Supplementary data for article: Milivojević, A.; Ćorović, M.; Carevic, M.; Banjanac, K.; Vujisić, L. V.; Velickovic, D.; Bezbradica, D. Highly Efficient Enzymatic Acetylation of Flavonoids: Development of Solvent-Free Process and Kinetic Evaluation. Biochemical Engineering Journal 2017, 128, 106–115. https://doi.org/10.1016/j.bej.2017.09.018.

Supplementary material for: [https://doi.org/10.1016/j.bej.2017.09.018]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2558

Development of SARS-CoV-2 N-protein specific capture ELISA

Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja za suzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostima ELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studije je bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih seruma za rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjam apsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testa proizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima i ze evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumi visokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog za ovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adherirani na dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli ite koncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu za kvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jim poliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe uje spektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikaciju N-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototip ELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ 10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekciju N-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela. Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u za kvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.The accurate diagnosis of people with suspected infection with the SARS-CoV-2 is essential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can be detected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens in biological fluids in ELISA or similar techniques using antibodies developed in animals. The aim of the study was the establishment of a quantitative polyclonal sera-based test for routine measurement of the concentration of SARS CoV-2 nucleocapsid protein using absorbance measurement in a standard 96-well microtiter plate. For the purposes of the test development, recombinant N protein was produced and used for the production of mice and rabbit antisera. Produced antisera were purified and titer was determined. High-affinity polyclonal N-protein specific antisera were used for N-protein specific ELISA test development. The test is based on mice polyclonal sera adhered to microtiter plate bottom for the capture of the N protein from the specimen. Various concentrations of the recombinant N-protein were used to generate a standard curve for protein quantification. The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera and anti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement. We have successfully developed the prototype ELISA for the quantification of N-protein with the detection limit being in the range of ng/mL. The average LOD value for the prototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was 10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for the detection of N-protein with affinity and specificity similar to, or better than commercial antibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence for quantification of the N-protein in protein-rich samples, similar to human sera.Poster: [https://cherry.chem.bg.ac.rs/handle/123456789/5362

Development of SARS-CoV-2 N-protein specific capture ELISA

Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja zasuzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostimaELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studijeje bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih serumaza rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjamapsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testaproizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima ize evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumivisokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog zaovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adheriranina dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli itekoncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu zakvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jimpoliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe ujespektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikacijuN-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototipELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekcijuN-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u zakvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.The accurate diagnosis of people with suspected infection with the SARS-CoV-2 isessential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can bedetected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens inbiological fluids in ELISA or similar techniques using antibodies developed in animals.The aim of the study was the establishment of a quantitative polyclonal sera-based test forroutine measurement of the concentration of SARS CoV-2 nucleocapsid protein usingabsorbance measurement in a standard 96-well microtiter plate. For the purposes of the testdevelopment, recombinant N protein was produced and used for the production of miceand rabbit antisera. Produced antisera were purified and titer was determined. High-affinitypolyclonal N-protein specific antisera were used for N-protein specific ELISA testdevelopment. The test is based on mice polyclonal sera adhered to microtiter plate bottomfor the capture of the N protein from the specimen. Various concentrations of therecombinant N-protein were used to generate a standard curve for protein quantification.The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera andanti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.We have successfully developed the prototype ELISA for the quantification of N-proteinwith the detection limit being in the range of ng/mL. The average LOD value for theprototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for thedetection of N-protein with affinity and specificity similar to, or better than commercialantibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence forquantification of the N-protein in protein-rich samples, similar to human sera.Abstract: [https://cherry.chem.bg.ac.rs/handle/123456789/5361

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Kinetics of the sunflower oil ethanolysis using CaO as catalyst

The ethanolysis of sunflower oil catalyzed by calcium oxide was studied in wider ranges of the reaction conditions: temperature 65-75°C, ethanol-to-oil molar ratio 6:1-18:1 and catalyst loading 10-20% in order to determine the reaction kinetics. The proposed kinetic model of the sunflower oil ethanolysis included the changing and first-order reaction mechanism with respect to triacylglycerols and fatty acid ethyl esters. The kinetic parameters were determined and correlated with the process variables. The Arrhenius equation could be applied to the reaction rate constant with the activation energy of 94.0 kJ/mol. The proposed kinetic model showed a good agreement with the experimental data with the mean relative percentage deviation of ±13% (based on 256 data points). [Projekat Ministarstva nauke Republike Srbije, br. III 45001

Highly efficient enzymatic acetylation of flavonoids: Development of solvent-free process and kinetic evaluation

