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Coumarins and triazoles are two interesting groups of heterocyclic compounds, both exhibiting a wide range of biological activities. The joining of these two scaffolds into a hybrid compound with the improved activities has already been reported. [1] In this research, 11 new hybrid compounds were synthesized, utilizing a deep eutectic solvent as an environmentally sustainable method of organic synthesis. Compounds were obtained in good yields through a one-step reaction of coumarinyl hydrazides and various substituted isothiocyanates, without the use of catalysts. [2] Since many commercially available fungicides are based on triazole derivatives, antifungal activity of 1, 2, 4-triazolyl coumarins was evaluated in vitro on two pathogenic fungi, Fusarium oxysporum and Fusarium culmorum. The tested compounds moderately inhibited the mycelial growth of F. oxysporum (35.0-57.5%), but were mostly ineffective against F. culmorum. Compound 1b, as the most potent growth inhibitor, was further analyzed computationally. A molecular docking analysis was performed to evaluate the binding mode of compound 1b with sterol 14 α-demethylase, the target enzyme of triazole based fungicides

Boja pokožice, pomološka i interna kvaliteta ploda predstavljaju jedan od ključnih i pokazatelja kvalitete u intenzivnoj proizvodnji jabuke. Istraživanje sa ciljem utvrđivanja utjecaja reflektivnog mulcha na poboljšanje i očuvanje interne kvalitete jabuke cv. FUJI Raku Raku u pre- i post-harvest periodu provedeno je 2020. godine u pokusnom nasadu jabuka (Istočna Hrvatska). Sorta je cijepljenja na podlogu M. N.9 NAKB T 339, uzgojnog oblika vitko vreteno s gustoćom sklopa 0.9 x 3.2 m. Pokus je postavljen po split-plot metodi (4 repeticije po 6 stabala). Tretmani su uključivali stabla ispod kojih je postavljena reflektirajuća folija i kontrolni tretman. Tretman folijom je postavljen 20 dana prije očekivane tehnološke berbe plodova s obije stane reda (istok i zapad). Pri berbi plodovi su odvajani po strani reda s kojeg su ubrani (istok i zapad) te su obavljena pomološka mjerenja (promjer, visina, masa, tvrdoća, Brixi i JŠI), intenzitet obojenosti plodova chromametrom po CIE L*a*b* sustavu te mjerenja internih pokazatelja kvalitete plodova (antocijani, fenoli i vitamin C). Navedeni interni pokazatelji kvalitete plodova mjereni su neposredno nakon berbe te su ponovljeni nakon 3 mjeseca skladištenja (hladnjača s kontroliranom atmosferom). Na razini cijelog pokusa između plodova ubranih s istočne i zapadne strane reda nije bilo značajne razlike u intenzitetu obojenosti plodova te internoj kvaliteti dok je koncentracija šećera i JŠI bila značajno veća na plodovima sa zapadne strane. Plodovi ubrani na istočnoj strani reda bili su jedino značajno veće mase. Vitamin C bio je značajno veći u plodovima analiziranim neposredno nakon berbe dok su fenoli bili značajno veći u plodovima analiziranim nakon skladištenja. Plodovi na tretmanu pod folijom rezultirali su značajno većim promjerom, visinom i masom te sadržajem antocijana i fenola. Također plodovi pod reflektirajućom folijom bili su tamniji i s više crvene, odnosno manje žute boje prema CIE L*a*b* sustavu (saturiraniji bojom) ; značajno niži L* i b*. u odnosu na plodove bez folije. Dobiveni rezultati istraživanja upućuju na veliki potencijal reflektirajuće folije u dostizanju, odnosno povećanju dopunske boje i interne kvalitete plodova. Standardiziranje ove dopunske pomotehničke mjere nameće se kao kvalitativno rješenje u prevladavanju nepovoljnih klimatskih prilika u periodu dozrijevanja.

