Cell cycle progression or translation control is not essential for vesicular stomatitis virus oncolysis of hepatocellular carcinoma.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bepsilon have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bepsilon are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future

GENDER DIFFERENCES IN PHYSICAL ACTIVITY AMONG THE UNIVERSITY STUDENTS IN THE VISEGRAD (V4) COUNTRIES

Introduction. Sedentary way of life has become a global phenomenon in the past decades. Therefore, the number of people with excess weight has doubled in the past 30 years. Besides this fact, it has been justified that more than half of the population is overweight. Even young adults are affected by the problem. It is an important issue because 60% of the overweight young people keep their excess body weight in later adulthood increasing the risk of different diseases. Material and method. Our study aims at assessing the differences between the health status and the physical activity among young people (secondary school and university students) in the Visegrad (V4) countries. Our current research examines the differences in the physical activity among university students regarding their sexes (n=2237). SPSS 22.00 software was used for statistical analysis. Results. According to the results, we found significant differences (p0.05). In Slovakia, we found significant differences between sexes in total MET/week and walking activities (MET/week) (p<0.001), thus, female students were found to be more active than males caused by the higher rates of walking activities of women. Conclusion. The V4 countries are not in an advantageous situation concerning physical activity in the European framework because only 21-35% of the population does sports once a week. According to our results, university students show a more positive picture on physical activity than the adult population. However, there are some specific risk groups. 43.8% of female and 57.3% male students can be considered as persons with high physical activity. Our findings may play a major role in the development of intervention programs targeting young people and in the concern of the differences between sexes. Furthermore, these results may call young people’s attention to health maintenance to preserve their fitness for getting better activity figures

Revisionsutskott - påverkas revisorns oberoende?

Bakgrund och problem: Revision har fått en ökad betydelse efter flera bolagsskandaler som har orsakat en stor brist på förtroende för bolagsstyrning och finansiell rapportering. Reaktioner till följd av dessa skandaler och krav från den internationella kapitalmarknaden har lett till implementeringen av Svensk Kod för Bolagsstyrning. Syftet med Koden är att bidra till förbättrad styrning av svenska bolag och därigenom öka förtroendet för svenskt näringsliv. Ett av kraven i Koden är att vissa noterade svenska företag skall införa revisionsutskott. Syftet med dessa utskott är att öka kommunikationen mellan revisorn och styrelsen samt att öka fokus på redovisnings- och revisionsfrågor. Inom utskottens ansvarsområde ligger även att fastställa riktlinjer för vilka andra tjänster än revision som bolaget får använda sina revisorer till. Syfte: Vi vill med denna uppsats undersöka om revisorns oberoende kommer att påverkas av revisionsutskotten och i så fall på vilket sätt. Avgränsningar: Respondenter av intresse för denna uppsats har varit revisorer på någon av de fyra stora revisionsbyråerna samt sakkunniga inom det behandlade ämnet. Kodens effekter för näringslivet i övrigt kommer inte att behandlas, utan endast de avsnitt som berör revisionsutskotten och revisorns oberoende. Oberoendet som utreds är i första hand oberoendet gentemot revisionsutskott och i andra hand oberoendet gentemot företagsledning och styrelse. Metod: Empirin för denna studie baseras på kvalitativ datainsamling genom intervjuer med revisorer och sakkunniga. Koden har varit en viktig källa för att förstå revisionsutskottens kontext och syfte. Tonvikten i denna uppsats ligger på den deskriptiva undersökningsansatsen även om vissa element kan sägas vara explorativa. Analys och slutsatser: Revisionsutskotten kommer främst att ha en positiv effekt på revisorns oberoende förutsatt att en praxis för utskotten upparbetas samt att ledamöterna i utskotten besitter den kompetens inom redovisning och revision som krävs. En annan slutsats är att ordet oberoende inte är lämpligt för att definiera förhållandet mellan revisorn och dennes uppdragsgivare då revisorn alltid kommer att vara beroende av lön eller motsvarande och då det är ett uppdragsförhållande. Förslag till fortsatt forskning: Det vore intressant att göra om uppsatsen när Koden varit igång ett tag. Vidare vore ett annat ämne att undersöka skillnaderna mellan revisorer och sakkunniga vad gäller synen på oberoende

Immortalized human hepatocytes, PH5CH8 cell line.

<p><b>A)</b> VSV growth in immortalized non-neoplastic hepatocytes (PH5CH8) was compared to HCC cell lines and primary human hepatocytes (PHH). Cells were infected with VSV-wt and the IFN-inducer mutant VSV-M51R at an MOI of 0.001 and titers were determined at different time-points post-infection as indicated. Data shown are the average of three independent experiments and error bars represent standard deviation. Significance of viral titers in PHH was calculated by comparison with titers in Huh-7 (** p<0.01). <b>B)</b> Proliferation of the cell lines: HepG2, Huh-7 and PH5CH8. Cells were plated at the concentration of 5×10³ cells per well, and their numbers were determined up to four days after plating by MTT proliferation assay. Data are representative of three independent experiments. <b>C)</b> Western blot showing the expression levels of cyclin D1 and CDK4 in HepG2, Huh-7 and PH5CH8 cells compared to PHH.</p

Cell type specificity of the CDK4 inhibitor.

