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U radu se analiziraju podaci dobiveni prvenstveno na temelju studija sva 82 predmeta u kojima je identificirano 177 žrtava trgovanja ljudima s ukupno 154 prijavljena počinitelja, odnosno osumnjičenika, za razdoblje 2016.–2020. u Hrvatskoj te 20 provedenih intervjua s osobama uključenim u sustav suzbijanja trgovanja ljudima u Hrvatskoj. Istraživanje je rezultiralo nizom preporuka i prijedloga intervencija u pravni okvir suzbijanja trgovanja ljudima koji se odnosena: objedinjavanje ili usklađivanje pravnog okvira trgovanja ljudima i ropstva; donošenje Zakona o suzbijanju trgovanja ljudima; uvođenjenezavisnog nacionalnog izvjestitelja za trgovanje ljudima; uključivanje žrtava trgovanja ljudima u Zakon o novčanoj naknadi žrtvama kaznenih djela; propisivanje hitnosti postupanja u predmetima trgovanja ljudima; propisivanje klauzule o nekažnjavanju žrtava trgovanja ljudima, kontinuirane edukacije o trgovanju ljudima na svim razinama i dr. Neki od razrađenih prijedloga općeg su karaktera, pa su primjenjivi i na druga kaznena djela, kao što je formuliranje dužnosti dosuđivanja imovinskopravnih zahtjeva kao pravila u Zakonu o kaznenom postupku, načela naknade štete žrtvama kaznenih djela i uvođenje izricanja posebnih obveza uz uvjetnu osudu kao pravila u Kaznenom zakonu.

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIlefor protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistantStaphylococcus aureusclinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance.

Cardiovascular pharmaceuticals (CVPs) are globally present in inland waters and have also been found in the sediment and plasma of fish from the Sava River, Croatia. Based on the previous research, CVPs amiodarone (AMI), ramipril (RAM), simvastatin (SIM), and verapamil (VER) have been selected for this study. Their effect has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) within the first 96 h of development. Upon exposure to environmentally relevant concentrations of tested CVPs (0.1, 1, and 10 μg/L) zebrafish survival and development as apparent from observed morphological abnormalities, heartbeat rates and changes in behavior, hatching success, larval length and oxidative stress level were monitored. The CVP causing the highest mortality and pathological changes was SIM (1 and 10 μg/L), which corresponds well with the observed effects during zebrafish exposure to CVPs' mixtures (4 and 40 μg/L). All pharmaceuticals affected cardiac function and decreased heart rate. SIM (1 μg/L), VER and RAM (10 μg/L) decreased larval length, while induced oxidative stress was recorded in the SIM- and VER-exposed specimens. Behavioral alterations of zebrafish were observed only in AMI-treated group (10 μg/L). Our amino acid sequence comparison and structural and docking analysis showed a highly conserved binding site between human and zebrafish HMG-CoA reductase for SIM and its main metabolite simvastatin acid. Using these ecotoxicological bioassays on a zebrafish model with particular emphasis on sublethal endpoints, the risk of CVPs, especially statins, for fish in inland waters has been identified.

Histamine levels in the human brain are controlled by rather peculiar metabolic pathways. In the first step, histamine is enzymatically methylated at its imidazole

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly- discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

Histamine levels in the human brain are controlled by rather peculiar metabolic pathways. In the first step, histamine is enzymatically methylated at its imidazole Nτ atom, and the produced N-methylhistamine undergoes an oxidative deamination catalyzed by monoamine oxidase B (MAO-B), as is common with other monoaminergic neurotransmitters and neuromodulators of the central nervous system. The fact that histamine requires such a conversion prior to oxidative deamination is intriguing since MAO-B is known to be relatively promiscuous towards monoaminergic substrates; its in-vitro oxidation of N-methylhistamine is about 10 times faster than that for histamine, yet this rather subtle difference appears to be governing the decomposition pathway. This work clarifies the MAO-B selectivity toward histamine and N-methylhistamine by multiscale simulations of the rate-limiting hydride abstraction step for both compounds in the gas phase, in aqueous solution, and in the enzyme, using the established empirical valence bond methodology, assisted by gas-phase density functional theory (DFT) calculations. The computed barriers are in very good agreement with experimental kinetic data, especially for relative trends among systems, thereby reproducing the observed MAO-B selectivity. Simulations clearly demonstrate that solvation effects govern the reactivity, both in aqueous solution as well as in the enzyme although with an opposing effect on the free energy barrier. In the aqueous solution, the transition-state structure involving histamine is better solvated than its methylated analog, leading to a lower barrier for histamine oxidation. In the enzyme, the higher hydrophobicity of N-methylhistamine results in a decreased number of water molecules at the active side, leading to decreased dielectric shielding of the preorganized catalytic electrostatic environment provided by the enzyme. This renders the catalytic environment more efficient for N-methylhistamine, giving rise to a lower barrier relative to histamine. In addition, the transition state involving N-methylhistamine appears to be stabilized by the surrounding nonpolar residues to a larger extent than with unsubstituted histamine, contributing to a lower barrier with the former.

