Investigation of antimicrobial activity and chromatographic behavior of endophytic fungi constituents using chemometric methods

Rezistencija bakterija na delovanje antibiotika predstavlja globalni problem. Na polju razvoja novih lekova iz prirodnih izvora, nedavno je prepoznat potencijal endofitnih gljiva zahvaljujući sposobnosti da proizvedu sekundarne metabolite različitih bioloških aktivnosti. Biosinteza ovih jedinjenja je pod velikim uticajem brojnih faktora koji se vezuju za izbor biljke domaćina, klimatske uslove, ishranu i prisustvo drugih mikroorganizama u okruženju. Velika baza podataka vezanih za aktivnost endofitnih gljiva prema patogenim bakterijama pretaživana je primenom metode analize glavnih komponenti sa ciljem pronalaženja obrazaca u podacima koji bi ukazali na manji broj pravih kandidata za novih razvoj lekova. Na ovaj način, osvetljen je antimikrobni karakter gljive Phomopsis species. In vitro testovima je potvrđeno da dihlormetanski ekstrakt gljive izolovane iz četina bora inhibira rast bakterija Escherichia coli i Staphylococcus aureus. Hromatografsko razdvajanje pojedinačnih jedinjenja ekstrakta optimizovano je primenom dizajna eksperimenata, a zatim je izvršeno izolovanje i karakterizacija njihove hemijske strukture korišćenjem masene spektrometrije i NMR spektroskopije. In silico metodama su definisani prediktori bioraspoloživosti i toksikološke aktivnosti jedinjenja (Z)-(Z)-2-acetoksiprop-1-en-1-il-3-(3-((E)-3,4-dihidroksipent-1-en-1- il)oksiran-2-il)akrilat i (Z)-(Z)-2-acetoksiprop-1-en-1-il-(3-((E)-4-hidroksi-3-oksopent-1-en-1- il)oksiran-2-il)akrilat. Za razliku od dosadašnih istraživanja koja su se ograničavala in vitro testovima antimikrobne aktivnosti endofitnih gljiva i razrešavanjem hemijske strukture izolovanih biomolekula, ova disertacija predstavlja proširenje prethodnih istraživanja primenom in silico metoda. Studija molekulskog dokinga omogućila je razumevanje mehanizama interakcije biomolekula sa receptorima koji pripadaju patogenim bakterijama uobičajeno multirezistentnim na antibiotike. Primenom veštačkih neuronskih mreža nagrađeni su pouzdani modeli koji ukazuju na vezu između hemijske strukture, parametara interakcije i afiniteta vezivanja za receptore na osnovu kojih je moguć razvoj novih hemijski srodnih antibiotika.Bacterial resistance towards antibiotics represents a global phenomenon. Potential of endophytic fungi as producers of secondary metabolites with wide spectra of different bioactivities in the field of drug discovery from natural resources has recently been introduced. The production of these compounds is under great impact of variety of factors related to the choice of plant host, climate conditions, nutrition and presence of other microorganisms in the same surrounding. Big data set comprising of indices of endophytic fungi antibacterial activity towards patogen bacteria was evaluated using principal component analysis with the aim to find patterns in data and to point out to a limited number of proper candidates for future pharmaceutical research. This resulted in highlightening of the antimicrobial character of Phomopsis species. In vitro tests proved that dichloromethane extract of endophytic fungi isolated from conifer needles inhibits the growth of Escherichia coli and Staphylococcus aureus. Chromatographic separation of individual components of extract was optimized using design of experiments followed by the isolation and chemical structure characterization using mass spectrometry and NMR spectroscopy. In silico methods were used to define the predictors of bioavaliability and toxicological activity of compounds (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3- (3-((E)-3,4-dihydroxypent-1-en-1-yl)oxiran-2-yl)acrylate and (Z)-(Z)-2-acetoxyprop-1-en-1- yl-3-(3-((E)-4-hydroxy-3-oxopent-1-en-1-yl)oxiran-2-yl)acrylate. Unlike up to date research outcomes limited to in vitro evaluation of antimicrobial activity of endophytic fungi and chemical structure elucidation of isolated biomolecules, this disertation represents an extension to previous investigations using in silico methods. The molecular docking study enabled the comprehensive understanding of the mechanisms underlying the interaction of biomolecules with receptors belonging to usually multidrug resistant bacterial pathogens. The artificial neural networks were used to build reliable models relating chemical structure, parameters of interaction and the binding affinity to receptors, thus providing the essence for future development of new chemically related antibiotics

