Half-sandwich ruthenium(II)-arene complexes: synthesis, spectroscopic studies, biological properties, and molecular modeling

In search for antitumor metal-based drugs that would mitigate the severe side-effects of cisplatin, Ru(II) complexes are gaining increasing recent interest. In this work, we report on the synthesis, characterization (1H- and 13C-NMR, FT-IR), and cytotoxicity studies of two new half-sandwich organometallic Ru(II) complexes of the general formula [Ru(η6-arene)(XY)Cl](PF6) where arene = benzene or toluene and XY = bidentates: dipyrido[3,2-a:2′,3′-c]phenazine (dppz) or 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip), which are bound to Ru(II) via two phenanthroline-N atoms in a characteristic “piano-stool” configuration of Ru(II)-arene complexes—as confirmed by vibrational and NMR spectra. In addition, cytotoxic studies were performed for similar half-sandwich organometallic [Ru(η6-p-cymene)(Me2dppz)Cl]PF6 complex (Me2dppz = 11,12-dimethyl-dipyrido[3,2-a:2′,3′-c]phenazine). This study is complemented with elaborate modeling with density functional theory (DFT) calculations, which provided insight into reactive sites of Ru(II) structures, further detailed by molecular docking on the B-DNA dodecamer, which identified binding sites and affinities: most pronounced for the [Ru(η6-benzene)(aip)Cl](PF6) in both A-T and G-C regions of the DNA minor groove. Cytotoxic activity was probed versus tumor cell lines B16, C6, and U251 (B16 mouse melanoma, C6 rat glioma, U251 human glioblastoma) and non-tumor cell line HACAT (HACAT normal human keratinocytes).This is the peer-reviewed version of the following article:Nikolić, S.; Grgurić-Šipka, S.; Djordjević, I. S.; Dahmani, R.; Dekanski, D.; Vidičević, S.; Tošić, J.; Mitić, D.; Grubišić, S. Half-Sandwich Ruthenium(II)-Arene Complexes: Synthesis, Spectroscopic Studies, Biological Properties, and Molecular Modeling. Journal of Coordination Chemistry 2019, 72 (1), 148–163. [https://doi.org/10.1080/00958972.2018.1553298].Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3753

Graphene Quantum Dots show protective effect in animal model of neuroinflammation

Background: Experimental autoimmune encephalomyelitis (EAE) is one of the most studied model of neuroinflammation, used to test immunomodulatory and antiinflammatory drugs. Graphene quantum dots (GQD) are oval graphite twodimensional sheets with a diameter <100 nm, one carbon atom thickness, with potential applications in biomedicine. Objective: To investigate the potential protective effect of GQD in EAE model. Methods: Female DA rats were immunized with spinal cord homogenate and Freund’s complete adjuvant. GQD treatment (10 mg/kg, ip) was administrated during the inductive, effector and both phases of a disease. MAP kinase (MAPK) and Akt activity in popliteal lymph nodes (PLN) and CNS was determined by western blot. Quantitative PCR and flow cytometry were used to examine the expression of proinflammatory cytokines and specific transcription factors while infiltration of GQD in cells/tissues was detected by transmission electron microscopy. GQD antiinflamatory/direct cytoprotective effect was analyzed on oligodendrocyte and neuron cell cultures by MTT assay. Data were analized by Mann Whitney test (p<0.05 was considered as statistical significant difference). Results: GQD administration, in all phases of EAE, significantly reduced clinical score of a disease. Clinical improvement correlates with increase in activity of ERK, p38 and Akt that is followed by reduction of Th1 cell response in PLN and infiltrated spinal coard T cells. Due to its capacity to infiltrate cells and tissues, GQD exhibits direct cytoprotective effect on CNS. Additionaly, GQD reduced the expression of proinflammatory cytokines in ConA stimulated lymphocytes. Conclusion: GQD alleviate EAE, through direct cytoprotective effect on CNS and inhibition of Th1 cell response.Poster Session: Neuroimmunoendocrine Interaction

The role of endoplasmic reticulum stress in the induction of experimental autoimmune encephalomyelitis in rats

