Ultrastructural morphometry of neurosecretory granules in the neuroblastomas of paediatric patients

Introduction: Peripheral neuroblastic tumours are among the most frequently detected tumours in paediatric patients. A correlation between the aggressiveness of these tumours and their clinical prognosis has been discovered. Neurosecretory granules containing neurotransmitters, an ultrastructural feature of neurons, were found in the cells of peripheral neuroblastic tumours, as well. Aim: The aim of this study was to compare the number of neurosecretory granules found in cells of tumours, with a favourable prognosis to the corresponding number in cells of tumours and with an unfavourable one with the use of transmission electron microscopy (TEM). Material and Methods: Ten tissue biopsies were obtained from paediatric patients diagnosed with peripheral neuroblastic tumours. The tumour samples were first frozen, then fixed in glutaraldehyde, and embedded in epoxy resin. Ultrastructural morphometric analysis was performed on ultrathin sections. Results: When observed under a transmission electron microscope (TEM), neurosecretory granules appear as round structures of a small diameter, with an electron dense centre surrounded by an electron lucent, peripheral halo. It was found that the cells of tumours with an unfavourable prognosis have more neurosecretory granules (141.2 ± 89.18 per 60 cells per sample, on average) in their cytoplasm than the cells of tumours with a favourable one (37.2 ± 41.17 per 60 cells per sample, on average). Conclusion: The larger number of neurosecretory granules found in cells of tumours with an unfavourable prognosis could potentially shed some light on the role these structures play in tumourigenesis of peripheral neuroblastic tumours

Higher percentage of in vitro apoptotic cells at time of diagnosis in patients with chronic lymphocytic leukemia indicate earlier treatment requirement: Ten years follow up

Introduction. Chronic lymphocytic leukemia (CLL) has an extremely variable clinical course. Biological reasons for that wide variation in clinical course and survival rates in CLL patients are not fully understood. Objective. The aim of the study was to evaluate the value of spontaneous apoptosis of CLL cells in vitro determined at presentation of disease, in prediction of treatment requirements and evolution of the CLL. Methods. Malignant B cells were isolated from the whole blood of 30 newly diagnosed CLL patients and cultured for 24 hours in RPMI-1640 medium supplemented with 10% of serum obtained from the same CLL patient. Cells were later fixed and processed for embedding in Epon, or cell smears were prepared and stained with TUNEL technique. Results. Ten-year follow-up revealed that patients with lower percentage of cells in apoptosis at presentation of disease had significant longer time treatment initiation (log rank test p<0.05). On the contrary, apoptosis of CLL cells was not shown to have significant impact on survival of patients (Kaplan Meier log rank test p>0.05). Conclusion. The results of this study emphasize the importance of apoptosis of CLL cells at the time of the initial diagnosis in pathobiology of this disease. [Projekat Ministarstva nauke Republike Srbije, br. 41025

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

Razvoj novih antidijabetičkih lekova na bazi polioksometalatnih nanoklastera

Zahvaljujući brojnim istraživanjima koja su ispitivala biološka svojstva strukturno različitih polioksometalata, došlo se do zapažanja da ova kompleksna neorganska jedinjenja, pored antimikrobnog i antitumorskog delovanja, mogu biti delotvorna u snižavanju hiperglikemije kod pacova sa eksperimentalno izazvanim dijabetesom. Stoga, cilj ove studije je bio da se ispitaju antidijabetički potencijal i mogući toksični efekti dva polioksovolframata:(NH4 )14[NaP5 W30O110]·31H2 O, {NaP5 W30} i K14[AgP5 W30O110]·22H2 O·6KCl, {AgP5 W30}. U cilju realizacije postavljenog cilja, korišćena su tri eksperimentalna modela: (1) antihiperglikemijska screening studija u kojoj je ispitivan uticaj jednokratne intraperitonealne primene {NaP5 W30} i {AgP5 W30} (5, 10 i 20 mg/kg) na snižavanje hiperglikemije kod dijabetičkih pacova, (2) akutna peroralna toksikološka studija koja je istraživala hepato- i nefrotoksične efekte odabranih heteropolivolframata kod zdravih pacova i (3) studija posvećena rasvetljavanju mogućih mehanizama antidijabetičkog delovanja heteropolivolframata. Rezultati screening studije su pokazali da su oba ispitivana heteropolivolframata efikasna u snižavanju hiperglikemije, s tim što se {NaP5 W30}, u odnosu na {AgP5 W30}, pokazao kao moćniji antihiperglikemijski agens. Rezultati biohemijskih parametara funkcije i patohistološka analiza jetre i bubrege korišćenjem konvencionalne svetlosne i transmisione elektronske mikroskopije pokazuju da dvonedeljna primena {NaP5 W30} i {AgP5 W30} (20 mg/kg) izaziva blagi do umereni stepen hepato- i nefrotoksičnosti kod zdravih životinja. U poslednjem eksperimentalnom protokolu, pokazano je da tronedeljna peroralna primena {NaP5 W30} (20 mg/kg) povećava koncentraciju insulina u serumu dijabetičkih pacova, što može biti jedan od mehanizama njegovog antidijabetičkog delovanja. Takođe, pokazano je da {NaP5 W30} ispoljava hepato-, nefro-, kardio- i neuroprotektivno dejstvo kod dijabetičkih pacova, što je procenjeno na osnovu analize: (1) relativne mase organa, (2) biohemijskih parametara funkcije, (3) parametara oksidativnog stresa u homogenatu tkiva, (4) aktivnosti acetilholinesteraze, Na+ /K+-ATPaze i ecto-ATPaza u sinaptozomima i (5) patohistoloških promena u tkivima korišćenjem konvencionalne svetlosne i transmisione elektronske mikroskopije. Stoga, {NaP5 W30} i {AgP5 W30} mogu se smatrati mogućim neinsulinskim lekovima-kandidatima u terapiji dijabetesa tipa 2, koji bi se podvrgli daljim pretkliničkim istraživanjima.Simpozijum „Stremljenja i novine u medicini“ Medicinskog fakulteta u Beogradu, Beograd, 04-08. decembra, 2023

