Investigation of water substitution at RuII complexes by conceptual density function theory approach.

In this paper, we investigated water exchange reactions and substitution of aqua RuII complexes of general formula [Ru(terpy)(N^N)(H2 O)]2+ (where N^N = ethylenediamine (en), 1,2-(aminomethyl)pyridine (ampy) and 2,2'-bipyridine (bipy)) by ammonia and thioformaldehyde. These reactions were studied in detail by applying conceptual density functional theory. This approach enabled us to gain further insight into the underlying reaction mechanism at the microscopic level (involving only direct participants of the reaction, without the influence of the solvent) and to put the concept of reaction mechanism on a quantitative basis. The course of the chemical reaction along the reaction coordinate ξ, is rationalized in terms of reaction energy, force, dipole moment, and reaction electronic flux (REF). The results yield and characterize the significant influence of an intermolecular hydrogen bond formed between the entering and the spectator ligand to the overall energy barrier of the reactions.

Circulating Endothelial Cells: A New Possible Marker of Endothelial Damage in Kawasaki Disease, Multisystem Inflammatory Syndrome in Children and Acute SARS-CoV-2 Infection.

BACKGROUND: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). METHODS: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. RESULTS: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. CONCLUSIONS: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Bis(triazinyl)pyridine complexes of Pt(II) and Pd(II): studies of the nucleophilic substitution reactions, DNA/HSA interactions, molecular docking and biological activity.

Four new complexes of Pt(II) and Pd(II), [Pd(L1)Cl]Cl 1, [Pd(L2)Cl]Cl 2, [Pt(L1)Cl]Cl 3 and [Pt(L2)Cl]Cl 4 (where L1 = 2,6-bis(5,6-diphenyl-1,2,4-triazin-3-yl)pyridine and L2 = 2,6-bis(5,6-dipropyl-1,2,4-triazin-3-yl)pyridine), were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The substitution reactions of 1-4 complexes with L-methionine (L-met), L-cysteine (L-cys) and guanosine-5'-monophosphate (5'-GMP), were studied spectrophotometrically at physiological conditions. Complexes with ligand L1 (1 or 3) were more reactive than those with ligand L2 (2 or 4) by a factor ranging up to 1.57 and 3.71, respectively. The order of reactivity of the nucleophiles was: L-met > L-cys > 5'-GMP. The interactions of complexes with calf thymus-DNA (CT-DNA) and human serum albumin (HSA) were studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. All studied complexes can interact with DNA through the intercalation and minor groove binding, where the latter was preferred. The binding constants (103 and 104 M-1) for the interaction of complexes with HSA indicate the moderate binding affinity of complexes 1-4 to protein. The trends in the experimental results of binding studies between complexes 3 and 4 with DNA and HSA were compared to those obtained from the molecular docking study. Biological evaluation of cytotoxicity of 1 and 2 on HCT-116 and MDA-MB-231 cell lines showed significant cytotoxic and prooxidative character, while 2 also exerted extraordinary selectivity towards colon cancer in comparison to breast cancer cells. The nucleophilic substitution reactions, DNA/HSA interactions, molecular docking and biological activity of bis(triazinyl)pyridine complexes of Pt(II) and Pd(II) were studied.

Impact of Clomiphene Citrate on the Steroid Profile in Dysmetabolic Men with Low Testosterone Levels.