Investigation of antimicrobial activity and chromatographic behavior of endophytic fungi constituents using chemometric methods

Rezistencija bakterija na delovanje antibiotika predstavlja globalni problem. Na polju razvojanovih lekova iz prirodnih izvora, nedavno je prepoznat potencijal endofitnih gljiva zahvaljujućisposobnosti da proizvedu sekundarne metabolite različitih bioloških aktivnosti. Biosinteza ovihjedinjenja je pod velikim uticajem brojnih faktora koji se vezuju za izbor biljke domaćina,klimatske uslove, ishranu i prisustvo drugih mikroorganizama u okruženju. Velika bazapodataka vezanih za aktivnost endofitnih gljiva prema patogenim bakterijama pretaživana jeprimenom metode analize glavnih komponenti sa ciljem pronalaženja obrazaca u podacima kojibi ukazali na manji broj pravih kandidata za novih razvoj lekova. Na ovaj način, osvetljen jeantimikrobni karakter gljive Phomopsis species. In vitro testovima je potvrđeno dadihlormetanski ekstrakt gljive izolovane iz četina bora inhibira rast bakterija Escherichia coli iStaphylococcus aureus. Hromatografsko razdvajanje pojedinačnih jedinjenja ekstraktaoptimizovano je primenom dizajna eksperimenata, a zatim je izvršeno izolovanje ikarakterizacija njihove hemijske strukture korišćenjem masene spektrometrije i NMRspektroskopije. In silico metodama su definisani prediktori bioraspoloživosti i toksikološkeaktivnosti jedinjenja (Z)-(Z)-2-acetoksiprop-1-en-1-il-3-(3-((E)-3,4-dihidroksipent-1-en-1-il)oksiran-2-il)akrilat i (Z)-(Z)-2-acetoksiprop-1-en-1-il-(3-((E)-4-hidroksi-3-oksopent-1-en-1-il)oksiran-2-il)akrilat. Za razliku od dosadašnih istraživanja koja su se ograničavala in vitrotestovima antimikrobne aktivnosti endofitnih gljiva i razrešavanjem hemijske struktureizolovanih biomolekula, ova disertacija predstavlja proširenje prethodnih istraživanjaprimenom in silico metoda. Studija molekulskog dokinga omogućila je razumevanjemehanizama interakcije biomolekula sa receptorima koji pripadaju patogenim bakterijamauobičajeno multirezistentnim na antibiotike. Primenom veštačkih neuronskih mreža nagrađenisu pouzdani modeli koji ukazuju na vezu između hemijske strukture, parametara interakcije iafiniteta vezivanja za receptore na osnovu kojih je moguć razvoj novih hemijski srodnih antibiotika.Bacterial resistance towards antibiotics represents a global phenomenon. Potential ofendophytic fungi as producers of secondary metabolites with wide spectra of differentbioactivities in the field of drug discovery from natural resources has recently been introduced.The production of these compounds is under great impact of variety of factors related to thechoice of plant host, climate conditions, nutrition and presence of other microorganisms in thesame surrounding. Big data set comprising of indices of endophytic fungi antibacterial activitytowards patogen bacteria was evaluated using principal component analysis with the aim to findpatterns in data and to point out to a limited number of proper candidates for futurepharmaceutical research. This resulted in highlightening of the antimicrobial character ofPhomopsis species. In vitro tests proved that dichloromethane extract of endophytic fungiisolated from conifer needles inhibits the growth of Escherichia coli and Staphylococcusaureus. Chromatographic separation of individual components of extract was optimized usingdesign of experiments followed by the isolation and chemical structure characterization usingmass spectrometry and NMR spectroscopy. In silico methods were used to define the predictorsof bioavaliability and toxicological activity of compounds (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3-(3-((E)-3,4-dihydroxypent-1-en-1-yl)oxiran-2-yl)acrylate and (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3-(3-((E)-4-hydroxy-3-oxopent-1-en-1-yl)oxiran-2-yl)acrylate. Unlike up to date researchoutcomes limited to in vitro evaluation of antimicrobial activity of endophytic fungi andchemical structure elucidation of isolated biomolecules, this disertation represents an extensionto previous investigations using in silico methods. The molecular docking study enabled thecomprehensive understanding of the mechanisms underlying the interaction of biomoleculeswith receptors belonging to usually multidrug resistant bacterial pathogens. The artificial neuralnetworks were used to build reliable models relating chemical structure, parameters ofinteraction and the binding affinity to receptors, thus providing the essence for futuredevelopment of new chemically related antibiotics