Aminoacyl-tRNA synthetases (AARS) activate amino acids and transfer them to cognate tRNAs. Some AARS cannot achieve the required specificity in the initial amino acid recognition at the synthetic site. Those AARS can incorrectly activate non-cognate amino acids and transfer them to tRNA. To correct the mistake and ensure faithful protein biosynthesis, these AARSs evolved a separate editing domain aimed to hydrolyze erroneously aminoacylated tRNAs (post-transfer editing). To understand what shaped the selectivity of the editing site and how it is related to the initial amino acid selectivity at the synthetic site, we used Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme.1-3 We investigated the synthetic and editing reactions of IleRS using a wide range of amino acids, including non-proteinogenic and synthetic: Val, Nva, Leu, Thr, Met, Ser, Ala, Abu, Nle, F2Abu and F3Abu. Among these, only Val and Nva were poorly discriminated at the synthetic site (i.e. well activated and transferred to the tRNA) and thus can be considered as threat to the fidelity of translation. To our surprise, tRNAs misaminoacylated with all tested amino acids were rapidly hydrolyzed at the editing domain. Thus, it appears that amino acid’s physicochemical features and how well it is rejected at the synthetic site does not pose a significant difference. Only the hydrolysis of cognate Ile-tRNAIle (misediting) was slow, suggesting that the need to keep the cognate product out of editing strongly shaped the specificity of the editing domain. Detailed kinetic analysis revealed that IleRS employs Thr246 and His333 for specific destabilization of Ile-tRNAIle hydrolysis – strategy also known as negative catalysis. Such design enabled IleRS to have broad substrate acceptance at the editing site whilst maintaining a high specificity towards preventing the futile post-transfer editing cycles. This was the first time such broad substrate specificity, paired with negative catalysis, was observed for an AARS, and as such, it marks a new moment in the understanding these vital and ancient enzymes.

Aminoacyl-tRNA synthetases (AARS) activate amino acids and transfer them to cognate tRNAs. Some AARS cannot establish the required specificity in the initial amino acid recognition at the synthetic site and thus may erroneously activate non-cognate amino acids and transfer them onto tRNA. To ensure the faithful protein biosynthesis, those aminoacyl-tRNA synthetases evolved a separate editing domain aimed to hydrolyse erroneously aminoacylated tRNAs (post-transfer editing). To understand what shaped the selectivity of the editing site we used Escherichia coli isoleucyl-tRNA synthetase as a model enzyme. We investigated the kinetics of synthetic and editing reactions of IleRS using a wide range of amino acids, from proteinogenic to synthetic: Val, Nva, Leu, Thr, Met, Ser, Ala, Abu (α-aminobutyrate), Nle (norleucine), F2Abu (γ, γ- difluoro-α-aminobutyrate) and F3Abu (γ, γ, γ- trifluoro-α-aminobutyrate). Among these, only Val and Nva were poorly discriminated (well activated and transferred to the tRNA) and thus can be considered as a threat to the fidelity of translation. To our surprise, tRNAs misaminoacylated with all tested amino acids were rapidly hydrolysed at the editing domain, showing that amino acid’s physicochemical features or how well they are discriminated at the synthetic site and consequently whether or not they can jeopardize the translational fidelity, does not make a significant difference. Only the hydrolysis of cognate Ile-tRNAIle (misediting) was slow, suggesting that the need to keep the cognate product out of editing strongly shaped the specificity of the editing domain. Detailed kinetic analysis revealed that isoleucyl-tRNA synthetase employs Thr246 and His333 for specific destabilization of Ile-tRNAIle hydrolysis (i.e. negative catalysis). Such design enabled isoleucyl-tRNA synthetase to have broad substrate acceptance at the editing site whilst maintaining a high specificity towards preventing the futile post-transfer editing cycles. This was the first time such broad substrate specificity, paired with negative catalysis, was observed for an AARS, and as such, it marks a new moment in the understanding of these vital and ancient enzymes. Živković I., et al. ; Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases ; Nucleic Acids Research, 50 (2022) 4029-4041.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor that recognizes the bacterial peptidoglycan fragment, muramyl dipeptide (MDP). Our group has synthesized and biologically evaluated desmuramyl peptides containing adamantane and its mannose derivatives. The most active mannosylated derivative, ManAdDMP (Man-OCH2-ᴅ- (1-Ad)Gly-ʟ-Ala-ᴅ-isoGln), is further characterized in silico within this study. We built intact model structures of the rabbit NOD2 protein, whose crystal structure lacks seven loops, and explored the binding of the ManAdDMP. Two main binding sites for ManAdDMP are located within the nucleotide-binding oligomerization domain (NOD) and C-terminal leucine-rich repeat (LRR) domains. Our analysis shows that dipeptide isoGln moiety of ManAdDMP significantly contributes to the binding, whereas mannose moiety interacts with modelled loop 7 which is a part of the NOD helical domain 2. The presented results point out the importance of loops 2 and 7 in ligand recognition that could be useful for further investigation of NOD2 activation/inhibition.

Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl- D-isoglutamine) is the smallest peptidoglycan fragment with adjuvant activity. MDP acts as a pathogen-associated molecular pattern and activates the NOD2 receptor. We have designed and prepared MDP analogs, desmuramyl peptides containing lypophilic 2-aminoadamantane-2- carboxylic acid, and furthermore modified it with mannose moieties. Here we present the design and synthesis of desmuramyl peptides containing 2- aminoadamantane-2-carboxylic acid, and its mono- and di-mannosylated derivatives. They exhibited an improved immunological activity in vivo in the mouse model, and the di-mannoslyated derivative showed to be the most active one. Therefore, molecular-dynamic simulations of NOD2 receptor complexes and manosylated desmuramyl peptides were performed. The intact structure of the NOD2 protein with seven loops, which were omitted in the experimentally solved structure, was modeled and the binding of synthesized mannose mimetics of MDP to NOD2 was investigated. Two potential binding sites have been identified, one located within the NBD domain and the other on the concave portion of the LRR surface of the NOD2 domain.

Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl- D-isoglutamine) is a conserved fragment of a bacterial cell walls and the smallest peptidoglycan fragment competent to trigger the immune response. MDP binds to NOD2 (nucleotide binding and oligomerization domain-containing protein 2) with affinity in the nanomolar range. Activation of NOD2 by MDP stimulates downstream signaling pathways and promote immune responses. Minor MDP structure modifications affect molecular recognition by NOD2, and thereby influence the modulation of the immune response. Here we present the synthesis of MDP analogs, desmuramyl peptides (DMPs) with incorporated lipophilic adamantly groups at the N-terminus of the dipeptide pharmacophore. The adamantly group was introduced in two ways, via 2-aminoadamantan- 2-carboxylic acid and via (R)-adamantylglycine. Molecular docking simulations of NOD2 receptor and prepared adamantly DMPs were also performed in order to investigate interactions established between DMPs and receptor protein.

The paper explains the importance and contribution of research to understanding and analyzing police and criminal justice data on hate crime. By identifying methodological, conceptual and logistical obstacles to such research it provides guidance for future research in the field.

Restorativna se pravda tijekom povijesti koristila za rješavanje konflikta između žrtve i počinitelja kaznenog dijela, ali se ona uglavnom izgubila odvajanjem kaznenog od građanskog prava u suvremenim kodifikacijama. Nakon toga 1970-ih godina počeo se razvijati suvremeni koncept restorativne pravde, koji nadopunjuje ili čak zamjenjuje kazneni postupak i kažnjavanje. Od 1999. godine do danas restorativna se pravda u kaznenim stvarima postupno afirmirala i u pravnom okviru Vijeća Europe, Ujedinjenih naroda i Europske unije. Hrvatski kaznenopravni okvir dopušta mogućnost primjene restorativne pravde u nizu segmenata kroz načelo svrhovitosti, sporazumijevanje s okrivljenikom, izricanje posebnih obveza, adhezijski postupak, mirenje u postupcima po privatnoj tužbi, po prijedlogu oštećenika ili sa supsidijarnim tužiteljem, tijekom izvršavanja kazne zatvora itd. Međutim praktična primjena restorativne pravde u tim segmentima još je uvijek jako rijetka, što upućuje na prepreke koje bi trebalo otkloniti kroz projekt Ministarstva pravosuđa i uprave RH „Unapređenje sustava sudskog mirenja“. Pritom kao uzori mogu poslužiti dva uspješna komparativna modela restorativne pravde u kaznenim stvarima koji su analizirani u ovom radu – norveški i nizozemski. U ovom je radu detaljno analiziran i jedan slučaj iz prakse koji otkriva praktične probleme i prednosti medijacije u kaznenim stvarima., Restorative justice has historically been used to resolve the conflict between victim and perpetrator, but it has largely been abandoned due to the separation of criminal from civil law in modern codifications. In the 1970s, the modern concept of restorative justice began to develop in order to complement or even replace criminal procedure and punishment. From 1999 until today, restorative justice in criminal matters has gradually been affirmed in the legal framework of the Council of Europe, the United Nations and the European Union. The Croatian criminal law framework allows the possibility of applying restorative justice in a number of segments through the principle of purposefulness, plea bargaining, the imposition of special obligations, the adhesion procedure, mediation in proceedings in private lawsuits, at the request of the injured party or with a subsidiary prosecutor, etc. The practical application of restorative justice in these segments is still very rare, which indicates the obstacles that should be removed through the project of the Ministry of Justice and Administration of the Republic of Croatia “Reinforcing the system of court-annexed mediation”. The paper analyses two successful comparative models of restorative justice in criminal matters – the Norwegian and Dutch ones – which can serve as examples. A case study in this paper reveals the practical problems and advantages of mediation in criminal matters.