Clomiphene citrate (CC) in male hypogonadism increases testosterone (T) and estrogen levels by stimulating pituitary gonadotropin release. Our group confirmed these hormonal changes in a randomized, cross-over, double-blind trial of CC versus placebo in addition to metformin, conducted in 21 obese dysmetabolic men with low T levels. However, we hypothesize that based on its mechanism of action, CC may directly or indirectly affect adrenal steroidogenesis. The aim of this sub-study was to better understand the changes in steroid levels and metabolism induced by CC treatment. We assessed 17α-hydroxypregnelone (17αOH-P5), dehydroepiandrosterone (DHEA), progesterone (P4), 17α-hydroxyprogesterone (17αOH-P4), androstenedione (A), T, dihydrotestosterone (DHT), estrone (E1), 17ß-estradiol (E2), 11-deoxycortisol (11 S), cortisol (F), and cortisone (E) by LC-MS/MS, and corticosteroid binding globulin (CBG) by ELISA, before and after each treatment. In addition, free-F and steroid product/precursor ratios were calculated. We observed a significant change in serum levels induced by CC compared with placebo for 17αOH-P4, DHT, T, E2, E1, F, E, and CBG, but not free-F. In addition, compared to placebo, CC induced higher 17αOH-P4/P4, E2/E1, 17αOH-P4/17αOH-P5, A/17αOH-P4, T/A, E1/A, F/11 S, and F/E ratios. Therefore, besides the CC stimulating effect on testis steroidogenesis, our study showed increased F, E, but not free-F, levels, indicating changes in steroid metabolism rather than adrenal secretion stimulation. The steroid profiling also revealed the CC stimulation of the Δ5 rather than the Δ4 pathway, thus indicating considerable testicular involvement in the increased androgen secretion.

Simultaneous Insertion of Two Ligands in gD for Cultivation of Oncolytic Herpes Simplex Viruses in Noncancer Cells and Retargeting to Cancer Receptors.

Insertion of a single-chain variable-fragment antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence to highly specific nonattenuated oncolytic agents. Clinical-grade virus production cannot rely on cancer cells. Recently, we developed a double-retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the noncancer producer Vero-GCN4R cell line and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double-retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more-advantageous detargeting strategies were devised so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of amino acids (aa) 35 to 39, 214 to 223, or 219 to 223 and replacement of the deleted sequences with one of the two ligands. The last two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best-performing recombinants carried a gD deletion as small as 5 amino acids and grew to titers similar to those exhibited by the singly retargeted R-LM113 and by the nonretargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effective in vitro and in vivoIMPORTANCE There is increasing interest in oncolytic viruses following the FDA and European Medicines Agency (EMA) approval of the oncolytic HSV OncovexGM-CSF and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in noncancer cells. Here, we describe a double-retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative and one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally disadvantageous detargeting modifications. The current and accompanying studies indicate how to best achieve the clinical-grade cultivation of retargeted oncolytics.

Dual Ligand Insertion in gB and gD of Oncolytic Herpes Simplex Viruses for Retargeting to a Producer Vero Cell Line and to Cancer Cells.

Oncolytic viruses gain cancer specificity in several ways. Like the majority of viruses, they grow better in cancer cells that are defective in mounting the host response to viruses. Often, they are attenuated by deletion or mutation of virulence genes that counteract the host response or are naturally occurring oncolytic mutants. In contrast, retargeted viruses are not attenuated or deleted; their cancer specificity rests on a modified, specific tropism for cancer receptors. For herpes simplex virus (HSV)-based oncolytics, the detargeting-retargeting strategies employed so far were based on genetic modifications of gD. Recently, we showed that even gH or gB can serve as retargeting tools. To enable the growth of retargeted HSVs in cells that can be used for clinical-grade virus production, a double-retargeting strategy has been developed. Here we show that several sites in the N terminus of gB are suitable to harbor the 20-amino-acid (aa)-long GCN4 peptide, which readdresses HSV tropism to Vero cells expressing the artificial GCN4 receptor and thus enables virus cultivation in the producer noncancer Vero-GCN4R cell line. The gB modifications can be combined with a minimal detargeting modification in gD, consisting in the deletion of two residues, aa 30 and 38, and replacement of aa 38 with the scFv to human epidermal growth factor receptor 2 (HER2), for retargeting to the cancer receptor. The panel of recombinants was analyzed comparatively in terms of virus growth, cell-to-cell spread, cytotoxicity, and in vivo antitumor efficacy to define the best double-retargeting strategy.IMPORTANCE There is increasing interest in oncolytic viruses, following FDA and the European Medicines Agency (EMA) approval of HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly checkpoint inhibitors. A strategy to gain cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. Cultivation of fully retargeted viruses is challenging, since they require cells that express the cancer receptor. We devised a strategy for their cultivation in producer noncancer Vero cell derivatives. Here, we developed a double-retargeting strategy, based on insertion of one ligand in gB for retargeting to a Vero cell derivative and of anti-HER2 ligand in gD for cancer retargeting. These modifications were combined with a minimally destructive detargeting strategy. This study and its companion paper explain the clinical-grade cultivation of retargeted oncolytic HSVs and promote their translation to the clinic.

Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins.

Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvß6 or αvß8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2 (human epidermal growth factor receptor 2) in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gBHER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins.

Synthesis, characterization, DFT study, DNA/BSA-binding affinity, and cytotoxicity of some dinuclear and trinuclear gold(III) complexes.

In this study, we have synthesized a series of dinuclear and trinuclear gold(III) complexes of the general formula [Au2(N-N)Cl6] (1-3) for dinuclear and [Au3(N-N)2Cl8]+ (4-6) for trinuclear compounds, respectively, in which N-N is a bidentate ligand (1,4-diaminobutane; 1,6-diaminohexane or 1,8-diaminooctane). These complexes were characterized by elemental analysis, molar conductivity, and spectroscopic techniques (IR, UV-Vis, 1H NMR, ESI-MS). We performed DFT calculations to get insight into the geometry of the studies complexes. DNA-binding studies were performed by UV-Vis spectrophotometry and fluorescence spectroscopy. The results of competitive reactions between gold(III) complexes and ethidium bromide (EB) towards DNA have shown that selected complexes can displace EB from DNA-EB adduct. In addition, these experiments confirm that polynuclear gold(III) complexes interact with DNA covalently or via intercalation. Furthermore, high values of binding constants of gold(III) complexes towards bovine serum albumin (BSA) protein indicate good binding affinity. In addition, redox stability of complexes in the presence of DNA/BSA was confirmed by cyclic voltammetry. Results of the interactions between gold(III) complexes with DNA/BSA were discussed in reference to molecular docking data obtain by Molegro virtual docker. The cytotoxic activity of synthesized gold(III) complexes was evaluated on human breast cancer cell line (MDA-MB-231), human colorectal cancer cell line (HCT-116), and normal human lung fibroblast cell line (MRC-5). All complexes dose-dependently reduced cancer and normal cells viabilities, with significant cytotoxic effects (IC50 < 25 µM) for trinuclear gold(III) complexes (4, 5) on HCT-116 cells.

New dinuclear palladium(II) complexes with benzodiazines as bridging ligands: interactions with CT-DNA and BSA, and cytotoxic activity.

Three new dinuclear Pd(II) complexes with general formula [{Pd(en)Cl}2(µ-L)](NO3)2 [L is bridging ligand quinoxaline (Pd1), quinazoline (Pd2) and phthalazine (Pd3)] were synthesized and characterized by elemental microanalyses, UV-Vis, IR and NMR (1H and 13C) spectroscopy. The interaction of dinuclear Pd1-Pd3 complexes with calf thymus DNA (CT-DNA) has been monitored by viscosity measurements, UV-Vis and fluorescence emission spectroscopy in aqueous phosphate buffer solution (PBS) at pH 7.40 and 37 °C. In addition, these experimental conditions have been applied to investigate the binding affinities of Pd1-Pd3 complexes to the bovine serum albumin (BSA) by fluorescence emission spectroscopy. In vitro antiproliferative and apoptotic activities of the dinuclear Pd(II) complexes have been tested on colorectal and lung cancer cell lines. All tested Pd(II) complexes had lower cytotoxic effect than cisplatin against colorectal cancer cells, but also had similar or even higher cytotoxicity than cisplatin against lung cancer cells. All complexes induced apoptosis of colorectal and lung cancer cells, while the highest antiproliferative effect exerted Pd2 complex.