Endoplazmatski retikulum (ER) predstavlja unutarćelijsku organelu sa brojnim funkcijama, pri čemu je jedna od najznačajnijih sinteza i obrada proteina. Nakupljanje nefunkcionalnih, pogrešno uvijenih ili neuvijenih proteina unutar lumena ER dovodi do nastanka ER stresa. Ukoliko dođe do nastanka ER stresa, ćelija pokušava da ga prevaziđe na različite načine, među kojima je i pokretanje UPR (unfolded protein response). Do pokretanja UPR dolazi nakon odvajanja šaperona ER, GRP78, od luminalnih domena tri transmembranska senzora ER stresa: IRE-1α, ATF6 i PERK. Aktivacija IRE-1α, PERK i ATF6 pokreće signalne kaskade koje za cilj imaju adaptaciju ćelije na stres i obnovu funkcije ER. Ukoliko obnova funkcije ER izostane, u cilju održavanja homeostaze, pokreće se programirana ćelijska smrt, apoptoza. Aktivacija IRE-1α dovodi do obrade iRNK za transkripcioni faktor XBP1 i do aktivacije MAP kinaza i NFƙB. Aktivirani ATF6 povećava ekspresiju gena za XBP1, dok PERK dovodi do fosforilacije, a time i inaktivacije eIF2α, što dalje dovodi do smanjene sinteze proteina. Fosforilacija eIF2α dovodi i do selektivne sinteze ATF4 koji, kod ireverzibilno oštećenih ćelija, aktivira CHOP koji indukuje apoptozu. ER stres i UPR imaju ulogu u patogenezi različitih bolesti, među koje spada i multipla skleroza (MS). Kako većina današnjih saznanja o MS potiče iz njenog animalnog modela, eksperimentalnog autoimunskog encefalomijelitisa (EAE), ciljevi ovog istraživanja su bili detekcija prisustva ER stresa u ćelijama imunskog sistema tokom indukcije EAE i ispitivanje efekata inhibicije ER stresa u ćelijama imunskog sistema na razvoj EAE. Imajući na umu razlike između pacova DA i AO soja u podložnosti za razvoj EAE, prisustvo ER stresa je prvo ispitivano u drenirajućim limfnim čvorovima imunizovanih DA i AO životinja, pri čemu je uočeno da kod oba soja pacova dolazi do aktivacije ER stresa. Indukcija ER stresa je bila nešto jača kod AO pacova, kod kojih je došlo do ranije aktivacije CHOP i fosforilacije eIF2α, dok je kod DA, ali ne i AO pacova, došlo do fosforilacije ERK kinaze. Hematoksilin-eozin bojenjem preseka drenirajućih limfnih čvorova uočeno je ranije i intenzivnije povećanje broja limfocita kod imunizovanih DA pacova. Pored toga, kod imunizovanih DA pacova uočeno je prisustvo atipičnih plazmocita, odnosno Mot ćelija sa Raselovim telima, što ukazuje na dilataciju ER u plazmocitima u ovom modelu EAE. Da se ER stres javlja u različitim tipovima ćelija unutar drenirajućih limfnih čvorova potvrđuje nalaz elektronske mikroskopije, gde se proširen lumen ER uočava u limfocitima, plazmocitima i makrofagima imunizovanih DA pacova. Ovaj nalaz je dodatno potvrđen i imunoblot analizom, gde se uočava porast markera ER stresa i u CD3+ i CD3- ćelijama. Pozitivna korelacija između markera ER stresa u mononuklearnim infiltratima kičmene moždine i kliničke aktivnosti bolesti (EAE skor 1-3) naglašava značaj aktivacije ER stresa i UPR u patogenezi EAE. Uticaj inhibicije ER stresa na klinički tok EAE ispitan je primenom hemijskog šaperona ursodeoksiholne kiseline (UDCA). Njena primena je dovela do poboljšanja kliničkog toka EAE, ali i do smanjene produkcije proinflamatornih citokina koji imaju značajnu ulogu u patogenezi EAE. UDCA je smanjila ekspresiju iRNK za efektorske citokine Th1 (IFNγ) i Th17 (IL-17) odgovora, kao i ekspresiju gena za transkripcione faktore i/ili citokine neophodne za diferencijaciju Th1 (T-bet, IL-12), odnosno Th17 ćelija (IL-23). Ekspresija gena za proinflamatorne citokine TNF i IL-1β je takođe bila snižena dejstvom UDCA. Nivoi markera ER stresa se nakon tretmana UDCA, za razliku od imunizovane grupe koja nije primila UDCA, nisu značajno razlikovali u odnosu na neimunizovane životinje, što sugeriše da je UDCA sprečila aktivaciju ER stresa kod imunizovanih DA pacova. Rezultati ove doktorske disertacije ukazuju da ER stres u ćelijama imunskog sistema ima značajnu ulogu tokom patogeneze EAE, kao i da inhibicija ER stresa dovodi do poboljšanja kliničkog toka EAE. Povoljan efekat inhibicije ER stresa je najverovatnije posledica smanjene produkcije encefalitogenih proinflamatornih i Th1/Th17 citokina, što bi moglo da ima važne implikacije za terapiju inflamatornih bolesti CNS.Endoplasmic reticulum (ER) is an intracellular organelle with multiple functions, with synthesis and processing of proteins being