Phytopathogenic fungi are one of the leading causes of plant diseases that reduce crop quality and cause huge yield losses. The control of fungal plant diseases is important for reducing or preventing these losses. The excessive use of available fungicides over a long period of time has increased the fungal resistance and become a major ecological issue, giving the incentive to find new antifungal agents. Quinazolinone is a common heterocyclic nitrogen scaffold, widely present in natural products. Its derivatives exert a variety of biological activities, such as antitumor, antiviral, antibacterial and antifungal. The aim of this study was to examine a potential antifungal activity of several structurally different, recently synthesized new quinazolinone derivatives using a computational approach. A molecular docking analysis has been performed on two fungal chitinases, type A (PDB ID: 2XUC) and B (PDB ID: 2IUZ), involved in chitin decomposition, cell wall degradation and remodeling. Molecular docking has shown that all tested compounds bind near the enzyme active sites. The most promissing compounds were N2, N6- bis(2-methyl-4- oxoquinazolin-3(4H)-yl)pyridine-2, 6-dicarboxamide for chitinase A, and 3-(4- methoxyphenyl)-2- methyl-6-nitroquinazolin-4(3H)- one for chitinase B.

Coumarin and its derivatives are well-known phytochemicals that have shown great potential in acetylcholinesterase (AChE) inhibition [1]. Novel coumarin derivatives have been tested as acethylcholinesterase (AChE) inhibitors by utilizing the Ellman's method [2]. Inhibition data were expressed as a logarithm of inhibition percentages. Molecular structures were optimized by PM3 method using Hyperchem software. Molecular descriptors have been calculated by ADMEWORKS ModelBuilder software. QSAR analysis for inhibition of AChE was performed on the 34 compounds in the training set. Due to the small number of compounds, they were not split in training and test sets. The best QSAR model obtained is: log inh.% = - 1.09 – 0.11 MATS7m + 1.84 MCB + 0.17 DIPZ (1) where MATS7m is Moran autocorrelation of lag 7, weighted by atomic masses ; MCB is Balabans topological index, and DIPZ is dipole moment on Z axes. The model has achieved the following statistical performance of the internal validation: R2 = 0.78 ; F = 37.26 ; Δk = 0.23 ; CCCtr = 0.88 ; CCCcv = 0.84 ; Q2loo = 0.71 ; Q2γscr = -0.18. Williams plot for applicability domain of model (1) is shown in Figure 1. Molecular descriptors in model (1) revealed that effect of inhibition of molecules decreased with: lower number of atoms higher mass at spatial lag 7, increasing branching, and small difference in electronegativity between two bonded atoms at the Z axes.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen responsible for the reduction of the crop quality and yield. One of the most efficient ways to prevent and control diseases caused by S. sclerotiorum is the use of chemical fungicides. However, their use led to the accumulation of residual fungicides in the soil, water, and agricultural products, as well as the appearance of fungicide-resistant forms of this phytopathogen. Therefore, there is a need for new, environmentally friendly compounds that would prove effective against S. sclerotiorum. This study aimed to derive a Quantitative Structure- Activity Relationship (QSAR) for the antifungal activity of 18 quinazolinone derivatives. Antifungal test against S. sclerotiorum was carried out according to the method of Siber et al. Antifungal activities of tested compounds, expressed as % inhibition, were converted in the form of the logarithm values (log % inhibition). The optimized 3D strictures of quinazolinone derivatives were used for the descriptor calculation. Considering the limited number of compounds, splitting into test and training set was not performed. The multiple linear regression QSAR models were obtained with a genetic algorithm (GA) using QSARINS 2.2.4. (University of Insubria, Varese, Italy). The best QSAR model was generated with three DRAGON descriptors: Mv, Mor09e and R6e. The model satisfied the fitting and internal validation criteria, confirming its stability and robustness: R2 = 0.91 ; R2adj = 0.89 ; F = 45.17 ; ΔK = 0.12 ; CCCtr = 0.95 ; Q2loo = 0.85 ; R2-Q2loo = 0.06 ; CCCcv = 0.92 ; R2Yscr = 0.18 ; Q2Yscr = -0.43. Molecular descriptors in obtained QSAR model revealed that quinazolinones of higher molecular weight (Mv), higher contribution of 3D distribution of electronegativity at 8 Å (Mor09e), and enhanced interactions of electronegative atoms at a topological distance 6 (R6e), have lower antifungal effects.