A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.

The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors.

Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand - a single-chain antibody (scFv) to the cancer-specific HER2 receptor - expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2-gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

Influence of acid-base equilibrium on interactions of some monofunctional coumarin Pd(II) complexes with biologically relevant nucleophiles-comprehensive kinetic study.

This aimed to develop a comprehensive theoretical protocol for examining substitution reaction processes. The researchers used a theoretical quantum-mechanical protocol based on the QM-ORSA approach, which estimates the kinetic parameters of thermodynamically favourable reaction pathways. This theoretical protocol was validated by experimentally investigating substitution mechanisms in two previously synthesised Pd(II) complexes: chlorido-[(3-(1-(2-hydroxypropylamino)ethylidene)chroman-2,4-dione)]palladium(II) (C1) and chlorido-[(3-(1-(2-mercaptoethylamino)-ethylidene)-chroman-2,4dione)]palladium(II) (C2), along with biologically relevant nucleophiles, namely L-cysteine (l-Cys), L-methionine (l-Met), and guanosine-5'-monophosphate (5'-GMP). Reactions were investigated under pseudo-first-order conditions, monitoring nucleophile concentration and temperature changes using stopped-flow UV-vis spectrophotometry. All reactions were conducted under physiological conditions (pH = 7.2) at 37 °C. The reactivity of the studied nucleophiles follows the order: l-Cys > l-Met > 5'-GMP, and the reaction mechanism is associative based on the activation parameters. The experimental and theoretical data showed that C2 is more reactive than C1, confirming that the complexes' structural and electronic properties greatly affect their reactivity with selected nucleophiles. The study's findings have confirmed that the primary interaction occurs with the acid-base species L-Cys, mostly through the involvement of the partially negative sulfur atom (87.2%). On the other hand, C2 has a higher propensity for reacting with L-Cys-, primarily through the partially negative oxygen atom (92.6%). The implementation of this theoretical framework will significantly restrict the utilization of chemical substances, hence facilitating cost reduction and environmental protection.

New Pd(II)-pincer type complexes as potential antitumor drugs: synthesis, nucleophilic substitution reactions, DNA/HSA interaction, molecular docking study and cytotoxic activity.

Two new complexes of Pd(II), [Pd(L1)Cl]Cl (Pd1) and [Pd(L2)Cl]Cl (Pd2), (where L1 = N2,N6-bis(5-methylthiazol-2-yl)pyridine-2,6-dicarboxamide and L2 = N2,N6-di(benzo[d]thiazol-2-yl)pyridine-2.6-dicarboxamide) were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The nucleophilic substitution reactions of complexes with L-Methionine (L-Met), L-Cysteine (L-Cys) and guanosine-5'-monophosphate (5'-GMP) were studied by stopped-flow method at physiological conditions (pH = 7.2 and 37 °C). Complex Pd1 was more reactive than Pd2 in all studied reactions, while the order of reactivity of the selected ligands was: L-Met > L-Cys > 5'-GMP. The interaction of complexes with calf thymus-DNA (CT-DNA) was studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. Both complexes interacted with DNA through intercalation and minor groove binding, where the latter was preferred. Additionally, the interaction of Pd1 and Pd2 complexes with human serum albumin (HSA) was studied employing fluorescence quenching spectroscopy. The results indicate a moderate binding affinity of complexes, with slightly stronger binding of the Pd1. Fluorescence competition experiments with site-markers (eosin Y and ibuprofen) for HSA were used to locate the binding site of Pd1 to the HSA. Additionally, the interaction with DNA and HSA was studied by molecular docking and the revealed results were in good agreement with the experimentally obtained ones. Pd1 complex exhibited cytotoxicity toward human (HCT116) and mouse cell lines (CT26) of colorectal cancer, mouse (4T1) and human (MDA-MB468) breast cancer lines and non-cancerous mouse mesenchymal stem cells (mMSC). In addition, Pd1 complex demonstrated significant selectivity towards cancer cells over non-cancerous mMSC, indicating a high potential to eliminate malignant cells without affecting normal cells. It induced apoptosis in CT26 cells, effectively arrested the cell cycle in the S phase, and selectively down-regulated cyclin D and cyclin E. Moreover, it can alter the expression of cell cycle regulators by increasing p21 and decreasing p-AKT. These findings confirm its ability to disrupt key tumor cell survival signals and suggest that the Pd1 complex is a potent candidate for effective cancer treatment.