<p><b>A)</b> PH5CH8 and Huh-7 cells were treated with DMSO or CDK4 inhibitor at different concentrations. Infection with VSV-wt was performed at an MOI of 0.1 for 24 hr. Viral titers were determined by TCID50. Data represent the average of three independent experiments ± standard deviation. <b>B)</b> Protein expression of CDK4, cyclin D1 and Akt in the lysates of the above described experiment was performed by Western blot analysis.</p

RNA interference assay for eIF2B epsilon (eIF2Bε).

<p><b>A)</b> Western blot analysis of mock-infected cultures was performed for each experiment to assess the efficiency of RNA silencing. <b>B)</b> HepG2, Huh-7 and PH5CH8 cells were transfected with siRNA for eIF2B<b>ε</b> at a concentration of 100 nM. As controls, cells were transfected in parallel with control siRNA or mock-transfected. At 72 hours post-transfection, cells were infected with VSV-wt using an MOI of 0.1 for 8 hours. Results are the average of three independent experiments and error bars indicate the standard deviation.</p

RNA interference assay for eIF4E and S6K.

<p>HCC and PH5CH8 cells were transfected with siRNA for eIF4E <b>A)</b> or S6K <b>B)</b> at a concentration of 100 nM. As controls, cells were transfected in parallel with siRNA scramble or mock-transfected. At 72 hours post-transfection, cells were infected with VSV-wt using an MOI of 0.1 and viral titers were determined 8 hours post-infection. Results are the average of three independent experiments, and error bars indicate the standard deviation. Mock-infected cultures were used to control the efficiency of the mRNA silencing by Western blot analyses. Western blot analysis was performed for each experiment.</p

VSV replication and cell-cycle progression.

<p><b>A)</b> HCC cells (HepG2 and Huh-7) and immortalized human hepatocytes (PH5CH8) were mock-treated (DMSO) or treated with different cell-cycle inhibitors: CDK4 inhibitor (CDK4); roscovitine (Rosco); Akt inhibitor (AKT IV); Ly294002 (Ly29); Aphidicolin (Aphid). Cultures were infected with VSV-wt at an MOI of 0.1 and viral titers were determined 24 hr post-infection by TCID 50. Data represent the average of 3 independent experiments. <b>B)</b> MTT proliferation assay in mock-treated or cell cycle inhibitor-treated cells. <b>C)</b> Analysis of cell cycle phases in Huh7 cells after treatment with cycle inhibitors: CDK4 inhibitor (CDK4); roscovitine (Rosco); Akt inhibitor (AKT IV); Ly294002 (Ly29); aphidicolin (Aphid). Samples were prepared in triplicate, and representative data from three independent experiments are shown (p<0.01). Typical FACS pattern of Huh7 cells after treatment with DMSO and LY294002 and PI staining is shown.</p

Rapamycin activity on VSV replication.

<p><b>A)</b> HepG2, Huh-7 and PH5CH8 cells were incubated overnight with increasing concentrations of rapamycin (20, 100 and 500 nM), or in the case of the controls, DMSO was added. VSV infection was performed at an MOI of 0.1 for 24 hours in the presence of fresh inhibitor. The viral titers shown are the average of three independent experiments. <b>B)</b> All cell lines were treated with 50 nM of rapamycin as described above and infected with VSV-wt at an MOI of 0.1. Viral titers were determined at 8 hours post-infection. The data represent at least two independent experiments ± standard deviation. <b>C)</b> Cell lysates of mock- and rapamycin-treated cells were analysed by Western blot for detection of the phosphorylated forms of kinase p70S6k and eIF4E and their corresponding base-line expression. <b>D)</b> MTT proliferation assay in mock-treated (DMSO) and rapamycin-treated (RAPA) cultures. Data represent the mean of at least three independent experiments ± standard deviation (* p< 0.05; *** p<0.001).</p

Effects of concomitant inhibition of mTOR and MNK on VSV proliferation.

<p>Cells were mock-treated (DMSO) or treated with rapamycin at 50 nm, MNK1 inhibitor at 20 µM, alone or together as indicated. <b>A)</b> Cell proliferation assays were performed using the MTT assay. Representative results of at least two independent experiments are shown. <b>B)</b> Western blot analysis of lysates obtained by PH5CH8 and HepG2 cell lines mock-treated (DMSO) or treated with rapamycin (RAPA), MNK inhibitor (MNK in) alone or in combination (RAPA+MNK in). The levels of S6K and eIF4E phosphorylated forms were monitored after inhibitor treatment. <b>C)</b> Cells were infected with VSV-wt at an MOI of 0.1 for 24 hours. Viral titers represent the mean ± standard deviation of three experiments.</p