Human dipeptidyl peptidase III (hDPP III) is a zinc‐hydrolase that cleaves dipeptides sequentially from the N‐ terminal of different bioactive peptides. Although the physiological role of hDPP III is not yet fully elucidated, its involvement in pathophysiological processes such as mammalian pain modulation and blood pressure regulation emphasizes the need to find new hDPP III inhibitors. Coumarins exhibit a variety of biological activities, and Schiff bases bearing coumarin moiety in conjunction with the imino group (-C=N-) form significant compounds in medicinal and pharmaceutical chemistry. To find new potential inhibitors of hDPP III, in this research we selected 6 coumarinyl Schiff bases to investigate their interactions with hDPP III, combining experimental and computational approaches. hDPP III activity was determined using the spectrophotometric assay, while molecular docking was performed by AutoDock Vina software to obtain information regarding the possible interactions of the strongest inhibitor and hDPP III binding site. The experimental results showed that all analyzed compounds have an inhibitory effect against hDPP III activity at the physiological concentration i.e., 100 μM. The strongest inhibition (87.9 %) has been obtained with compound 5. Docking predicts that 5 interacts with amino acid residues of hDPP III mostly in the region of inter-domain cleft by forming hydrogen bonds with Ile386, His568 and Arg572, π – interactions with Phe109, Pro387 and Ala388, and numerous van der Waals interactions. Most of these residues are found to be constituents of the hDPP III substrate binding subsites S1, S1’, S2, S2’ and S3’. The results of this study provide an insight into the coumarinyl Schiff bases inhibitory activity and interactions toward hDPP III.

In plant biochemistry and physiology, coumarins are known as antioxidants, enzyme inhibitors and precursors of toxic substances. Nuclear magnetic resonance (NMR) spectra are primary sources of molecular structural data. NMR provides detailed information about the local environment of the atom which can be used to determine the atomic connectivity, stereochemistry, and molecular conformation. For many years the molecular structure has been determined by NMR spectroscopy and chemical shifts are determined manually with the help of computer programs. However, recent progress in computational chemistry and chemo/bioinformatics opened the possibility for the prediction of chemical shifts (especially those of 1H and 13C nuclei) of new chemicals. We analyzed the accuracy of three available chemoinformatics methods developed for the prediction of 1H and 13C chemical shifts based on deep neural networks CASCADE [1], an older prediction method based on classical neural networks NMRshiftDB [2, 3], and group-contribution method in ChemDraw [4]. The mean absolute errors (MAEs) in the prediction of NMR shifts of four newly synthesized coumarins [5] by CASCADE, NMRshiftDB and ChemDraw are (respectively) 0.39, 0.65 and 0.32 ppm for 1H, and 1.5, 6.5 and 2.3 ppm for 13C atoms, shoving relatively big differences between these prediction methods.

Prior to comercialization of plant protection products, active components must be evaluated regarding their impact on the enviroment, animal and human health. Toxicity research data must be obtained in a laboratory environment by animal testing for long-term and short-term health effects. The REACH (Registration, Evaluation, and Authorization of Chemicals) guidelines implemented by the European Parliament restrict the overusage of animals in testing, and suggest the application of computational softwares in preliminary toxicity predictions. The aim of this research is to estimate the toxicity of the 10 newly synthetized flourinated pyrazole derivatives as potential compounds in plant protection treatments, as they show great prominence as phosphodiesterase inhibitors, making them possibly applicable as future pesticides. The software used to predict the compound toxicity is the T.E.S.T. (Toxicity Estimation Software Tool) software. Toxicity parameters that were evaluated are: • the lethal doses for rats (oral rat LD50) ; • aquatic toxicity (doses for growth inhibition of Tetrahymena pyriformis and lethal doses that kills half of fathead minnow (Pimephales promelas) ; • mutagenicity of compound that induces revertant colony growth of Salmonella typhimurium ; • bioaccumulation factor (the ratio of the concentration of a chemical in the tissue of an aquatic organism (fish) to its concentration in water.

