The impact of diabetes mellitus on long-term mortality in patients with STEMI after primary PCI

Introduction: Primary percutaneous coronary intervention (PPCI) is a gold standard in treating patients with acute myocardial infarction (AMI) with ST Elevation Myocardial Infarction (STEMI). Aim: The aim of this study is to evaluate the impact of diabetic status three year survival in patients with STEMI treated with PPCI on admission. Material and methods: We analyzed 499 STEMI patients treated with primary PCI, from a prospective, electronic database of the Clinical Center of Serbia Catheterization Laboratory -that contains patient records from 2009 to 2016. Patients were divided into 2 groups: patients with diabetes mellitus (DM) and those without DM. Results: Our results showed that 416 (83,4%) patients did not have DM and 83 (16,6%) had DM. There was a statistically significant difference in mortality rate among two groups after 30 days and 3 years after intervention (DM 13,3% vs without DM 4,1%, p=0,003 after 30 days; DM 21,7% vs without DM 10,6%, p=0,010 after 3 years). However, when adjusted for other baseline risk factors, DM was not an independent predictor of 3-year mortality (HR=1,275, 95% CI 0,652-2,494, p=0,478). Conclusion: Patients with DM had higher mortality rates in both short-term and long-term follow-ups after PPCI

Spray drying of camel milk induces protein aggregates and Maillard reaction products formation

Camel milk (CM) powders are nutritious food with many health benefits. We investigated physicochemical properties of CM proteins upon spray drying at six inlet temperatures (190°C - 250°C). Electrophoretic and spectrophotometric analysis revealed occurrence of Maillard reaction upon spray drying. Size exclusion chromatography showed increase in protein Mw and aggregates formation. Spray drying inlet temperatures exerted significant effects on the properties of CM powder proteins. Project was supported by the GA No.172024 of Ministry of Education, Science and Technological Development

Role of resveratrol in prevention and control of cardiovascular disorders and cardiovascular complications related to COVID-19 disease: Mode of action and approaches explored to increase its bioavailability

Resveratrol is a phytoalexin produced by many plants as a defense mechanism against stress-inducing conditions. The richest dietary sources of resveratrol are berries and grapes, their juices and wines. Good bioavailability of resveratrol is not reflected in its high biological activity in vivo because of resveratrol isomerization and its poor solubility in aqueous solutions. Proteins, cyclodextrins and nanomaterials have been explored as innovative delivery vehicles for resveratrol to overcome this limitation. Numerous in vitro and in vivo studies demonstrated beneficial effects of resveratrol in cardiovascular diseases (CVD). Main beneficial effects of resveratrol intake are cardioprotective, anti-hypertensive, vasodilatory, anti-diabetic, and improvement of lipid status. As resveratrol can alleviate the numerous factors associated with CVD, it has potential as a functional supplement to reduce COVID-19 illness severity in patients displaying poor prognosis due to cardio-vascular complications. Resveratrol was shown to mitigate the major pathways involved in the pathogenesis of SARS-CoV-2 including regulation of the renin-angiotensin system and expression of angiotensin-converting enzyme 2, stimulation of immune system and downregulation of pro-inflammatory cytokine release. Therefore, several studies already have anticipated potential implementation of resveratrol in COVID-19 treatment. Regular intake of a resveratrol rich diet, or resveratrol-based complementary medicaments, may contribute to a healthier cardio-vascular system, prevention and control of CVD, including COVID-19 disease related complications of CVD

SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children

RT-PCR remains the primary method of diagnosing SARS-CoV-2. Serological diagnosis of COVID-19 is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false negative when antibody concentrations fall below detection limit. Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. Antigen-based tests show high specificity, but sensitivity is relatively low. For development and further improvement of both serological and antigen-based tests, availability of recombinantly producted SARS CoV-2 antigens is needed and during the periods of high demend in the pandemic, it was a limited factor both for research and diagnostic tests production by national producers of tests. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified 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 beta-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. 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). Application of serological ELISA for seropositivity detection in 200 allergic children in comparison to non-allergic did not show a bias towards any of the tested groups. Furthermore, we have optimized production of rfNP at a large scale to raise specific antibodies to N-protein in rabbit and mice and develop an in house ELISA for quantification of N protein in biological fluids. Test was clinically validated in 200 PCR positive patients and showed a sensitivity of around 50%

Electrophoretic and mass spectrometry-based characterization of soluble fraction of camel milk proteins upon freeze and spray drying treatment