Extremity dosimetry for exposed workers in positron emission tomography in Bosnia and Herzegovina.

Occupational exposure in Bosnia and Herzegovina is regulated by the national regulation on radiation protection for occupational and public exposure. All radiation workers are required to be monitored using whole body passive thermoluminescent dosemeters and, in case of non-uniform external exposures, by dosemeters that would indicate dose to the most affected body parts. Exposed workers are almost exclusively employed in the medical field, and some of them work in nuclear medicine departments where they handle unsealed radioactive sources. Introduction of the positron emission tomography-computed tomography (PET-CT) in two largest clinical centers in the country was expected to cause the increase of equivalent doses to hands received by staff handling the positron emitting radionuclides. Hence, routine monitoring of finger doses became a necessity. The purpose of this study was to evaluate the available data on monitoring with ring dosemeters during PET-CT procedure in two hospitals in Bosnia and Herzegovina and compare them with other practices in the nuclear medicine department, as well as with the results of monitoring in other countries. In general, results confirm that effective doses, as well as equivalent doses to hands, are well below annual dose limits. Finger dosemeters have been proven to be an invaluable asset in the incidental situations that sometimes occur in nuclear medicine departments. Different number of patients and differences in injection methodologies are identified as a possible source of differences between doses in two hospitals. Overall, routine evaluation of doses to hands provides a sound basis for possible optimization processes, as well as confirmation of good practices.

New ruthenium(II) complexes with quinone diimine and substituted bipyridine as inert ligands: synthesis, characterization, mechanism of action, DNA/HSA binding affinity and cytotoxic activity.

This paper presents the synthesis and structural characterization of a series of new ruthenium(II) complexes 1-7, with the general formula mer-[RuL3(N-N)Cl]Cl, where L is 2,2':6',2''-terpyridine (tpy) or 4'-(4-chlorophenyl)-2,2':6',2''-terpyridine (Cl-Ph-tpy) and N-N is o-benzoquinonediimine (o-bqdi), 2,3-naphthoquinonediimine (nqdi), 4,4'-dimethyl-2,2'-bipyridine (dmbpy) or 2,2'-bipyridine-4,4'-dicarboxylic acid (dcbpy). The kinetic results showed that the ligand substitution reactions of new Ru(II)-polypyridyl complexes with biomolecules were affected by different substituents and the aromaticity of meridional tridentate and bidentate spectator ligands as well as by the nature of the entering nucleophile. The reactivity of the complexes increases in the order: dmbpy < dcbipy < nqdi < o-bqdi. In addition, quantum chemical calculations were performed to support the interpretation and discussion of the experimental data. Furthermore, combining ethidium bromide (EB) and Hoechst 33258 (2-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl)benzimidazo-2-yl]-benzimidazole) fluorescence assay results implied that 1-7 might interact with calf thymus DNA through partial intercalation and/or minor groove binding. The human serum albumin (HAS)-fluorescence binding studies involving the site markers, eosin Y, as a marker for site I of subdomain IIA, and ibuprofen, as a marker for site II of subdomain IIIA, showed that Ru(II) compounds bind to both sites with moderately strong affinity (Kb = 104-106 M-1). Moreover, these DNA/HSA experimental results were confirmed by molecular docking. Complexes 2, 5 and 6 exerted good to strong and highly selective cytotoxic activity against breast adenocarcinoma (MDA-MB 231), colorectal carcinoma (HCT116) and cervix adenocarcinoma (HeLa). Depending on their structure and cell line, the complexes acted differently in terms of their influence on autophagy, the cell cycle and the engaged apoptotic pathway.

