the wound healing processes in vivo.

[Biomarkers of system inflammation in local and diffuse peritonitis]. / Markery sistemnogo vospaleniia pri mestnom i rasprostranennom peritonite.

In cases of any acute surgical abdominal disease the progression of purulent inflammation can lead to local or diffuse peritonitis. The indicators of the degree and specificity of the inflammatory response in blood such as cytokine concentration, neutrophil activity, plasma antioxidant capacity (thiols concentration) could be considered as potential predictors of complications. The luminol-dependent chemiluminescence (CL) response of blood activated by the phorbol ester (PMA), and the concentration of cytokines IL-6, IL-8, IL-10, myeloperoxidase (MPO) and thiols in plasma were measured in patients with uncomplicated condition (group 1, n=8), local peritonitis (group 2, n=9) or diffuse peritonitis (group 3, n=9) at admission to surgery (before surgical operation, b/o), immediately after surgical operation (a/o) and a day after surgery (1 day) as well as in healthy volunteers (norm, n=12). In all time-points the cytokines and MPO concentrations measured by ELISA, in group 3 were higher than in healthy volunteers and in patients in groups 1 and 2. Blood CL demonstrated a more than 5-fold increase above the normal values in all patients, and was also higher in group 2 as compared to group 1 (b/o and a/o). Patients in group 3 had shown both maximum and minimum of CL values, which could be a consequence of neutrophil priming or exhaustion ("immune paralysis"), respectively. The same patients' plasma exhibited low thiol concentration (≤30% vs normal values). In patients with fatal outcomes (group 3, n=2) within a day after surgery, either a decrease of the CL to zero values concurrently with elevated IL-8 and IL-6 concentrations and low thiol levels was observed, or CL exceeded normal values more than 20 times with concurrent complete exhaustion of the plasma thiol pool. No clear dependency between the plasma parameters and neutrophil activity was found. Hence a parameter set for prognosis and/or early diagnosis of infectious complications in acute abdominal pathology should include different biomarkers of the inflammatory response: cytokine profile (IL-6, IL-8, IL-10), MPO and neutrophil activity, antioxidant plasma capacity (e.g., total thiols concentration).

The plutonium isotopes and strontium-90 determination in hot particles by characteristic X-rays.

This paper reports the developed non-destructive methods for the plutonium isotopes and strontium-90 content determination in hot particles and other samples. The proposed methods are based on the measurement of the characteristic X-rays accompanying the decay of these radionuclides. For hot particles of NPP accident origin, the proposed method's error limits are 10-15% for hot particles (samples) with activity above 100 Bq and 15-20% for hot particles (samples) with activity less than 100 Bq. For explosive particles, the determination accuracy is 10-15% for activity more than 5 Bq and 20-30% for 0.1-5 Bq activity. The accuracy of the proposed method for determining 90Sr in samples with its specific content of more than 104 Bq/sample is 5%, with ~102 Bq/sample its content is 15-20%. The cost of one sample measurement and the processing time of these methods are significantly reduced compared to traditional studies. The proposed methods are reasonably simple measurement methods and can be carried out even in the field condition. They open up new possibilities for the quick search and study of hot particles and environmental samples.

Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death.

High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these "partial" intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented.

Myeloperoxidase-Induced Oxidation of Albumin and Ceruloplasmin: Role of Tyrosines.

Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.

[Experimental and clinical studies of mechanisms of the anti-aging effects of chemical compounds in Astragalus membranaceus (review).]

Astragalus membranaceus (webbed astragalus) for more than two millennia has been used in traditional Chinese medicine as a means of slowing down the aging process and increasing longevity. The article analyzes the data of experimental and clinical studies of recent years, identifying possible mechanisms of the anti-aging effects of the plant. It is noted that the extract or various groups of chemical compounds of this plant activate telomerase, inhibit the processes of replicative senescence, have antioxidant activity, have a neuroprotective effect, affect age-related macular degeneration, skin photoaging and alopecia.

[Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus]. / Neitrofily kak istochnik faktorov, uvelichivaiushchikh prodolzhitel'nost' fazy vospaleniia ranevogo protsessa u bol'nykh sakharnym diabetom 2-go tipa.

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

SNCA variants and alpha-synuclein level in CD45+ blood cells in Parkinson's disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder. Impaired metabolism of alpha-synuclein (SNCA) and its aggregation are implicated in PD pathogenesis. SNCA has been identified as a highly significant genetic risk loci associated with the sporadic form of PD in across populations in GWAS and replicative studies. In this study we conducted a genetic analysis of five SNCA single nucleotide polymorphisms (SNPs) (rs356219, rs2619364, rs11931074, rs2583988, rs356168) in 458 PD patients and 353 from North-West region of Russia. We also assessed an association of studied SNPs with alpha-synuclein levels in homogeneous cell fraction of CD45+ blood cells in PD patients and controls. An association with PD was shown for SNPs rs356219, rs11931074, rs356168. After correction for covariates the significant association with the disease only for rs11931074 and rs356168 was shown. Alpha-synuclein level in peripheral blood CD45+ cells was significantly increased in PD patients compared to control subjects (р = 0.02). The effect of SNCA rs356219 and rs356168 on CD45+ alpha-synuclein level in PD patients and control groups was shown. At the same tame the increase of CD45+ alpha-synuclein level in PD patients was revealed only in risk allele carriers as for rs356219 and rs356168 SNPs. Therefore, our study was the first that demonstrated the increased level of alpha-synuclein in CD45+ blood cells in PD patients and showed that it could be influenced by SNCA rs356168 and rs356219. In conclusion we confirmed the significance of the SNCA locus in the PD development.

