PHYSICAL ACTIVITY AND DIETARY HABITS AMONG SECOND YEAR STUDENTS OF THE SCHOOL OF MEDICINE, UNIVERSITY OF BELGRADE

The aim of this study was to investigate the physical activity and dietary habits of second year medical students of the School of Medicine, University of Belgrade. The level of physical activity and dietary habits were also investigated according to other factors: gender, sports activity before and after attending college and students’ self-assessment related to their physical activity level. All second year medical students (490 students: 155 male and 355 female) were asked to participate in the study by filling out questionnaires during one week in the 2016/17 school year. They filled out a demographic questionnaire, a shorter version of International Physical Activity Questionnaire (IPAQ) as well as a food frequency questionnaire comprising 13 indicator variables. The Mann-Whitney U test was used to test the overall differences between male and female students, while a Correlation Analysis was investigated using the Spearman correlation coefficient. There is a statistically significant difference in sport habits between both male and female students, before and after enrolling in college. The Spearman correlation coefficient analysis showed that there is a moderate positive correlation between the levels of physical activity calculated from the IPAQ questionnaire with sports activity habits of the students after enrolling in college (0.344) as well as with self-assessment of the level of physical activity by the students (0.440). Second year medical students have good dietary habits that could be responsible for their adequate body composition

Expression and functional activity of nucleoside transporters in human choroid plexus

Abstract Background Human equilibrative nucleoside transporters (hENTs) 1-3 and human concentrative nucleoside transporters (hCNTs) 1-3 in the human choroid plexus (hCP) play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. Methods Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR); for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E) and the quantification cycle (Cq) were calculated. The uptake of [3H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. Results RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E-Cq value being only about 40 fold less that the E-Cq value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [3H]inosine by the CP samples was linear and consisted of an Na+-dependent component, which was probably mediated by hCNT3, and Na+-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), when used at a concentration of 0.5 μM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 μM NBMPR, a finding that suggested the involvement of hENT2 in uptake. Conclusion Transcripts for hENT1-3 and hCNT3 were detected in human CP; mRNA for hENT3, an intracellularly located nucleoside transporter, was the most abundant. Human CP took up radiolabelled inosine by both concentrative and equilibrative processes. Concentrative uptake was probably mediated by hCNT3; the equilibrative uptake was mediated only by hENT2. The hENT1 transport activity was absent, which could suggest either that this protein was absent in the CP cells or that it was confined to the basolateral side of the CP epithelium.</p

Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [¹⁴C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture

Abstract Background Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture. Methods To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts. Results and conclusion CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 ± 9 Ω cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion. However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [14C] adenosine in the donor chamber was mostly present as [14C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [14C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.</p

HPLC chromatograms of the investigated extracts of old man’s beard and usnic acid.

<p>Supercritical CO₂ extract (E1); ether fraction of Soxhlet extract (E2); ethanol fraction of Soxhlet extract (E3); macerate (E4).</p

The effects of supercritical CO₂ extract of old man’s beard (E1) and usnic acid (UA) on the induction of autophagy.

<p>On the left, the intracellular acidification was assessed by flow cytometry following acridine orange staining of B16 (A) and C6 (B) cells and is represented by histograms of mean fluorescence FL3/FL1 intensity increase under the 24 h treatment with UA and E1 (25 μg/mL). On the right, the fluorescent micrograph of tumor cells stained with acridine orange indicates the presence of acidic cytoplasmic vesicles in B16 (A) and C6 (B) cells treated with the most active extract-E1. Relative increase in FL3/FL1 fluorescence (left) represents means ± SD values from at least three independent experiments (*p<0.05 refers to control as untreated cells).</p

The effects of supercritical CO₂ extract of old man’s beard (E1) and usnic acid (UA) on the cell cycle progression of B16 (A) and C6 (B) cells.

<p>Numbers on histograms represents the percentage of cells in appropriate phase of cell cycle (sub G₀, G₁, S and G₂) under the treatment with UA (25 μg/mL) and the most active extract-E1 (25 μg/mL) for B16 (A) and C6 (B) cells.</p

The effects of supercritical CO₂ extract of old man’s beard (E1) and usnic acid (UA) on the ROS production.

<p>B16 cells (A) and C6 cells (B) were incubated with E1 and UA (25 μg/mL) and ROS production was measured after 24 h by flow cytometry. On the left, relative increase in DHR fluorescence (compared to control as untreated cells) under the tumor cells treatment with E1 and UA (concentrations are expressed in μg/mL) is presented as mean ± SD values from three independent experiments (*p<0.05 refers to control-untreated cells). On the right, representative histograms of mean fluorescence intensity increase under the 24 h treatment with most active extract-E1 for both B16 (A) and C6 (B) cells.</p

Cytotoxic dose dependent action of old man’s beard extracts/usnic acid.

<p>B16 mouse melanoma cells and C6 rat glioma cells were incubated with different concentrations of samples: usnic acid (UA), supercritical CO₂ extract (E1), ether fraction of Soxhlet extract (E2), ethanol fraction of Soxhlet extract (E3) and macerate (E4). The cell viability was assessed after 24 h by using acid phosphatase test. The results are presented as means ± SD values of triplicate observations from a representative of three independent experiments (*p<0.05 refers to control-untreated cells).</p

Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-alpha (HIF-1 alpha) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain. (C) 2015 Elsevier Inc. All rights reserved.Ministry of Education, Science and Technological Development of Serbia {[}III 41031, III 41025