Objective: Camel milk is highly nutritious food with numerous health benefits proposed. Demand for camel milk has increased worldwide.Production of camel milk powders facilitate its transport, prolonge shelf-life, and also offer an attractive additive for various food products. In this study we characterized proteins of soluble fraction of freeze/spray dried camel milk powders. Material and Methods: Whole camel milk powders were prepared by spray drying treatment at six different inlet temperatures (190°C - 250°C) or by freeze drying. The soluble protein fractions upon the treatments were analysed by combination of electrophoretic techniques and circular dichroism. Freeze dried camel milk and spray dried at 250°C were analysed by mass spectrometry. Results: SDS-PAGE revealed non-uniform increase in Mw of major protein bands, while native electrophoresis revealed non-uniform decrease in pI values with increased inlet temperature of spray drying. That indicated occurence of the Maillard reaction. Far-UV circular dichroism spectra showed no differences in secondary structures between freeze and spray dried samples. Mass spectrometry identified α-lactalbumin, glycosylation-dependant cell adhesion molecule 1 (GLYCAM1), immunoglobulin heavy chain, peptidoglycan recognition protein and camel serum albumin as dominant proteins in soluble fraction of camel milk powders. Carboxymethyl-lisyne (CML), well known marker of Maillard reaction in food analysis, was detected on GLYCAM1 and on immunoglobulin heavy chain. Conclusions: Our results indicate glycation of camel milk proteins via Maillard reaction upon spray drying treatment which further may affect techno-functional properties of camel milk powders, their shelf-life and nutritional value. Acknowledgments: This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 172024. The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 810752

Physicochemical characterization of soluble proteins of whole camel milk powders produced by spray drying treatment at high temperatures

Objective. Camel milk is highly nutritious food with numerous health benefits proposed. Demand for camel milk has increased worldwide. Production of camel milk powders facilitate its transport, prolonge shelf-life, and also offer an attractive additive for various food products. In this study we characterized proteins of soluble fraction of freeze/spray dried camel milk powders. Material and Methods. Whole camel milk powders were prepared by spray drying treatment at six different inlet temperatures (190°C - 250°C) or by freeze drying. The soluble protein fractions upon the treatments were analysed by combination of electrophoretic and spectroscopic techniques. Functional properties, such as antioxidant activity and protein solubility were assessed. Results. SDS-PAGE revealed non-uniform increase in Mw of major protein bands, while native electrophoresis revealed non-uniform decrease in pI values with increased inlet temperature of spray drying. That indicated attachement of lactose moieties to NH2- group of proteins via non-enzymatic Maillard reaction. Spectrophotometric analysis showed formation of intermediate Maillard reaction products (increased absorbance at 294 nm) and no detectable late Maillard reaction products formation. Far-UV circular dichroism spectra showed no differences in secondary structures between freeze and spray dried samples. Antioxidant activity and protein solubility were increased with increase in inlet temperature. Conclusions. Our results showed that spray drying treatment promoted non-enzymatic glycation of camel milk proteins. Glycation of food proteins affects their technofunctional properties, shelf-life and nutritional value. Thus, optimization of spray drying parametars is essential for production of high quality camel milk powders. Acknowledgements: This research work was funded the Ministry of Education and Science of the Republic of Serbia, GA No. OI172024, Ghent University Global Campus, Belgian Special Research Fund BOF StG No. 01N01718, Serbian Academy of Sciences and Arts Project F-26. The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 810752

Prognostic Value of Mitral Regurgitation in Patients with Primary Hypertrophic Cardiomyopathy

Background and Objectives: Mitral valve pathology and mitral regurgitation (MR) are very common in patients with hypertrophic cardiomyopathy (HCM), and the evaluation of mitral valve anatomy and degree of MR is important in patients with HCM. The aim of our study was to examine the potential influence of moderate or moderately severe MR on the prognosis, clinical presentation, and structural characteristics of HCM patients. Materials and Methods: A prospective study examined 176 patients diagnosed with primary asymmetric HCM. According to the severity of the MR, the patients were divided into two groups: Group 1 (n = 116) with no/trace or mild MR and Group 2 (n = 60) with moderate or moderately severe MR. All patients had clinical and echocardiographic examinations, as well as a 24 h Holter ECG. Results: Group 2 had significantly more often the presence of the obstructive type of HCM (p < 0.001), syncope (p = 0.030), NYHA II class (p < 0.001), and atrial fibrillation (p = 0.023). Also, Group 2 had an enlarged left atrial dimension (p < 0.001), left atrial volume index (p < 0.001), and indirectly measured systolic pressure in the right ventricle (p < 0.001). Patients with a higher grade of MR had a significantly higher E/e' (p < 0.001) and, as a result, higher values of Nt pro BNP values (p < 0.001) compared to Group 1. Kaplan-Meier analysis demonstrated that the event-free survival rate during a median follow-up of 88 (IQR 40-112) months was significantly higher in Group 1 compared to Group 2 (84% vs. 45% at 8 years; log-rank 20.4, p < 0.001). After adjustment for relevant confounders, the presence of moderate or moderately severe MR remained as an independent predictor of adverse outcomes (HR 2.788; 95% CI 1.221-6.364, p = 0.015). Conclusions: The presence of moderate or moderately severe MR was associated with unfavorable long-term outcomes in HCM patients

