In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).     

Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.     

Diet of Eurasian lynx <Emphasis Type="Italic">Lynx lynx</Emphasis> in the northern Dinaric Mountains (Slovenia and Croatia)

The Eurasian lynx Lynx lynx (Linnaeus 1758) is an opportunistic predator that usually selects the smallest ungulate available. Its diet varies considerably among different regions; therefore it is important to study lynx diet in different parts of the species’ range. We studied the diet of lynx from the endangered Dinaric population in Slovenia and Croatia by analyzing lynx scats, prey remains, and stomach contents. Dinaric lynx mainly killed European roe deer Capreolus capreolus (0.64 frequency of occurrence, 79% of all consumed biomass), which were used more frequently during winter and spring. Ungulates were killed more often by adult males than by lynx of other age and sex groups. In contrast to studies from other regions, lynx in the northern Dinaric Mountains also frequently fed on the edible dormice Glis glis (0.18 frequency of occurrence, 7% of all consumed biomass). This large rodent appears to be an important alternative prey, especially for females and young lynx, and was the reason for the highest use of rodents reported so far for the Eurasian lynx. Edible dormice in Dinaric forests have highly variable numbers of active animals. Seasonal and possibly annual variation in dormouse availability obviously affects lynx diet. This is a rare example where variability in the availability of the alternative prey and not the preferred prey leads to the dietary shift. This study confirms the opportunistic nature of Eurasian lynx and the regional variability of its diet.     

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).     

Fungicidal properties of individual components of copper–ethanolamine-based wood preservatives

The importance of copper–ethanolamine-based wood preservatives is increasing. These preservatives usually consist of copper as a fungicide, ethanolamine as a fixative, and secondary fungicides (boron, triazoles) and other additives (water repellents, fixatives, wax emulsions, etc.). Questions arise as to how each of these ingredients interacts with wood-decay fungi, and whether there are any synergistic effects between the components. In order to elucidate these questions, Norway spruce wood specimens were impregnated with five different aqueous solutions consisting of one single component only and of complete formulation of five different concentrations. These specimens were exposed to two brown-rot fungi, Antrodia vaillantii and Gloeophyllum trabeum, as well as to the white-rot fungus Trametes versicolor for 8 weeks according to mini block procedure. In parallel, petri dishes with nutrient medium containing different quantities of ingredients and of complete wood preservative were inoculated with the same fungal species, and their growth was compared with growth on media without chemicals. The results showed that both experimental methods give similar results. In general, there was no synergistic effect determined. Ethanolamine did not decrease fungicidal properties of the system, while on the other hand octanoic acid has a positive effect on the growth of brown-rot fungi. The minimal effective concentration of tested copper–ethanolamine preservative was determined by the minimum effective concentration of the most fungi-toxic ingredient.     

Empagliflozin on top of metformin treatment improves arterial function in patients with type 1 diabetes mellitus

Background

Deteriorated arterial function and high incidence of cardiovascular events characterise diabetes mellitus. Metformin and recent antidiabetic drugs, SGLT2 inhibitors, reduce cardiovascular events. We explored the possible effects of empagliflozin’s effect on top of metformin treatment on endothelial function and arterial stiffness parameters in type 1 diabetes mellitus (T1DM) patients.

Methods

Forty T1DM patients were randomised into three treatment groups: (1) empagliflozin (25 mg daily), (2) metformin (2000 mg daily) and (3) empagliflozin/metformin (25 mg daily and 2000 mg daily, respectively). The fourth group received placebo. Arterial function was assessed at inclusion and after 12 weeks treatment by: endothelial function [brachial artery flow-mediated dilation (FMD), reactive hyperaemia index (RHI)], arterial stiffness [pulse wave velocity (PWV) and common carotid artery stiffness (β-stiffness)]. For statistical analysis one-way analysis of variance with Bonferroni post-test was used.

Results

Empagliflozin on top of metformin treatment significantly improved endothelial function as did metformin after 12 weeks of treatment: FMD [2.6-fold (P?<?0.001) vs. 1.8-fold (P?<?0.05)] and RHI [1.4-fold (P?<?0.01) vs. 1.3-fold (P?<?0.05)]. Empagliflozin on top of metformin treatment was superior to metformin in improving arterial stiffness parameters; it significantly improved PWV and β-stiffness compared to metformin [by 15.8% (P?<?0.01) and by 36.6% (P?<?0.05), respectively]. Metformin alone did not influence arterial stiffness.

Conclusion

Empagliflozin on top of metformin treatment significantly improved arterial stiffness compared to metformin in T1DM patients. Endothelial function was similarly improved in all treatment groups. Empagliflozin seems to possess a specific capacity to decrease arterial stiffness, which could support its cardioprotective effects observed in large clinical studies.Trial registration Clinical trial registration: NCT03639545

     

The golden jackal (<Emphasis Type="Italic">Canis aureus</Emphasis>) in Bosnia and Herzegovina: density of territorial groups,population trend and distribution range

In this article, we present the results of the first systematic surveys of golden jackals in Bosnia and Herzegovina (B&H). The population status and distribution of the golden jackal (Canis aureus) in B&H was largely unknown, and the few existing literature records mention their presence at only five localities in the country. We interviewed managers of all the hunting grounds in B&H and reviewed available jackal hunting records from 2000 to 2016. In total, we collected 212 records of legally harvested jackals. We observed an increasing trend of harvested jackals in B&H (on average 35% annual increase) during this same period. Using the acoustic (playback) method, we confirmed the presence of 80 territorial jackal groups along six transects covering 3081 km². We estimated density to be a minimum of 0.33 groups/10 km² in northern B&H and 0.10 groups/10 km² in central B&H. Jackal groups were located at significantly lower altitudes with respect to available area along the transects. We present a distribution map of confirmed jackal occurrences in B&H, which indicates that the core area of jackal distribution in the country is currently located along the Sava River and its tributaries in the northern part of B&H. Jackals are sporadically present in the rest of the country, where gray wolves (Canis lupus) probably limit their presence. In total, jackal presence has been detected in 19% (109 out of 586) of 10?×?10 km grid cells covering the country. The primary factor driving the expanding population of jackals in northern B&H appears to be immigration of jackals from Croatia and Serbia.     

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.