Acetylcholinesterase (AChE) is an enzyme responsible for the metabolism of acetylcholine (ACh), the neurotransmitter controlling motor activities in nematodes. Inhibition of AChE results in accumulation of ACh at neuromuscular junctions and leads to paralysis, which makes AChE a good target enzyme for the control of parasitic nematodes. AChE inhibitors used to control parasitic nematodes are mainly organophosphates and carbamates, however, due to their toxicity, they have been withdrawn or have restricted usage. Halogenated pyrazoline derivatives exhibit various biological activities, among them antibacterial, antifungal, and antimicrobial. A series of fluorinated pyrazolines was chosen for the investigation of possible AChE inhibition, using Ellman’s protocol modified for microplate reader. All tested compounds exhibited moderate to good inhibitory effect against AChE at 50 μM concentration, with compound 9 (Fig. 1) having the highest inhibition of 79.5 %. A molecular docking study was performed to gain insight into the binding interaction of compound 9 with AChE (PDB: 2c5g). Results show that compound 9 interacts with amino acid residues located in the peripheral active site of AChE, blocking the narrow passage to the catalytic site. The effect of fluorinated pyrazolines was also tested on two entomopathogenic nematodes, Heterorhabditis bacteriophora, and Steinernema feltiae. Entomopathogenic nematodes are beneficial nematodes used in insect pest management programs because of their resilience to pesticide exposure. Since these nematodes mostly have been unaffected by tested compounds, it indicates their potential use as plant protection agents.

Human dipeptidyl peptidase III (hDPP III) is a zinc‐hydrolase that cleaves dipeptides from the N‐ terminal of different bioactive peptides. Quinazolinones belong to a diverse class of nitrogen-containing heterocyclic compounds, derivatives of which exhibit a wide range of biological activities. In this research, we selected 10 previously synthesized [1] structuraly different quinazolinone derivatives (QDs) to investigate their potential to inactivate hDPP III activity combining in vitro and in silico approach. The experimental results showed that all analyzed QDs (at the physiological concentration i.e., 100 μM) have inhibitory effect against hDPP III activity. The strongest inhibition (% inh. = 81.9) has been obtained with compound QD8. Molecular docking revealed that hDPP III interacts mostly by hydrogen bonds and hydrophobic interactions with QD8. Among important intermolecular interactions are those with amino acid residues F443, Q446, V447, H568 and R572 that are constituents of hDPP III substrate binding subsites S1, S1’, S2’, and S3’.

Reliable representation of the systems with transition metals is still a weak point of the force field based molecular modeling methods. We tested different strategies for modeling the zinc ion in classical molecular dynamics simulations based on nonpolarizable (nonbonded, hybrid bonded/nonbonded and cationic dummy atom models) as well as on polarizable (Drude-2013) potentials using human dipeptidyl peptidase III (DPP III) as a template. In the experimentally determined human DPP III structure the zinc ion is mostly tetrahedrally coordinated by two histidines, glutamate and one water or the substrate/inhibitor molecule. The quantum mechanics - molecular mechanics (QMMM) calculations showed an exchange of the four- and five-coordinated zinc ion during the reaction. Since neither of the strategies based on the nonpolarizable potential showed good agreement with experimental findings and the results of QMMM calculations, and the simulations utilizing the polarizable potential turned out to be extremely expensive, we modified the approach of Yang et al. [1] by extending the region used in quantum mechanical (QM) calculations based parametrization procedure. Namely, the zinc coordinated water molecule and the residues constituting the second metal ion coordination sphere were included in QM calculations resulting with a new set of the zinc ion parameters, so called 4-ligand-extended parameters, to be used within the AMBER force fields ff12SB and ff14SB. These parameters enabled realistic modeling of the active site configuration in different DPP III orthologues as well as in distant peptidases like thermolysin, neprilysin and aminopeptidase N.