Monolacunary Wells-Dawson Polyoxometalate as a Novel Contrast Agent for Computed Tomography: A Comprehensive Study on In Vivo Toxicity and Biodistribution

Polyoxotungstate nanoclusters have recently emerged as promising contrast agents for computed tomography (CT). In order to evaluate their clinical potential, in this study, we evaluated the in vitro CT imaging properties, potential toxic effects in vivo, and tissue distribution of monolacunary Wells–Dawson polyoxometalate, α2-K10P2W17O61.20H2O (mono-WD POM). Mono-WD POM showed superior X-ray attenuation compared to other tungsten-containing nanoclusters (its parent WD-POM and Keggin POM) and the standard iodine-based contrast agent (iohexol). The calculated X-ray attenuation linear slope for mono-WD POM was significantly higher compared to parent WD-POM, Keggin POM, and iohexol (5.97 ± 0.14 vs. 4.84 ± 0.05, 4.55 ± 0.16, and 4.30 ± 0.09, respectively). Acute oral (maximum-administered dose (MAD) = 960 mg/kg) and intravenous administration (1/10, 1/5, and 1/3 MAD) of mono-WD POM did not induce unexpected changes in rats’ general habits or mortality. Results of blood gas analysis, CO-oximetry status, and the levels of electrolytes, glucose, lactate, creatinine, and BUN demonstrated a dose-dependent tendency 14 days after intravenous administration of mono-WD POM. The most significant differences compared to the control were observed for 1/3 MAD, being approximately seventy times higher than the typically used dose (0.015 mmol W/kg) of tungsten-based contrast agents. The highest tungsten deposition was found in the kidney (1/3 MAD—0.67 ± 0.12; 1/5 MAD—0.59 ± 0.07; 1/10 MAD—0.54 ± 0.05), which corresponded to detected morphological irregularities, electrolyte imbalance, and increased BUN levels

Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors.

We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling

The assessment of maturation changes of humic coal organic matter - Insights from closed-system pyrolysis experiments

The artificial maturation changes of the humic coal organic matter (OM) from late diagenesis to the semianthracite stage of coalification were investigated using the closed-system pyrolysis. Two extracted coal samples, a subbituminous coal 26C (with a random vitrinite reflectance Rr = 0.42%) and a high volatile bituminous coal 30C (Rr = 0.56%), were pyrolysed during 4 hat two different temperatures, 250 degrees C and 400 degrees C. The applied petrographic and organic geochemical analyses showed that the humic coal OM is sensitive to the artificial thermal stress. A good correlation between the changes in petrographic characteristics and the derived liquid products was observed. The contents of all vitrinite macerals, as well as total vitrinite, increased during the thermal maturation. The abundance of liptinite macerals decreased during the maturation and they practically disappeared after 4 h of the pyrolysis at 400 degrees C. The total inertinite contents increased after the pyrolysis at 250 degrees C, yet to be decreased after the pyrolysis at 400 degrees C. The content of organic carbon increased from coals to the solid residues at 400 degrees C, whereas the contents of total hydrogen and sulphur decreased. The yields of gases, liquid products and solid residues at respective temperatures were very similar for both coals, regardless of the rank. As expected, the pyrolysis at 400 degrees C gave higher gas and liquid yields than at 250 degrees C. Regarding the liquid pyrolysates, more pronounced changes were observed in the distribution of aromatic compounds than in saturated biomarkers. The distributions of the aromatics indicate that apart from cracking and isomerisation (the formation of thermodynamically more stable isomers), the reactions of dehydrocyclization, condensation (the formation of more stable conjugated systems) and aromatization, as well as reactions involving H2S, elemental sulphur and reactive phenyl radicals released during the pyrolysis, also play an important role. It was shown that bentonitic clay plays a more important role in the subsequent aromatization of the OM than the initial maturity of the coal OM. n-Alkane parameters were useful at lower maturities (up to similar to 0.75% Rr), whereas sterane, naphthalene and phenanthrene maturity ratios are applicable at higher maturities ( gt 0.75% Rr). Hopane parameters showed variable sensitivities over the investigated maturity range (0.5-1.8% Rr). The most sensitive maturity parameters in the wide range of the humic OM maturity (0.5-1.8% Rr) are vitrinite reflectance, phenylnaphthalene ratio, methylchrysene index 2 and a parameter proposed in this study, based on the distribution of methylfluorenes (MF): (2- + 3-MF) / (1- + 4-MF). Generally, maturity parameters based solely on the isomerisation reactions of alkylaromatics, which do not include alkylation/dealkylation processes, are more appropriate for the humic OM, particularly at higher maturities. (C) 2016 Elsevier B.V. All rights reserved

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