Amazonian wildfires in 2019 have raised awareness about rainforest burning due to increased emissions of particulate matter and carbon. In the context of these emissions, by-products of lignin thermal degradation (i.e. methoxyphenols) are often neglected. Methoxyphenols entering the atmosphere may form intermediates with currently unknown reaction mechanisms and toxicity. This study for the first time provides a comprehensive insight into the impact of lignin degradation products [guaiacol, catechol], and their nitrated intermediates [4-nitrocatechol, 4,6-dinitroguaiacol, 5-nitroguaiacol] on zebrafish Danio rerio. Results revealed 4-nitrocatechol and catechol as the most toxic, followed by 4,6DNG > 5NG > GUA. The whole-organism bioassay integrated with molecular modeling emphasized the potential of methoxyphenols to inhibit tyrosinase, lipoxygenase, and carbonic anhydrase, consequently altering embryonic development (i.e. affected sensorial, skeletal, and physiological parameters, pigmentation formation failure, and non-hatching of larvae). The whole-organism bioassay integrated with in silico approach confirmed the harmful effects of lignin degradation products and their intermediates on aquatic organisms, emphasizing the need for their evaluation within ecotoxicity studies focused on aquatic compartments.

Additional file 1. Detailed description of parameterization process.Description of optGM parameterization scheme and preparation of PUB pept dataset.

Additional file 5. Details of GDMIN and optGM comparison.Description of procedure, values of quality metrics and correlation graphs.

We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP+ binding domain and possesses acidic N- terminal extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide- binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP+. By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP+ cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP+ indicating that its core protein structure is not just preserved but it binds NADP+ even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP+ cofactor ; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP+ has significantly higher interaction occupancy thus binds NADP+ with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP+ and hydrogen bond between Ser244 and phosphate of NADP+.

Two extracellular enzymes of the SGNH hydrolase superfamily reveal highly homologous 3D structures, but act on different substrates ; one is a true phospholipase A1 from Streptomyces albidoflavus (SaPLA1, EC: 3.1.1.32, PDB code: 4HYQ), whereas the promiscuous enzyme from Streptomyces rimosus (SrLip, EC: 3.1.1.3, PDB code: 5MAL) exhibits lipase, phospholipase, esterase, thioesterase, and Tweenase activities. To understand binding modes of phospholipid and triglyceride substrates in both enzymes and their chain-length preferences, we opted for a computational approach based on in silico prepared enzyme-substrate complexes. Our results pinpointed subtle amino acid differences in the hydrophobic pockets which accommodate the acyl chain attached to sn-1 position of glycerol to be responsible for the chain length preference. Slight differences in the binding grooves of SaPLA1 and SrLip, which accommodate the acyl chain attached to sn-3 position are responsible for exclusive phospholipase and both phospholipase/lipase activities of these two enzymes, respectively. The results of modelling correlate with the experimentally obtained kinetic parameters given in the literature and are important for protein engineering that aims to obtain a variant of enzyme, which would preferably act on the substrate of interest.

The empirical valence bond (EVB) method is a multiscale model for complex chemical systems, which represents an effective way to study enzyme reactions and perform an analysis of the catalytic effects of enzymes, and in particular, for comparing reactions in different environments.

Additional file 6. Details of empirical charge method comparison.Description of procedure, values of quality metrics and correlation graphs.