Metallopeptidases of the M49 family are proteolytic enzymes that contain highly conserved HEXXXH motif and whose catalytic activity depends on the zinc cation [1]. Dipeptidyl peptidase III (DPP III) is the best characterized member of this family with distribution detected in almost all forms of life. The physiological role of DPP III has not been precisely determined, however it is assumed that it is involved in intracellular protein catabolism, mammalian pain modulatory system, and in the defense against oxidative stress. Flavonoids belong to the group of polyphenolic compounds that are ubiquitous in plants. They are important component of human diet and seem to have various pharmacological actions [2]. In this study, we selected 15 dietary flavonoids belonging to four different structural subclasses to investigate their potential inhibitory activity (at the physiological concentration i.e., 100 μM) against human DPP III. Inhibition of DPP III activity was determined using the spectrophotometric method [3] with minor modifications. For that purpose, purified recombinant human DPP III was briefly preincubated with a flavonoid in buffered solution and the enzymatic reaction was started with a Arg-Arg β- naphthylamide as a substrate. After the incubation time the reaction was stopped and the absorbance at 530 nm was measured. The percentage of enzyme inhibition (% inh.) was calculated by comparing the enzymatic activity without (normal activity) and with a flavonoid (inhibited activity). The results, obtained by in vitro assay showed that all analyzed dietary flavonoids have inhibitory effect against human DPP III hydrolytic activity at given concentration. The strongest inhibition (% inh. > 90) has been obtained with galangin, genistein, fisetin, kaempferol, and luteolin. A slightly lower inhibition (% inh. 50-90) has been observed for 3, 6- dihydroxyflavone, 3, 7- dihydroxyflavone, 6- hydroxyflavone, apigenin, morin and quercetin, while 3-hydroxyflavone, chrysin, flavone and flavanone have shown an inhibition of less than 50%. These data indicate that the presence and exact distribution of different structural groups on dietary flavonoids are important for their inhibitory properties. [1] N. D. Rawlings, F. R. Morton, C. Y. Kok, J. Kong, and A. J. Barrett Nucleic Acids Res 36 (2008) D320– D325. [2] S. Kumar, A. K. Pandey, Sci. World J. (2013) 162750. [3] M. Abramić, M. Zubanović, Lj. Vitale, Biol. Chem. Hoppe-Seyler 369 (1988) 29–38.

The quality and quantity of light are important factors for the production of quality seedlings of vegetables. Effective portion of the spectrum for photosynthesis is in the red and blue light, however, in the production of seedlings additional LED lighting that emits light just in the range of those wavelengths are often used. The aim of this study was to determine the effect of red, blue and combinations of red and blue LED lights and fluorescent lights on morphological characteristics, SPAD index and the content of chlorophyll, carotenoids and phenol in the leaves of pepper (Capsicum annuum L.) in two terms of measurements during the growth. The highest content of chlorophyll, carotenoids, SPAD index and the largest leaf area was established in the treatment red + blue LED illumination. The lowest values of the width and length of the leaves were found in plants grown under red LED light while the lowest total plant height determined in plants grown under fluorescent lights. From these results we conclude that additional LED lighting using a combination of light from the blue and red parts of the spectrum can positively affect the growth and development of seedlings of pepper. Further research is needed to determine the most favourable ratio of before mentioned wavelengths of the spectrum through the stages of growth and development as well as for individual varieties, because numerous studies confirm different response of certain varieties in the same plant species.

Human dipeptidyl-peptidase III (h.DPP III) is a zinc-exopeptidase that hydrolysis dipeptides from the N-terminus of its substrates. Our study presents the first insight into the reaction mechanism of h.DPP III ; determined on the model and real systems. The Glu451-assisted water addition on amide carbon followed by nitrogen inversion are shown to be the rate- determine steps with the activation energies in a good agreement with the experimental results for the Leu-enkephalin hydrolysis. We found that precisely defined geometry of the enzyme binding site puts an additional restrains on the tetrahedral intermediate and stimulates the forward reaction towards final hydrolytic product. Namely, differently from the model, the N-inversion is in concerted fashion followed by favourable hydrogen bonding with Glu451 that immediately “locks” the system into the configuration where reversion to the enzyme-substrate complex is hardly achievable. Therefore we propose that the functional significance of DPP III is dual: to lower the energy barrier of the peptide hydrolysis and to suppress the reverse reaction. Based on the determined mechanism and protein dynamics we were able to rationalize the experimentally determined activity of several h.DPP III mutants detected in human cancers.

