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Molecular characterization of Dobrava-Belgrade hantavirus in Serbia, 2007-2011
Get PDFBackground: Hantaviruses are etiological agents of emerging zoonotic diseases worldwide, including hemorrhagic fever with renal syndrome (HFRS). A number of hantavirus species is known to be present in Europe. In Serbia, existing data on hantavirus presence and prevalence rely in serological findings. In this study, molecular analysis was performed in order to characterize HFRS causing hantaviruses in Serbia. Methods: Sixty four serum samples of HFRS cases, previously found seropositive to anti-hantaviral anti-bodies, were included in the study. Partial hantaviral L and S segments were PCR amplified producing 390nt and 598nt amplicons, respectively, in parallel with human beta-actin mRNA as external reverse transcription positive control. Hantavirus specific PCR products were DNA sequenced in both direction and the obtained sequences phylogenetically confirmed and analyzed. Results: PCR detection of hantavirus L and S genome segments was positive in 18/64 and 11/64 tested samples, respectively. Positive PCR results involved samples obtained from different locations, mostly from central and southern parts of Serbia. All the obtained sequences were identified as Dobrava-Belgrade virus (DOBV). In the phylogenetic analysis sequences from Serbia tended to cluster in distinctive, geographically related clusters. Conclusions: Our findings indicate DOBV as the main HFRS causing hantavirus in Serbia, the site of its initial isolation. (C) 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Specificity of IgA isotypes is defined by the location and the nature of bacterial antigenic determinants
No full textThe breakthrough in microbiota analysis calls for the thorough analysis of the steadystate antibody reactivity to different microorganisms. The location of the antibodies
plays a key role in determining host-microorganism relationship. Here we compared
the level of specificity of serum and salivary IgA antibodies towards different bacterial
antigens. Enzyme-linked immunosorbent assays targeting four designated antigens –
whole Escherichia coli cells - urinary infection clinical isolate, whole Lactobacillus
rhamnosus LA68 cells, Escherichia coli O55:B5 lipopolysaccharide (LPS) and
Staphylococcus aureus peptidoglycan (PGN) were performed. The tested population
comprised 14 healthy adults with serum and saliva samples collected at the same time.
Both total IgA and IgA subclasses were analyzed and compared. The selected dilutions
of serum and saliva used were such that there were effectively 10 x more serum
antibodies than salivary ones in the assay. Notwithstanding, extremely high correlations
in salivary IgA were obtained between very distant bacterial species such as L.
rhamnosus and E. coli, as well as towards isolated components of microorganisms (r =
0.7-0.96), implying high cross-reactivity and/or low specificity. For serum IgA, the
reactivity was profoundly different. Serum IgA differentiates G + from Gmicroorganisms, i.e. serum IgA towards E. coli correlated best with serum IgA towards
LPS (0.86), and serum IgA against L. rhamnosus correlated best with the anti-PGN
IgA2 response (0.88).
Serum IgA response against E. coli cells consisted primarily of IgA1 response, whereas
serum response to L. rhamnosus cells consisted primarily of IgA2 response. For
isolated components, serum IgA against LPS was guided by IgA2 response and towards
PNG by IgA1 response. The explanation of this phenomenon exceeds the capacity of
this study and confirms the delicacy, specificity and orchestration of the immune
system
Fluoroquinolone-resistant Achromobacter xylosoxidans clinical isolates from Serbia: high prevalence of the aac-(6)-Ib-cr gene among resistant isolates
No full textThe aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H+ conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6)-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6)-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6)-Ib-cr gene present a platform for emergence of more resistant strains
Comparative study of raw and thermally treated peanut major allergen post- translational modifications (PTMs)
Get PDFIntroduction. Peanut allergy affects a large portion of world population causing reactions rangingfrom mild to severe. Major peanut allergen IgE epitopes are well characterized but little is knownabout their post-translational modifications (PTM) and how they are affected by thermaltreatment. PTM profile may differ between raw and thermally treated peanut, which could affectits allergic potential depending on type, size and position of modifications.Objective. Our aim was to analyse and compare PTM profiles of 4 major peanut allergens - Ara h 1,Ara h 2, Ara h 3 and Ara h 6, as well as their amounts in raw and roasted samples using bottom-upproteomics methods.Methodology. Full peanut protein extracts (both thermally treated and non-treated) were digestedin gel and in solution, and analysed by a Top10 nLC-MS/MS method by LTQ Orbitrap XL (ThermoFisher Scientific Inc., Germany). Within the extracts major allergens - Ara h 1, Ara h 2, Ara h 3 andAra h 6 were identified, label free quantified (LFQ) and searched for PTMs by Peaks X software(Bioinformatics solutions Inc.I, Canada). Epitope sequences were acquired from the ImmuneEpitope Database (IEDB www.iedb.org).Main findings. LFQ results show that there is no significant change in the amountsof any of thestudied allergens between raw and roasted extracts.Out of the 4 allergens Ara h 6 is modified in thehighest portion, with respect to the protein size: 15% and 12% of its positions are modified in rawand roasted sample, respectively. Total of 21 modifications were quantified between the twopreparations, with oxidation (M), methylation (K,R) and dethiomethylation affecting the largestnumber of peptides.Conclusions. Peanut allergen epitopes are indeed carriers of PTMs that differ in pattern andquantity between treated and non-treated extracts. The in silico discovered PTMs could affectprotein digestibility and allergenicity. Further investigation is necessary in order to fully understandthe impact protein modifications could have on their allergenic potential
Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed
Get PDFThe aim of this research was to improve nutritive value of fishmeal-based feed by lactobacilli in order to achieve satisfactory nutrient availability needed to support fish development. Feed was solid-state treated at a laboratory scale with the combination of Lactobacillus paracaseisubsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 in different experimental settings, which included the variation of strain ratio, total lactobacilli concentration, percentage of moisture and duration of incubation. Short peptides, soluble proteins, phospho-, neutral and unsaturated lipids were quantified. Differences among treated and control feeds were evaluated by Student t-test, while Gaussian process regression (GPR) modeling was employed to simulate the incubation process and define the optimal treatment combination in the context of overall feed nutritional profile. Treatment duration was shown to be the critical determinant of final outcome, either as single factor or via interaction with strain ratio. Optimal nutrient balance was achieved with 12 h incubation period, 260% moisture, 75:25 and 50:50 BGHN14:BGT10 ratios and 200 mg of lactobacilli per g of dry feed. This study should serve as the basis for large-scale tests which would simulate on-farm production of both fishmeal-based and unconventional, lower cost aquafeed with added value
In-depth quantitative profiling of post-translational modifications of Timothy grass pollen allergome in relation to environmental oxidative stress
Get PDFAn association between pollution (e.g., from traffic emissions) and the increased prevalence of respiratory allergies has been observed. Field-realistic exposure studies provide the most relevant assessment of the effects of the intensity and diversity of urban and industrial contamination on pollen structure and allergenicity. The significance of in-depth post-translational modification (PTM) studies of pollen proteomes, when compared with studies on other aspects of pollution and altered pollen allergenicity, has not yet been determined; hence, little progress has been made within this field. We undertook a comprehensive comparative analysis of multiple polluted and environmentally preserved Phleum pratense (Timothy grass) pollen samples using scanning electron microscopy, in-depth PTM profiling, determination of organic and inorganic pollutants, analysis of the release of sub-pollen particles and phenols/proteins, and analysis of proteome expression using high resolution tandem mass spectrometry. In addition, we used quantitative enzyme-linked immunosorbent assays (ELISA) and immunoglobulin E (IgE) immunoblotting. An increased phenolic content and release of sub-pollen particles was found in pollen samples from the polluted area, including a significantly higher content of mercury, cadmium, and manganese, with irregular long spines on pollen grain surface structures. Antioxidative defense-related enzymes were significantly upregulated and seven oxidative PTMs were significantly increased (methionine, histidine, lysine, and proline oxidation; tyrosine glycosylation, lysine 4-hydroxy-2-nonenal adduct, and lysine carbamylation) in pollen exposed to the chemical plant and road traffic pollution sources. Oxidative modifications affected several Timothy pollen allergens; Phl p 6, in particular, exhibited several different oxidative modifications. The expression of Phl p 6, 12, and 13 allergens were downregulated in polluted pollen, and IgE binding to pollen extract was substantially lower in the 18 patients studied, as measured by quantitative ELISA. Quantitative, unrestricted, and detailed PTM searches using an enrichment-free approach pointed to modification of Timothy pollen allergens and suggested that heavy metals are primarily responsible for oxidative stress effects observed in pollen proteins
Predictors of health related-quality of life among elderly with disabilities
No full textAim There have been no previous studies of health-related quality of life (HRQOL) among adults aged 65 years and older with disabilities in Serbia. The aim of study was to identify predictors of HRQOL in the context of sociodemographic characteristics, disability aetiology, comorbid diseases, indices of comorbidities, realization of social rights, and domestic violence. Methods The cross-sectional study involved 275 consecutive elderly outpatients with disabilities. They were recruited by general practitioners at Primary Health Centers in Belgrade, Serbia, from March to May 2015. Data were acquired through face-to-face interviews and general practitioners' charts, while the generic 36-item Short Form Health Survey was used to assess HRQOL. In statistical analysis, Student's t-test, Z-test, Spearman's correlation test, and both univariate and multivariate linear regression were performed. Results Multivariate analysis revealed that a higher Functional Comorbidity Index (beta = -0.194, P lt 0.01) and not asking for realization of right to assistance and care financial benefits (beta = -0.142, P lt 0.05) were predictors of a