Serological Elisa test development at the INEP Institute

The COVID-19 diagnostic tools are categorized into two main groups of Nucleic Acid (NA)- based and protein-based tests. To date, nucleic acid-based detection has been announced as the gold-standard strategy for coronavirus detection; however, protein-based tests are promising alternatives for rapid and large-scale screening of susceptible groups. During the first months, no rapid and reliable detecting tool was readily available to adequatly respond to the requirement of massive testing. The aim was to develope cost-effective, sensitive and rapid screening mechanisms for the detection of immune response to SARS-CoV2 (which causes COVID-19), based on the principle of ELISA. The institute INEP has developed tests for detection of IgM and IgG SARS-CoV-2 specific antibodies based on S and N proteins of virus intended to monitor different phases of natural infection, ELISA test for IgG detection in naturale infection based on the use of exclusively domestic components of the ELISA kit (including proteins produced in Serbia, Faculty of Chemistry) and a test specifically designed to monitor the effects of immunization (determination of IgG antibodies specific for the RBD domain of S protein). The tests were independently validated at the relevant laboratories in the country and abroad, and compared to an FDA/WHO approved tests of a major test producers. All testing for validation was carried out on samples collected before COVID19 (negative controls), PCR confirmed COVID19 samples (positive controls) and potencialy cross-reactive samples (other pathogens and autoimmune diseases). The antibody tests showed high levels of sensitivity and specificity and extremely low background noises. The kits are extremely stable, have a shelf life of 1 year and opened kits are usable up to 3 months at storing temperatures of 5°

Supplementary data for the article: Perusko, M.; Ghnimi, S.; Simovic, A.; Stevanovic, N.; Radomirovic, M.; Gharsallaoui, A.; Smiljanic, K.; Van Haute, S.; Stanic-Vucinic, D.; Cirkovic Velickovic, T. Maillard Reaction Products Formation and Antioxidative Power of Spray Dried Camel Milk Powders Increases with the Inlet Temperature of Drying. LWT 2021, 143, 111091. https://doi.org/10.1016/j.lwt.2021.111091.

Supplementary material for: [https://doi.org/10.1016/j.lwt.2021.111091]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4332]Related to accepted version: [https://cherry.chem.bg.ac.rs/handle/123456789/4333

Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus

Нови корона вирус (SARS CoV-2) који се појавио у Вухану 2019. године припада групи једноланчаних РНК вируса [1]. Представља нови инфективни агенс за хуману популацију и веома је брзо детектован у великом броју земаља. Узрочник је респираторних инфекција које могу да буду праћене и веома тешком клиничком сликом. Брзо ширење, одсуство имунитета на овај вирус и одсуство поузданих тестова за детекцију вируса у тренутку избијања пандемије су болест изазвану овим вирусом брзо претворили у здравствени и друштвени проблем највишег приоритета на глобалном нивоу. Иако су највеће биотехнолошке компаније убрзано почеле са развојем и масовном производњом дијагностичких тестова и вакцина, њихова доступност у тренуцима највеће потражње је и даље недовољна, а цене истих су лимитирајући фактор за бољу контролу болести и ширења пандемије [2]. Развој сопствених и одржива производња тестова и вакцина за COVID-19 су од великог друштвеног значаја. Важан предуслов за одрживу производњу тестова је доступност рекомбинантних антигена вируса и могућност производње истих на великој скали за потребе производње домаћих тестова. Овим техничким решењем се описује добијање два кључна антигена новог корона вируса рекомбинантном технологијом и њихова примена у серолошком ЕЛИСА тесту који производи Институт за примену нуклеарне енергије, ИНЕП, као и за добијање реагенаса за детекцију антигена новог корона вируса (специфичних антитела). У првој фази, оптимизоване су секвенце протеина које су подигле осетљивост постојећих серолошких тестова. Иновативност нашег приступа се огледа и у разрађеним експерименталним протоколима за добијање рекомбинантних протеина нуклеокапсида на великој скали, као и у солубилној форми, што олакшава поступак пречишћавања. Избор фрагмента нуклеокапсида који се хетеролого експримира у солубилној форми, а специфично детектује антитела и генерише јак имуни одговор током имунизације животиња (имуногеност) на основу прегледа познатих епитопских секвенци је кључна иновација овог техничког решења. Ово је први пример успешно примењеног рекомбинатног протеина произведеног у Србији у дијагностичком тесту који је регистрован код Агенције за лекове и медицинска средства Републике Србије (број решења 515-02-02370-21-002), а који је примену нашао и на међународном нивоу.Tehničko rešenje je priznato u kategoriji M82 - novo tehničko rešenje (metoda) primenjeno na nacionalnom nivou. U prilogu se nalazi odluka Matičnog naučnog odbora za biotehnologiju i poljoprivredu