Студија молекулског докинга са биомолекулима изолованим из ендофитних гљива

Recently, growing interest has been devoted to the investigation of compounds with antimicrobial activity due to rising cases of resistance of mic-robes to known therapies. A reliable and versatile source of novel drug disco-very was recently found among endophytic fungi. Hitherto, the research usu-ally enclosed the in vitro evaluation of antimicrobial activity and chemical structure elucidation of biomolecules extracted from fungal material. There-fore, this research was designed as an extension to previous investigations of endophytic fungi growing on conifer needles by means of conducting a mole-cular docking study. The in silico methods were used with the main goal to make a contribution to the understanding of the mechanisms underlying the interaction of biomolecules isolated from fungus Phomopsis species and eight different types of receptors that belong to usually multidrug resistant bacterial pathogens. The results revealed valuable interactions with receptors 3G7B (Staphylococcus aureus’s gyrase B), 1F0K (1.9 Å structure of Escherichia coli’s transferase) and 1SHV (Klebsiella pneumoniae’s SHV-1 β-lactamase) thus pointing out the receptors that trigger antibiotic response upon activation by the most potent compounds 325-3, 325-5, phomoenamide and phomol. These findings also recommended further discovery of novel potent and broad-spectrum antibiotics based on the structure of selected molecules.У последње време, као одговорна повећање резистенције микроорганизама на познату терапију, све већа пажња се поклања истраживању једињења са антимикробном активношћу. Ендофитне гљиве су недавно представљене као поуздан и богат извор за развој нових лекова. До сада, истраживања су се углавном ограничавала на in vitro процену антимикробне активности и разоткривање хемијске структуре биомолекула изолованих из материјала гљива. Из тог разлога, ово истраживање је осмишљено као проширење претходно спроведених испитивања ендофита које расту на иглицама четинара путем in silico студије молекулског докинга. Главни циљ употребе in silico метода је био да се направи прилог разумевању механизама који стоје иза интеракције биомолекула изолованих из гљиве Phomopsis species са осам различитих типова рецептора који припадају патогеним бактеријама у обичајеном ултирезистентних на лекове. Резултати су указали на важне интеракције са рецепторима 3G7B (Staphylococcus aureus гиразаБ), 1F0K (структура Escherichia Coli трансферазе величине 1,9 Å) и 1SHV (SHV-1 β-лакта-маза Klebsiella pneumoniae) указујући на тај начин на рецепторе путем којих се започиње антибиотски одговор након активације најпотентнијим једињењима, 325-3, 325-5, фомо-енамидом и фомолом. Овим открићем се такође препоручује будући развој нових моћних антибиотика са широким спектром деловања базиран на структури изабраних молекула

N-terminal pro-b-type natriuretic peptide in patients with hypertensive heart disease

Patients with hypertensive heart disease have elevated concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP). The aim of our study was to evaluate NT-proBNP in patients with long-standing hypertension and in patients with signs of hypertensive cardiomyopathy. The study included three groups of 50 subjects: healthy persons (Control Group), patients with hypertension and normal left ventricular systolic function (Group 1) and patients with long-standing hypertension and signs of hypertensive cardiomyopathy with impaired left ventricular systolic function (Group 2). Our results show a very good correlation (Pearson's test) between NT-proBNP in Group 1 and Group 2 and C-reactive protein (Group 1: r = 0.8424; Group 2: r = 0.6650), systolic blood pressure (Group 1: r = 0.7213; Group 2: r = 0.4856), diastolic blood pressure (Group 1: r = 0.4282; Group 2: r = 0.3989) and ejection fraction (Group 1: r = -0.7390; Group 2: r = 0.9111). ROC analysis revealed that the AUC between the Control Group and Group 1 for NT-proBNP (0.912) was not significantly different (p>0.05) from the AUC for systolic (0.924) and diastolic pressure (0.937). A cut-off value for NT-proBNP of 5.89 pmol/L can be used to reliably distinguish patients of Group 1 from the Control Group, and a cut-off value of 21.67 pmol/L reliably separates patients from Group 1 and Group 2 (in both cases, the AUC is 1.000). Patients in Group 2 who belonged to the II and III New York Heart Association (NYHA) class had significantly higher levels of NT-proBNP than those in NYHA class I (ANOVA test, p=0.001). These data suggest that NT-proBNP is a useful biomarker for distinguishing patients with long-standing hypertension who are at risk of heart failure, allowing optimization and proper treatment of these patients

Correlation of N-terminal pro-B-type natriuretic peptide with clinical parameters in patients with hypertension