lower scores Mental Composite Score (F = 9.262; P lt 0.001). Not asking for realization of right to assistance and care financial benefits (beta = -0.187, P lt 0.01), congestive heart failure (beta = -0.123, P lt 0.05), and stroke (beta = -0.120, P lt 0.05) were predictors of a lower Physical Composite Score (F = 7.169, P lt 0.001). Conclusions This study provides valuable data for better understanding the underlying factors associated with the HRQOL of elderly persons with disabilities, and currently, these are the only available data of their kind in Serbia. National authorities could identify predictors of HRQOL as a basic starting point for improving the social welfare and health-care systems. Better prevention and management of clinical factors, increased access to social services, and enhanced delivery of social services will improve the ageing process and HRQOL of this vulnerable population
Sexual dimorphism in rat thymic involution: a correlation with thymic oxidative status and inflammation
No full textThe study investigated mechanisms underlying sex differences in thymic involution in Dark Agouti rats. Adverse effects of aging on thymus were more pronounced in males than in females. Thymi from old males exhibited more prominent: (i) fibro-adipose degeneration which correlated with greater intensity of thymic oxidative stress and enhanced thymic TGF- and IL-6 expression and (ii) decline in thymopoiesis, as suggested by the number of the most mature CD4+CD8-/CD4-CD8+ single positive (SP) TCRhigh thymocytes. The greater accumulation of adipose tissue in old male thymus was linked with greater age-related increase in thymic expression of PPAR and STAT3, a transcription factor regulating the expression of PPAR downstream genes, in male than in female rats. In aged thymi of both sexes the early CD4-CD8- double negative (DN) stage of thymocyte development was affected, so relative accumulation of the least mature CD45RC+CD2- cells followed by decreased frequency of their DN and CD4+CD8+ double positive (DP) TCR- descendants was observed. Additionally, in old males, because of the increased thymic expression of Nur77, a nuclear receptor involved in negative selection, and decreased CD90 (a negative regulator of thymocyte selection threshold) MFI on DP TCRint thymocytes, less efficient positive/more efficient negative selection was found. Moreover, in male rats, thymocyte post-selection differentiation/maturation was skewed towards CD4-CD8+ SP TCRhigh cells compared with age-matched females, reflecting, at least partly, greater IL-15 expression in their thymi. The study indicated mechanisms underlying sex-based differences in age-related thymic changes and consequently necessity of sex-specific approaches in designing strategies to rejuvenate thymus
17β-Estradiol and genistein affect macrophage inflammatory cytokine production during aging in sex-specific manner
Get PDFAging differently affects the expression of estrogen receptors alpha and beta
(ERα and ERβ) and Toll-like receptors (TLR4) on peritoneal cavity cells of
male and female rats. We explored the involvement of ERα and ERβ in the in
vitro treatment of LPS-stimulated peritoneal macrophages with 17β-estradiol
(which stimulates both receptors) or genistein (which is predominantly an ERβ
agonist) on inflammatory cytokine secretion from young (3 months old) and
middle-aged (16 months old) female and male AO rats. Aging diminished the
proportion of TLR4+ cells and secretion of IL-1β and IL-6 in macrophages
from female rats while the effect on male rat macrophages was opposite. 17βestradiol increased IL-1β secretion by middle-aged females’ macrophages via
ERα, and suppressed it in cells from young females via ERβ. Genistein-induced
decrease of IL-1β in macrophages from all experimental groups was probably
mediated by ERβ. 17β-estradiol augmented IL-6 secretion by cells from all
experimental groups via ERα while genistein diminished it in all females’ and
in middle-aged male rats’ macrophages by activating ERβ. However, genistein
increased IL-6 secretion from macrophages of young male rats via ERα.
Although 17β-estradiol and genistein stimulated secretion of macrophage
inflammatory cytokines via ERα and suppressed it probably via ERβ, their
modulatory actions were determined by aging-induced changes in macrophage
ERs expression and possible ERα / ERβ interactions (Supported by Ministry of
Education, Science and Technological development, Republic of Serbia, Grant
No 175050)
Benzo[4,5]thieno[2,3-d]pyrimidine phthalimide derivative, one of the rare noncompetitive inhibitors of dipeptidyl peptidase-4
No full textA small library of benzo[4,5]thieno[2,3-d]pyrimidine phthalimide and amine derivatives was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4). The phthalimide derivatives exhibited better activity than the amine precursors, with 2-(2-(3-chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)isoindoline-1,3-dione (compound 14) as the most effective inhibitor (IC50 = 34.17 +/- 5.11 mu M). The five most potent selected inhibitors did not show cytotoxicity to a greater extent on Caco-2 cells, even at a concentration of 250 mu M. Compound 14 is considered as a novel representative of the rare noncompetitive DPP-4 inhibitors. Molecular docking and dynamics simulation indicated the importance of the Tyr547, Lys554, and Trp629 residues of DPP-4 in the formation of the enzyme-inhibitor complex. These observations could be potentially utilized for the rational design and optimization of novel (structurally similar, with phthalimide moiety, or different) noncompetitive DPP-4 inhibitors, which are anyway rare, but favorable in terms of the saturation of substrate competition