We employed hydrogen/deuterium exchange reaction combined with molecular dynamics (MD) simulations to investigate conformational dynamics and ligand binding within the M49 enzyme family. Six, two- domain study dipeptidyl peptidase III (DPP III), orthologues, human, yeast, three bacterial and one moss were studies. According to the results all orthologues seems to be quite compact with protected regions located within the two domains core and with the overall flexibility profile consistent with semi-closed conformation as the dominant protein form in solution. Furthermore by comparing HDX data obtained for unliganded protein and its tynorphin complex it was found that tynorphin binds within the inter-domain cleft in all orthologues, but in different orientations. Docking combined with MD simulations revealed details of the protein ligand interactions on human, yeast and three bacterial DPPs III. H- bond analysis revealed that the interdomain active site cleft is more protected in the complexes than in apo enzyme and enabled interpretation of conformational changes noticed at regions distant from the binding site.

Milk production by adding milk powder or selling reconstituted milk powder as raw, pasteurized or UHT milk is one of the serious problems facing the dairy industry today. Adulteration, besides creating unfair market competition, misleads the consumer. The development of methods, in order to prevent product market failures that do not comply with legal regulations, is one of the key challenges in the protection of society. The aim of this paper was to modify the spectrophotometric method for determining the adulteration of drinking milk. The method includes degreasing of milk, coagulation of milk proteins by adding acetic acid, heating the formed curd in the presence of phosphomolybdic acid resulting in its blue staining. The intensity of the blue colour is proportionally increased by the addition of reconstituted milk powder and is measured spectrophotometrically at 620 nm. For semi- quantitative determination of the amount of added milk powder the calibration milk samples with known composition, produced from a mixture of raw or pasteurised or UHT milk with 0 ; 5 ; 15 ; 35 and 65 % (v/v) reconstituted milk powder were prepared, respectively upon analysed type of milk. The curd of unadulterated milk samples coloured greenish while the curd of adulterated milk samples with added milk powder coloured light to dark blue depending on the percentage of added milk powder. The molecular modelling methods were used to predict the clarification of staining mechanism of the formed curd and phosphomolybdic acid. The applied and modified spectrophotometric method was found to be suitable for the detection of milk adulteration by the addition of milk powder

Pesticides are toxic chemicals aimed for the destroying pest on crops. Since pesticides with similar properties tend to have similar biological activities, toxicity may be predicted from structure. Their structure feature and properties are encoded my means of molecular descriptors. Molecular descriptors can capture quite simple two-dimensional (2D) chemical structures to highly complex three- dimensional (3D) chemical structures. Quantitative structure-toxicity relationship (QSTR) method uses linear regression analyses for correlation toxicity of chemical with their structural feature using molecular descriptors. Molecular descriptors were calculated using open source software PaDEL and in-house built PyMOL plugin (PyDescriptor). PyDescriptor is a new script implemented with the commonly used visualization software PyMOL for calculation of a large and diverse set of easily interpretable molecular descriptors encoding pharmacophoric patterns and atomic fragments. PyDescriptor has several advantages like free and open source, can work on all major platforms (Windows, Linux, MacOS). QSTR method allows prediction of toxicity of pesticides without experimental assay. In the present work, QSTR analysis for toxicity of a dataset of mixtures of 5 classes of pesticides comprising has been performed. A good number of molecular descriptors were calculated followed by extensive objective and subjective feature selection to avoid redundant descriptors. For model building, the dataset was divided into training (80%) and test (20%) sets. A QSAR model built using three easily interpretable descriptors was subjected to extensive internal and external validation. The QSAR model is statistically robust with R2 = 0.872, Q2 = 0.844, CCCex = 0.845. The analysis revealed that lipophilicity, frequency of occurrence of hydrogen within 3 Å from phosphorus, and the presence of two benzene rings with –CH2– group as linker have good correlation with the toxicity of the pesticides.