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton's jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

Synthesis, characterization, biomolecular interactions, molecular docking, and in vitro and in vivo anticancer activities of novel ruthenium(III) Schiff base complexes.

In order to discover new anticancer drugs, novel ruthenium(III) complexes [Ru(L)Cl(H2O)], where L is tetradentate Schiff base bis(acetylacetone)ethylendiimine (acacen, 1), bis(benzoylacetone)ethylendiimine (bzacen, 2), (acetylacetone)(benzoylaceton)ethylendiimine (acacbzacen, 3), bis(acetylacetone)propylendiimine (acacpn, 4), bis(benzoylacetone)propylendiimine (bzacpn, 5) or (acetylacetone)(benzoylaceton)propylendiimine (acacbzacpn, 6), were synthesized. The complexes 1 - 6 were characterized by elemental analysis, molar conductometry, and by various spectroscopic techniques, such as UV-Vis, IR, EPR, and ESI-MS. Based on in vitro DNA/BSA experiments, complexes 2 (bzacen) and 5 (bzacpn) with two aromatic rings showed the highest DNA/BSA-activity, suggesting that the presence of the aromatic ring on the tetradentate Schiff base ligand contributes to increased activity. Moreover, these two compounds showed the highest cytotoxic effects toward human, A549 and murine LLC1 lung cancer cells. These complexes altered the ratio of anti- and pro-apoptotic molecules and induced apoptosis of A549 cells. Further, complexes 2 and 5 reduced the percentage of Mcl1 and Bcl2 expressing LLC1 cells, induced their apoptotic death and exerted an antiproliferative effect against LLC1. Finally, complex 5 reduced the volume of mouse primary heterotopic Lewis lung cancer, while complex 2 reduced the incidence and mean number of metastases per lung. Additionally, molecular docking with DNA revealed that the reduced number of aromatic rings or their absence causes lower intercalative properties of the complexes in order: 2 > 5 > 6 > 3 > 4 > 1. It was observed that conventional hydrogen bonds and hydrophobic interactions contribute to the stabilization of the structures of complex-DNA. A molecular docking study with BSA revealed a predominance of 1 - 6 in binding affinity to the active site III, a third D-shaped hydrophobic pocket within subdomain IB.

Role of π-acceptor effects in controlling the lability of novel monofunctional Pt(II) and Pd(II) complexes: crystal structure of [Pt(tripyridinedimethane)Cl]Cl.

The kinetics and mechanism of substitution reactions of novel monofunctional [Pt(tpdm)Cl](+) and [Pd(tpdm)Cl](+) complexes (where tpdm = tripyridinedimethane) and their aqua analogues with thiourea (tu), L-methionine (L-met), glutathione (GSH), and guanosine-5'-monophosphate (5'-GMP) were studied in 0.1 M NaClO(4) at pH = 2.5 (in the presence of 10 mM NaCl for reactions of the chlorido complexes). The reactivity of the investigated nucleophiles follows the order tu > l-met > GSH > 5'-GMP. The reported rate constants showed the higher reactivity of the Pd(II) complexes as well as the higher reactivity of the aqua complex than the corresponding chlorido complex. The negative values reported for the activation entropy as well as the activation volume confirmed an associative substitution mode. In addition, the molecular and crystal structure of [Pt(tpdm)Cl]Cl was determined by X-ray crystallography. The compound crystallizes in a monoclinic space group C2/c with two independent molecules of the complex and unit cell dimensions of a = 38.303(2) Å, b = 9.2555(5) Å, c = 27.586(2) Å, ß = 133.573(1)°, and V = 7058.3(8) Å(3). The cationic complex [Pt(tpdm)Cl](+) exhibits square-planar coordination around the Pt(II) center. The lability of the [Pt(tpdm)Cl](+) complex is orders of magnitude lower than that of [Pt(terpyridine)Cl](+). Quantum chemical calculations were performed on the [Pt(tpdm)Cl](+) and [Pt(terpyridine)Cl](+) complexes and their reactions with thiourea. Theoretical computations for the corresponding Ni(II) complexes clearly demonstrated that π-back-bonding properties of the terpyridine chelate can account for acceleration of the nucleophilic substitution process as compared to the tpdm chelate, where introduction of two methylene groups prevents such an effective π-back bonding.