Solubility and stability of flavonoid glycosides, valuable natural constituents of cosmetics and pharmaceuticals, could be improved by lipase-catalyzed acylation. Focus of this study was on development of eco-friendly process for the production of flavonoid acetates. By using phloridzin as model compound and triacetin as acetyl donor and solvent, 100% conversion and high productivity (23.32 gl(-1) day(-1)) were accomplished. Complete conversions of two other glycosylated flavonoids, naringin and esculin, in solvent-free system were achieved, as well. Comprehensive kinetic mechanism based on two con-secutive mono-substrate reactions was established where first one represents formation of flavonoid monoacetate and within second reaction diacetate is being produced from monoacetate. Both steps were regarded as reversible Michaelis-Menten reactions without inhibition. Apparent kinetic parameters for two consecutive reactions (V-m constants for substrates and products and Km constants for forward and reverse reactions) were estimated for three examined acetyl acceptors and excellent fitting of experimental data (R-2 gt 0.97) was achieved. Obtained results showed that derived kinetic model could be applicable for solvent-free esterifications of different flavonoid glycosides. It was valid for entire transesterification course (72 h of reaction) which, combined with complete conversions and green character of synthesis, represents firm basis for further process development

Sinteza CaO nanešenog na Al2O3 od prekursora kalcujum acetata i njegova primena u transesterifikaciji suncokretovog ulja

Transesterification of sunflower oil was carried out over CaO loaded onto γ-Al2O3 as heterogeneous base catalysts. The solid base catalysts were syn­thesized by the modified wet impregnation of calcium acetate monohydrate onto neutral γ-Al2O3, calcined at different temperatures in an inert atmosphere and characterized by FESEM, XRPD and FTIR techniques. The catalyst con­taining 5.5 wt. % CaO supported on γ-Al2O3 calcined at 700°C exhibited the highest FAME yield at mild reaction conditions (60°C, atmospheric pressure, methanol-to-oil molar ratio of 9:1 and 0.51% of active species-CaO by weight of oil). The best CaO/γ-Al2O3 catalyst showed a similar or lower catalytic activity than some other CaO-based catalysts at a smaller amount of the catalyst (at least two times less than in other studies).Transesterifikacija jestivog suncokretovog ulja izvršena je korišćenjem CaO nanešenog na γ-Al2O3 kao heterogenog baznog katalizatora. Katalizator je si-ntetisan korišćenjem modifikovane metode mokre impregnacije gde je kalcijum acetat monohidrat kao precursor nanešen na neutralnu γ-Al2O3. Karakterizacija katalizatora je urađena korišćenjem sledećih metoda: FESEM, XRPD i FTIR. Na katalitičku aktivnost, a samim tim i prinos metil estara masnih kiselina, utiču morfologija i sastav katalizatora pripremljenog kalcinacijom na različitim temperatu­rama. U reakciji transesterifikacije suncokretovog ulja pri umerenim reakcionim uslovima (60°C, molski odnos metanola:ulja 9:1 i količina CaO 0,51% u odnosu na masu ulja) katalitički najaktivniji je katalizator sa 5,5 mas. % CaO nanešenih na nosač i kalcinisan na 700°C. U poređenju sa literaturnim rezultatima drugih katalizatora baziranih na CaO, primenjenim u sličnim reakcionim uslovima CaO/ γ-Al2O3 je pokazao sličnu ili nižu katalitičku aktivnost. Razlog tome je zato što je u ovom radu korišćena najmanje dva puta manja količina katalizatora po eksperimentu u odnosu na istraživanja sa kojima je vršeno poređenje

Tehnološki postupak dobijanja metil-estara masnih kiselina iz suncokretovog ulja u prisustvu sirovog biodizela kao kosolventa i kalcijum-oksida kao katalizatora : tehničko rešenje

Tehnološki postupak dobijanja metil-estara masnih kiselina iz suncokretovog ulja u prisustvu sirovog biodizela kao kosolventa i CaO kao katalizatora testiran je na laboratorijskom nivou. Istraživanje je urađeno radi validacije tehnološkog procesa, određivanja prinosa i sastava MEMK, kao i mogućnosti ponovnog korišćenja izdvojenog katalizatora u reakciji, bez ikakvog dodatnog tretmana prečišćavanja ili kalcnacije. Definisan tehnološki postupak je šaržnog tipa i obuhvata sedam faza jednog ciklusa: aktivacija katalizatora, metanoliza suncokretovog ulja, izdvajanje katalizatora centrifugiranjem, uparivanje metil-estarske faze (tj. sirovog biodizela) i sušenje biodizela