Speciation of actinides in groundwater samples collected near deep nuclear waste repositories.

Actinide binding to colloidal particles of different nature was studied under oxic and anoxic conditions of an underground nuclear waste disposal site using successive micro- and ultrafiltration techniques. According to the actinide redox speciation, under oxic conditions they were present in high oxidation states except for plutonium, for which a significant part was found in the tetravalent state. In case of the anoxic conditions, the share of An (IV) was proportional to the total U(IV) concentration. This indicated formation of intrinsic U(IV) hydroxocolloids, which bound other actinides. Formation of the intrinsic actinide colloids was proven by the secondary ion mass spectrometry (SIMS) with the submicron resolution. In contrast, under the oxic conditions uranium and plutonium were sorbed by natural colloids (amorphous hydrous ferric oxide and Mn oxides).

Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines.

Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme.

[THE SPECIES COMPOSITION OF AGENTS OF COMMUNITY-ACQUIRED INFECTIONS OF URINARY TRACTS AND SENSITIVITY OF MAIN UROPATHOGENS TO ANTIBACTERIAL PREPARATIONS IN MOSCOW IN 2013].

The article presents the results of analysis of spectrum of agents of community-acquired infections of urinary tracts in Moscow The prevalence of resistance these infections to antibiotics applied in treatment of the mentioned pathology are considered.

[Non-convulsive status epilepticus with a phenomenon of transitory amnesia].

This article describes clinical cases of 13 year old boy with two non-convulsive status epilepticus which had transient epileptic amnesia as a clinical implication. Status EEG pattern in form of diffuse epileptic activity "benign epileptiform discharge of childhood" type was registered.

[Quantitative determination of HBsAg in blood sera and hepatitis B virus covalently closed circular DNA in liver tissue as markers of chronic viral hepatitis B activity].

AIM: Quantitative evaluation of HBV covalently closed circular DNA (cccDNA) content in liver tissue of patients with moderately active CHBV course compared with inactive HBsAg carriers as well as establishment of a possible link between HBV cccDNA in liver cells and HBsAg level in blood sera in these groups of patients. MATERIALS AND METHODS: Patients (n = 34) with CHBV diagnosis were examined for levels ofALT, HBsAg (qualitatively and quantitatively), anti-HBcor IgG, anti-HBe IgG, anti-HCV IgG+IgM, anti-HDV IgG+IgM, HBV DNA in qualitative and quantitative variant. Liver biopsy was carried out in all the patients. HBV DNA was determined in liver tissue by Pollicino T. et al. (2004). RESULTS: Based on HBV DNA PCR, the patients were allocated to a group of inactive HBsAg carriers (n = 16) and CHBV (n = 18) of moderate activity. Viral load in CHBV patients had a mean of 540 +/- 230 IU/ml. ALT level in carriers was comparatively lower than in patients with CHBV. HBsAg level in blood of inactive carriers was significantly lower, 940 +/- 259 IU/ml against 2559 +/- 982 IU/ml in patients with CHBV (p < 0.05). The quantity of cccDNA per 1 cell in inactive HBsAg carriers--0.15 +/- 0.14, and in patients group of CHBV with moderate activity--1.71 +/- 1.32 (p = 0.034). CONCLUSION: The method of quantitative determination of HBV cccDNA in liver tissue of patients was worked out. Differences in quantitative content of HBsAg in blood sera of inactive carriers and CHBV patients with moderate activity reflect changes in the extent of hepatocyte infection by HBV.

[Myeloperoxidase-induced biodegradation of single-walled carbon nanotubes is mediated by hypochlorite].

Broad prospects for the use of single-walled carbon nanotubes (SWNTs) in medicine and biotechnology raise the concerns about both their toxicity, and the mechanisms of biodegradation and excretion from the body. SWNTs biodegradation as a result of catalytic activity of myeloperoxidase (MPO) was shown in the isolated MPO system as well as in the suspension of neutrophils [Kagan V.E., et al., 2010]. In the present study we analyzed the ability of different MPO-produced oxidants to participate in the modification and degradation of SWNTs. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that myeloperoxidase, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in the degradation of carbon nanotubes. The biodegradation of SWNTs in the model system can also be caused by free radicals generated as a result of heme degradation and, to a lesser extent, by active oxoferryl intermediates of peroxidases. Our experiments showed that in the presence of blood plasma, peroxidase intermediates or free radical products of heme degradation were unable to initiate biodegradation of carbon nanotubes, only the generation of hypochlorite by MPO can cause the biodegradation of carbon nanotubes in vivo. Titration of SWNTs suspension containing plasma with hypochlorite at high concentrations resulted in the decrease in the optical absorbance of the suspension indicating the degradation of nanotubes. Our results clearly indicate that hypochlorite can serve as a main oxidizing agent which is able to modify and degrade nanotubes in the sites of inflammation and in the phagosomes.