Background/Aim. Identification of patients with arterial hypertension and a possible onset of heart failure by determining the concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP) enables timely intensification of treatment and allows clinicians to prescribe and implement optimal and appropriate care. The aim of this study was to evaluate NT-proBNP in patients with longstanding hypertension and in patients with signs of hypertensive cardiomyopathy. Methods. The study involved 3 groups, with 50 subjects each: "healthy" persons (control group), patients with hypertension and normal left ventricular systolic function (group 1) and patients with longstanding hypertension and signs of hypertensive cardiomyopathy with impaired left ventricular systolic function (group 2). We measured levels of NT-proBNP, C-reactive protein and creatinine according to the manufacturer's instructions. All the patients were clinically examined including physical examination of the heart with blood pressure, pulse rate, electrocardiogram (ECG) and echocardiogram. Results. Our results showed that the determined parameters generally differed significantly (Student's t-test) among the groups. The mean (+/- SD) values of NT-proBNP in the control group, group 1 and group 2 were: 2.794 (+/- 1.515) pmol/L, 9.575 (+/- 5.449) pmol/L and 204.60 (84,93) pmol/L, respectively. NT-proBNP correlated significantly with the determined parameters both in the group 1 and the group 2. In the group 1, the highest correlation was obtained with C-reactive protein (r = 0.8424). In the group 2, the highest correlation was obtained with ejection fraction (r = -0.9111). NT-proBNP showed progressive increase in proportion to the New York Heart Association (NYHA) classification. The patients in the- group 2 who belonged to the II and III NYHA class had significantly higher levels of NT-proBNP than those in the NYHA class I (ANOVA test,p = 0.001). Conclusion. The obtained results suggest that NT-proBNP is a useful biomarker in the treatment of patients with longstanding hypertension who are at risk for heart failure

A Comparative Study on Mental Disorder Conceptualization: A Cross-Disciplinary Analysis

The conceptualization of mental disorders varies among professionals, impacting diagnosis, treatment, and research. This cross-disciplinary study aimed to understand how various professionals, including psychiatrists, psychologists, medical students, philosophers, and social sciences experts, perceive mental disorders, their attitudes towards the disease status of certain mental states, and their emphasis on biological versus social explanatory attributions. A survey of 371 participants assessed their agreement on a variety of conceptual statements and the relative influence of biological or social explanatory attribution for different mental states. Our findings revealed a consensus on the need for multiple explanatory perspectives in understanding psychiatric conditions and the influence of social, cultural, moral, and political values on diagnosis and classification. Psychiatrists demonstrated balanced bio-social explanatory attributions for various mental conditions, indicating a potential shift from the biological attribution predominantly observed among medical students and residents in psychiatry. Further research into factors influencing these differing perspectives is necessar

Detekcija in ocena imunogenosti strukturnih variant rekombinantnih monoklonskih protiteles

Background: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple, or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immune response in organism. Methods: Doctoral dissertation includes proposed detection and characterization of aggregated as well as other modified forms of monoclonal antibodies (mAb) by using selected chromatographic and spectrometric methods. Additionally, the selected mAb’s aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells’ (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immune response of peripheral blood mononuclear cell (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMC to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. Results and conclusion: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays could be used as a platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products, which contain monoclonal antibodies as active pharmaceutical ingredients.Ozadje: Razvoj novih rekombinantnih biotehnoloških izdelkov se je močno razširil v sodobno farmacijo in medicino. Ker so biološke rekombinantne molekule občutljive in kompleksne beljakovine, je njihova funkcija močno odvisna od njihove strukture. Poleg njihove učinkovitosti lahko biološka zdravila povzročijo neželene učinke, kot je imunogenost. Zato je odkrivanje in karakterizacija strukturnih variant beljakovin bistvenega pomena pri razvoju in nadzoru kakovosti terapevtskih proteinov, ki bi lahko sprožili imunogeni odziv v organizmu. Metode: Doktorska disertacija vključuje predlagano detekcijo in karakterizacijo agregiranih in drugih modificiranih oblik monoklonskih protiteles (mAb) z uporabo izbranih kromatografskih in spektrometričnih metod. Poleg tega smo vzorce agregatov in modificiranih strukturnih variant monoklonskih protiteles izpostavili testu z nezrelimi dendritičnimi celicami (DC), ki izhajajo iz monocitov, za spremljanje aktiviranih celic DC in ugotavljanje potencialne imunogenosti strukturnih variant monoklonskega protitelesa. Ovrednotili smo tudi potencialni prirojeni imunogenski odziv kulture mononuklearnih celic periferne krvi (PBMC) v stiku z agregati mAb, in sicer z merjenjem vnetnega citokinskega odziva pri zgodnji izpostavitvi PBMC-jev različnim strukturnim oblikam monoklonskega protitelesa in z določanjem učinka agregatov mAb na proliferacijo dolgoživeče kulture PBMC-jev. Rezultati in sklep: Vse razvite in predlagane analitske metode in imunološka in vitro testiranja s celicami DC in PBMC bi se lahko uporabljala kot platforma za komplementarno analitsko karakterizacijo in določitev potenciala za imunogenost za vse biofarmacevtske izdelke, ki vsebujejo monoklonska protitelesa kot aktivne farmacevtske sestavine