one of the most significant. Accumulation of non-functional, misfolded or unfolded proteins within the ER lumen leads to ER stress. Tthe cell tries to overcome ER stress in various ways, including the initiation of UPR (unfolded protein response). UPR starts after the dissociation of the ER chaperone GRP78 from the luminal domains of three transmembrane ER stress sensors: IRE-1α, ATF6 i PERK. Activation of IRE-1α, PERK and ATF6 initiates signaling cascades, which aim to adapt the cell to stress and restore the ER function. If ER function is not restored, in order to maintain homeostasis, programmed cell death (apoptosis) is initiated. Activation of IRE-1α leads to the processing of mRNA for the transcription factor XBP1, as well as activation of MAP kinases and NFƙB. Activated ATF6 increases the gene expression for XBP1, while PERK leads to phosphorylation and thus the inactivation of eIF2α, which further leads to decrease in protein synthesis. Phosphorylation eIF2α also leads to selective synthesis of ATF4, which in irreversibly damaged cells activates CHOP that leads to the initiation of apoptosis. ER stress and UPR play a role in pathogenesis of various diseases, including multiple sclerosis (MS). As the majority of current knowledge about MS stems from its animal model, experimental autoimmune encephalomyelitis (EAE), the goals of this study were detection of ER stress response in the immune system cells during EAE induction, as well as examination of the effect that ER stress inhibition in immune system has on the EAE development. Keeping in mind the differences between the DA and AO strain rats in susceptibility to EAE development, the presence of ER stress was first investigated in the draining lymph nodes of immunized DA and AO animals, where it was observed that ER stress is activated in both rat strains. The ER stress response was somewhat stronger in AO rats, with earlier CHOP activation and eIF2α phosphorylation in, while ERK phosphorylation occurred in DA, but not AO rats. Hematoxylin-eosin staining of draining lymph nodes sections revealed an earlier and more prominent lymphocyte proliferation in immunized DA rats. Furthermore, the presence of atypical plasma cells, i.e. Mott cells with Russell bodies, was observed in immunized DA rats, indicating ER dilatation in plasma cells in this experimental model of EAE. ER stress in different types of cells within the draining lymph nodes was confirmed by electron microscopy findings showing dilated ER lumen in lymphocytes, plasmocytes and macrophages of immunized DA rats. This finding was additionally confirmed by immunoblot analysis, where an increase in ER stress markers was observed in both CD3+ and CD3- cells. The positive correlation between ER stress markers in spinal cord-infiltrated mononuclear cells and the clinical activity of the disease (EAE score 1-3) highlights the significance of ER stress and UPR induction in the pathogenesis of EAE. The effect of ER stress inhibition on the clinical course of EAE was investigated using the chemical chaperone ursodeoxycholic acid (UDCA). In addition to improvingthe EAE clinical score, UDCA application also led to a decrease in the production of proinflammatory cytokines that play a significant role in the pathogenesis of EAE. UDCA reduced the expression of mRNA for the effector cytokines of Th1 (IFNγ) and Th17 (IL-17) response, as well as gene expression of transcription factors and/or cytokines necessary for the differentiation of Th1 (T-bet, IL-12) and Th17 cells (IL-23). Gene expression of the proinflammatory cytokines TNF and IL-1β was also decreased. The levels of ER stress markers in UDCA-treated group, in contrast to immunized rats that did not receive UDCA, were not significantly different in comparison to non-immunized animals, suggesting that UDCA prevented the activation of ER stress in EAE. The results of this doctoral dissertation indicate that ER stress in the immune system cells plays a significant role in the pathogenesis of EAE, as well as that the inhibition of ER stress leads to an improvement of the EAE clinical course. The positive effect of ER stress inhibition is most probably mediated by decreased production of encephalitogenic proinflammatory and Th1/Th17 cytokines which might have important implications for the therapy of CNS inflamatory diseases