Synthesis, characterization, antitumor potential, and investigation of mechanism of action of copper(ii) complexes with acylpyruvates as ligands: interactions with biomolecules and kinetic study.

Considering the urgency of finding a cure for vicious diseases such as tumors, we have synthesized and characterized a small series of new copper(ii) complexes with biologically important ligands such as acylpyruvate. In addition to this, we used another four copper(ii) complexes, with ligands of the same type to examine the antitumor potential. The antitumor potential of the copper(ii) complexes was examined on three tumor cell lines and one normal human cell line using the MTT assay. All seven tested complexes showed very good cytotoxic effects. Two copper complexes that showed the best antitumor potential were selected for further testing that showed the best potential for potential application in the future. The mechanism of activity of these complexes was examined in detail using tests such as cell cycle, ROS level, oxidative DNA damage, and proteins related to hypoxia analysis. In addition, we examined the binding abilities of these complexes with biomolecules (Guo, Ino, 5'-GMP, BSA, and DNA). The results showed that the tested compounds bind strongly to DNA molecules through intercalation. Also, it has been shown that the tested compounds adequately bind to the BSA molecule, which indicates an even greater potential for some future application of these compounds in clinical practice.

Synthesis, characterization, DNA interactions and biological activity of new palladium(II) complexes with some derivatives of 2-aminothiazoles.

Newly palladium(II) complexes (C1, C2) with derivatives of 2-aminothiazoles (L1 = 2-amino-6-methylbenzothiazole, L2 = 2-amino-6-chlorobenzothiazole), general formula [PdL2Cl2] were synthesized and characterized by elemental microanalyses, IR, NMR spectroscopy and X-ray spectroscopy in case of [Pd(L2)2Cl2]. The kinetic of the substitution reactions of complexes and the nucleophiles, such as guanosine-5'-monophosphate (5'-GMP), tripeptide glutathione (GSH) and amino acid L-methionine (L-Met), were studied by stopped-flow technique. The complex C2 was always more reactive, while the order of the reactivity of the nucleophiles, due to the associative mode of the reaction, was L-Met > GSH > 5'-GMP. In order to determine the type of interactions between palladium(II) complexes and calf thymus DNA (CT-DNA), we used electronic absorption spectroscopy, viscosity measurements, and fluorescence spectroscopic studies, while interactions with bovine serum albumin (BSA) were determined only with fluorescence spectroscopic studies. The observed results confirmed that both complexes bound to DNA by groove binding. The significantly strong interaction with BSA, especially for complex C2, was also observed. In vitro cytotoxic activity was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MB-468, HCT116 and mesenchymal stem cells (mMSC). C1 complex showed higher cytotoxic activity against CT26 cell line. Flow cytometry analysis showed that C1 stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule and decelerated proliferation by decreasing Cyclin-D and increasing expression of P21. In vitro antimicrobial activity for ligands and corresponding palladium(II) complexes was investigated by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. Tested compounds exhibited selective and moderate activity.