Associations of Common Variants in HFE and TMPRSS6 Genes with Hepcidin-25 and Iron Status Parameters in Patients with End-Stage Renal Disease

Background. Influence of TMPRSS6 A736V and HFE (C282Y and H63D) polymorphisms on serum hepcidin-25 levels and iron status parameters in end-stage renal disease (ESRD) patients stratified according to gender has not been previously investigated. In addition, we aimed to evaluate the diagnostic accuracy of the parameters to separate iron-deficiency anemia (IDA) from anemia of chronic disease. Materials and Methods. Iron status parameters and genetic analysis were performed in 126 ESRD patients and in 31 IDA patients as the control group. Results. ESRD patients had significantly higher ferritin and hepcidin-25 ( lt 0.001) relative to IDA patients. Cut-off values with the best diagnostic accuracy were found for hepcidin 9.32ng/mL, ferritin 48.2g/L, transferrin saturation 16.8%, and MCV 81fL. Interaction between gender and HFE haplotypes for the hepcidin-25 and ferritin levels in ESRD patients (p=0.005, partial eta squared=0.09; p=0.027, partial eta squared=0.06, respectively) was found. Serum transferrin was influenced by the combined effect of gender and TMPRSS6 A736V polymorphism in ESRD patients (p=0.002, partial eta squared=0.07). Conclusion. Our findings could contribute to the further investigation of mechanisms involved in the pathophysiology and important gender-related involvement of the TMPRSS6 and HFE polymorphisms on anemia in ESRD patients

Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production, suitable for serological diagnosis. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified by immobilized metal ion affinity chromatography and strong cation exchange chromatography after which it was analyzed by Mass and CD spectrometry and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.Book of Abstract

Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests

Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique

Traditional maceration method was used for the extraction of polyphenols from chokeberry (Aronia melanocarpa) dried fruit, and the effects of several extraction parameters on the total phenolics and anthocyanins contents were studied. Various solvents, particle size, solid solvent ratio and extraction time have been investigated as independent variables in two level factorial design. Among examined variables, time was not statistically important factor for the extraction of polyphenols. The optimal extraction conditions were maceration of 0.75 mm size berries by 50% ethanol, with solid solvent ratio of 1:20, and predicted values were 27.7 mg GAE/g for total phenolics and 0.27% for total anthocyanins. Under selected conditions, the experimental total phenolics were 27.8 mg GAE/g, and total anthocyanins were 0.27%, which is in agreement with the predicted values. In addition, a complementary quantitative analysis of individual phenolic compounds was performed using HPLC method. The study indicated that maceration was effective and simple technique for the extraction of bioactive compounds from chokeberry fruit

Spatial distribution and vertical migration of Cs-137 in soils of Belgrade (Serbia) 25 years after the Chernobyl accident

In this study, the specific activity of Cs-137 was determined by gamma-ray spectrometry in 72 surface soil samples and 11 soil profiles collected from the territory of Belgrade 25 years after the Chernobyl accident. Based on the data obtained the external effective gamma dose rates due to Cs-137 were assessed and geographically mapped. The influence of pedogenic factors (pH, specific electrical conductivity, cation exchange capacity, organic matter content, soil particle size and carbonate content) on the spatial and vertical distribution of Cs-137 in soil was estimated through Pearson correlations. The specific activity of Cs-137 in surface soil samples ranged from 1.00 to 180 Bq kg(-1), with a mean value of 29.9 Bq kg(-1), while in soil profiles they ranged from 0.90 to 58.0 Bq kg(-1), with a mean value of 15.3 Bq kg(-1). The mean external effective gamma dose at 1 m above the ground due to Cs-137 in the soil was calculated to be 1.96 nSv h(-1). Geographic mapping of the external effective gamma dose rates originating from Cs-137 revealed much higher dose rates in southern parts of Belgrade city and around the confluence of the Sava and Danube. Negative Pearson correlation coefficients were found between pH, cation exchange capacity and Cs-137 specific activity in surface soil. There were positive correlations between organic matter and Cs-137 specific activity in surface soil; and between specific electrical conductivity, organic matter, silt content and Cs-137 specific activity in soil profiles

Chokeberry polyphenols preservation using spray drying: effect of encapsulation using maltodextrin and skimmed milk on their recovery following in vitro digestion

Aim: Microencapsulation of chokeberry extracts was performed in order to improve functionality, stability, and bioavailability of extracted polyphenols. Methods: Chokeberry fruits and juice by-product (waste) extracts were spray-dried by using two carriers, maltodextrin and skimmed milk. Morphological and physicochemical characteristics of the obtained powders were analysed. In vitro simulated digestion model was used as an indicator of polyphenolics bioavailability. Results: The moisture content varied between 3.39 and 4.61%, zeta potential had negative values (35-39 mV), maltodetrin powders were smaller (4.27-5.12 mu m) compared to skimmed ones (8.50-11.01 mu m). All microparticles exhibited high encapsulation efficiency of total polyphenols and anthocyanins (73-97% and 63-96%, respectively). For both extract types, maltodextrin powders released higher phenolics content compared to skimmed milk. During in vitro digestion, maltodextrin exhibited a higher protective effect on both active compounds. Conclusion: Taking into account the obtained results, chokeberry polyphenols stability might be improved using spray drying technique, and maltodextrin showed better properties

Trace element distribution in surface soils from a coal burning power production area: A case study from the largest power plant site in Serbia

The content of trace elements (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb and Zn) in surface soils in the area surrounding the largest coal-fired power plant in Serbia was determined to assess the contribution of emissions to pollution. Analysis of mutual associations between the trace elements and their correlation with soil particle size fractions indicated anthropogenic origin at most sampling sites. Enrichment factor analysis confirmed these findings. Common patterns in trace element concentrations of the analysed soils were identified by hierarchical cluster analysis. Explanatory spatial analysis, used for characterization and mapping of spatial variability patterns, revealed the highest concentrations of trace elements in areas in predominant wind directions

Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection and for assessment of immunological response after the vaccination. Nucleocapsid (N) protein is the most abundant virus protein and strong immunogen. The aim was develop efficient, low-cost production of N protein large fragment and to characterize it with bottom-up, high-resolution tandem mass spectrometry and immunologically. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form and purified by several chromatographic steps and was subjected to SDS-PAGE and in-gel digested with trypsin. rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower. Identity of rfNP was confirmed with high scores and peptide coverage above 80%. Estimation from the value of areas under ion extracted chromatographic curves is that only up to 0,03% of the total band protein quantity belongs to host proteins, while rfNP share is well above 99,9%, resulting in highly pure nucleocapsid protein preparation.Book of Abstract