Microdistribution of 241Am in structures of submerged macrophyte Elodea canadensis growing in the Yenisei River.

A submerged macrophyte of the Yenisei River, Elodea canadensis, was used to study the microdistribution of the artificial radionuclide (241)Am among different components of the plant. The total amount of (241)Am added to the experimental system was 1850+/-31 Bq/L. The total amount of (241)Am accumulated by the plants was 182 Bq per sample, or 758,333+/-385 Bq/kg dry mass. It has been found that the major portion of (241)Am accumulated by E. canadensis, up to 85%, was bound to solid components of the cells. It is observed that the microdistribution of (241)Am within different components of the submerged plant E. canadensis was not uniform. (241)Am distribution vary depending on the age of the leaf blades, the state of the cells and morphological features of the plant stem.

[Effect of ischemic preconditioning in ischemic insult by estimating the damage to low-density lipoproteins of rat blood by spectroscopic methods].

It has been shown by optical and spectral methods that ischemic preconditioning in ischemic insult induces a protective action on brain tissues, which is registered by an decrease in the damage to low-density lipoproteins after ischemic insult.

[A study of the effect of ceruloplasmin and lactoferrin on the chlorination activity of leukocytic myeloperoxidase using the chemiluminescence method].

The chlorination activity of free myeloperoxidase and myeloperoxidase bound with ceruloplasmin or with both ceruloplasmin and lactoferrin has been studied by luminal-dependent chemiluminescence. It was shown that the addition of hydrogen peroxide to the "myeloperoxidase + Cl- + luminal" system is accompanied by a fast flash of light emission. In the absence of myeloperoxidase or Cl-, the flash intensity was considerably reduced. The inhibitor of myeloperoxidase NaN3, the HOCl scavengers taurine and methionine, and guaiacol, a substrate for peroxidation cycle of myeloperoxidase, prevented luminescence. These results suggest that the generation of luminescence was due to the halogenating activity of myeloperoxidase, and hence, the flash light sum may serve as a measure of chlorination activity of myeloperoxidase. The activity of myeloperoxidase was suppressed by ceruloplasmin. Lactoferrin exhibited no significant influence on the myeloperoxidase activity, nor did it prevent the inhibitory effect of ceruloplasmin when they both were combined with myeloperoxidase. These data were confirmed using alternative approaches for evaluating the myeloperoxidase activity, namely, the assessment of peroxidation activity and the taurine chlorination assay. It is noteworthy that the inhibitory effect of ceruloplasmin on chlorination and peroxidation activities of myeloperoxidase is seen with the latter, traditional approaches only if ceruloplasmin is present in a large excess relative to myeloperoxidase, whereas the chemiluminescence method allows the detection of the inhibitory effect of ceruloplasmin using lower proportions of the protein with respect to myeloperoxidase, which are close to the stoichiometry of the myeloperoxidase/ceruloplasmin and the myeloperoxidase'ceruloplasmin'lactoferrin complexes.

[Determination of antibiotics using luminescent Escherichia coli and serum].

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.

Role of myeloperoxidase-mediated modification of human blood lipoproteins in atherosclerosis development.

The mechanism of interaction of hypochlorite and hypobromite formed in myeloperoxidase catalysis with lipids of human blood low-density lipoprotein is described. Both agents react with unsaturated lipids via two mechanisms: molecular (with the formation of mainly chloro- or bromohydrins and lysophospholipids) and free-radical (paralleled by lipid peroxidation). These reactions modify physicochemical properties of low-density lipoproteins and disorder their lipid-transporting function thus initiating early stages of atherosclerosis development.

pH-dependent regulation of myeloperoxidase activity.

The balance between peroxidase and chlorinating activities of myeloperoxidase (MPO) is very important for the enhancement of antimicrobial action and prevention of damage caused by hypochlorite. In the present paper, the peroxidase and chlorinating activities have been studied at various pH values. The possibility of using neutrophil protein solution for the evaluation of MPO activity has been demonstrated. It is shown that at neutral pH MPO had higher affinity to peroxidase substrate guaiacol: at pH 7.4, chloride ions did not compete with guaiacol up to the concentration of 150 mM. At acidic pH, chlorinating activity of MPO dominates: only hypochlorite production can be detected at equal chloride and guaiacol concentrations of 15 mM. However, horseradish peroxidase does not exhibit any difference in activity in the presence of chloride ions even at acidic pH values. It was demonstrated by MALDI-TOF mass-spectrometry that the amount of hypochlorite produced is sufficient to modify phospholipids (with formation of Cl- and Br-hydrins and lyso-derivatives) only at acidic pH (5.0). Thus, in the presence of phenolic peroxidase substrate, MPO chlorinating activity can be displayed at acidic pH only. It can lead to elimination of hypochlorite production in normal tissues at neutral pH (7.4) and its enhancement in phagosomes where the pH range is 4.7-6.0.