Hemometrijski prozor ka antibakterijskoj aktivnosti biomolekula izolovanih iz endofitnih gljiva

Drug resistance, especially bacterial antibiotic resistance, is recognized as a global phenomenon. Potential of endophytic fungi as producers of secondary metabolites with wide spectra of different bioactivities in the field of drug discovery has recently been introduced. The production of these compounds is under the great impact of the variety of factors related to the choice of plant host, climate conditions, nutrition, presence of other microorganisms in the same surrounding, etc. Due to a large number of different indices of endophytic fungi antibacterial activity, critical evaluation with the assistance of data mining analytical methods was performed. The activity towards several pathogen bacteria of endophytes species growing on different plant hosts, deciduous trees as well as herbaceous plants spread worldwide in different climatic zones together with the taxonomy of endophytes was taken into consideration. The principal component analysis was used to explore formed big data set and through finding patterns in data, to point out to a limited number of proper candidates for future pharmaceutical research. The antimicrobial character of Phomopsis species was highlighted and future perspectives in their therapeutic use were projected.Rezistencija na lekove, posebno rezistencija bakterija na delovanje antibiotika, smatra se globalnim problemom. Na polju razvoja novih lekova nedavno su se pojavile endofitne gljive zahvaljujući sposobnosti da proizvedu sekundarne metabolite sa širokim spektrom različitih bioloških aktivnosti. Biosinteza ovih jedinjenja je pod velikim uticajem brojnih faktora koji se vezuju za izbor biljke domaćina, klimatske uslove, ishranu, prisustvo drugih mikroorganizama u istom okruženju itd. Pošto je u literaturi primećen veliki broj različitih indikatora antibakterijske aktivnosti endofitnih gljiva, izvršena je njihova kritička procena korišćenjem analitičkih metoda pretrage podataka. Razmatrana je aktivnost prema nekoliko sojeva patogenih bakterija koje su pokazale endofitne gljive koje rastu na različitim biljkama domaćinima, listopadnom drveću i zeljastim biljkama širom sveta i u različitim klimatskim uslovima, kao i taksonomska rasprostranjenost vrsta endofitnih gljiva. Istraživanje velike grupe podataka je vršeno pomoću analize glavnih komponenata (eng. Principal component analysis) sa ciljem pronalaženja obrazaca u podacima koji bi ukazali na manji broj pravih kandidata za dalji razvoj lekova. Osvetljen je antimikrobni karakter Phomopsis vrsta, a takođe su procenjeni i budući pravci u njegovoj terapijskoj primeni

Inter-laboratory verification of European pharmacopoeia monograph on derivative spectrophotometry method and its application for chitosan hydrochloride

Inter-laboratory verification of European pharmacopoeia (EP) monograph on derivative spectrophotometry (DS) method and its application for chitosan hydrochloride was carried out on two generation of instruments (earlier GBC Cintra 20 and current technology TS Evolution 300). Instruments operate with different versions of Savitzky-Golay algorithm and modes of generating digital derivative spectra. For resolution power parameter, defined as the amplitude ratio A/B in DS method EP monograph, comparable results were obtained only with algorithm's parameters smoothing points (SP) 7 and the 2nd degree polynomial and those provided corresponding data with other two modes on TS Evolution 300 Medium digital indirect and Medium digital direct. Using quoted algorithm's parameters, the differences in percentages between the amplitude ratio A/B averages, were within accepted criteria (+/- 13%) for assay of drug product for method transfer. The deviation of 1.76% for the degree of deacetylation assessment of chitosan hydrochloride, determined on two instruments, (amplitude D-1(202); the 2nd degree polynomial and SP 9 in Savitzky-Golay algorithm), was acceptable, since it was within allowed criteria (+/- 2%) for assay deviation of drug substance, for method transfer in pharmaceutical analyses