In Sicilia sbarcati 800 migranti, altri seicento in arrivo. Ad Augusta i 24 morti dell'ultimo naufragio, repubblica.it, 27/08/2014

"I dispersi al largo della Libia sarebbero un centinaio. Tra i profughi approdati a Pozzallo c'era anche un gatto. Salvata una bimba di 14 mesi aggrappata a una tavola C'erano anche 81 donne e 133 minori tra i 449 migranti siriani, palestinesi ed egiziani arrivati a Pozzallo, nel ragusano, le cui operazioni di sbarco sono terminate intorno alle due della scorsa notte. Con il gruppo degli immigrati c'era anche un gatto, sottoposto poi a visita veterinaria per verificare che non fosse portatore..

Prognostic value of presepsin (soluble CD14-subtype) in diagnosis of ventilator-associated pneumonia and sepsis in trauma patients

Background/Aim. Presepsin (soluble CD14-subtype) is a fragment of CD14 produced in response to bacterial infections and a novel biomarker of pneumonia, sepsis and septic shock. The aim of this study was to compare sensitivity and specificity of persepsin, soluble CD14-subtype (sCD14-ST) with other biomarkers: procalcitonine (PCT), C-reactive protein (CRP) and leukocyte count (Le) in mechanically ventilated injured patients, as a marker of pneumonia, sepsis and septic shock. Methods. The prospective study was undertaken in trauma and surgery intensive care unit of the Emergency Center, the Clinical Center of Serbia from January to April 2013. The study included 39 trauma patients requiring mechanical ventilation, and who developed one of the following inclusion criteria: Systemic Inflammatory Response Syndrome (SIRS), ventilator associated pneumonia (VAP), sepsis and/or septic shock. On admission Acute Physiology and Chronic Health Evaluation II (APACHE II) Score and Injury Severity Score (ISS) were calculated. Seventy-two measurements of four biomarkers (presepsin, PCT, CRP and Le) were performed in 39 patients at the moments of diagnosis of SIRS, VAP, sepsis and/or septic shock (21 when SIRS diagnosis was established, 21 after the diagnosis of VAP, 18 at the moment of diagnosis of sepsis and the remaining 12 measurements were conducted while diagnosing the septic shock). The Sequential Organ Failure Assessment (SOFA) score was calculated at these points as well. Results. Patients were mainly severely injured (mean ISS = 24.2) and had moderately severe medical condition at admission (mean Apache II score, 14.5). Presepsin concentration significantly differed among all the four groups, except between sepsis and septic shock. The strongest positive correlation of presepsin evinced with PCT (r = 0.741, p < 0.001). The sCD14-ST indicated better performance in diagnosis of both VAP (AUC = 0.909) and sepsis (AUC = 0.899), compared to PCT (AUCs: 0.863, 0.885, respectively), CRP (AUCs: 0.703, 0.677, respectively) and Le (AUCs: 0.668, 0.700, respectively). Conclusion. This study revealed that sCD14-ST is a reliable biomarker for distinguishing sepsis severity. It also showed a good correlation with the infection development as well as worsening in injured patients. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no.175046

Graphene quantum dots show protective effect on a model of experimental autoimmune encephalomyelitis

29th Congress of the European-College-of-Neuropsychopharmacology (ECNP), Sep 17-20, 2016, Vienna, Austri

Graphene quantum dots inhibit T cell-mediated neuroinflammation in rats

We investigated the therapeutic capacity of nano-sized graphene sheets, called graphene quantum dots (GQD), in experimental autoimmune encephalomyelitis (EAE), an animal model of immune-mediated central nervous system (CNS) damage. Intraperitoneally administered GQD (10 mg/kg/day) accumulated in the lymph node and CNS cells of Dark Agouti rats in which EAE was induced by immunization with spinal cord homogenate in complete Freund's adjuvant. GQD significantly reduced clinical signs of EAE when applied throughout the course of the disease (day 0–32), while the protection was less pronounced if the treatment was limited to the induction (day 0–7 post-immunization) or effector (from day 8 onwards) phase of the disease. GQD treatment diminished immune infiltration, demyelination, axonal damage, and apoptotic death in the CNS of EAE animals. GQD also reduced the numbers of interferon-γ-expressing T helper (Th)1 cells, as well as the expression of Th1 transcription factor T-bet and proinflammatory cytokines tumor necrosis factor, interleukin-1, and granulocyte-macrophage colony-stimulating factor in the lymph nodes and CNS immune infitrates. The protective effect of GQD in EAE was associated with the activation of p38 and p42/44 mitogen-activated protein kinases (MAPK) and Akt in the lymph nodes and/or CNS. Finally, GQD protected oligodendrocytes and neurons from T cell-mediated damage in the in vitro conditions. Collectively, these data demonstrate the ability of GQD to gain access to both immune and CNS cells during neuroinflammation, and to alleviate immune-mediated CNS damage by modulating MAPK/Akt signaling and encephalitogenic Th1 immune response. © 2018 Elsevier Lt