Differentiation of human Embryonic Stem Cells (hESC) into neural progenitors as a tool to study both the pathways during early brain development and the neuroteratogenic effecys of ethanol

Differentiation of human Embryonic Stem Cells (hESC) into neural progenitors as a tool to study both the pathways during early brain development and the neuroteratogenic effects of ethanol Thesis: Jelena Kostic The main objective of this work is to use human neuroprogenitors (hNPs) cells from hESC as a tool to study the cellular and molecular events involved in early human neural development under physiological conditions and to study the teratogenic effects of ethanol during the initial formation of the CNS. Specific objectives include: Objectives - Development of an in vitro protocol of derivation of human neural progenitors (hNPs) from hESCs, which could mirror early stages of human brain development - Characterize the in vitro culture by evaluating the gene and protein expression of human Neuroprogenitors (hNPs) in culture during their proliferation and differentiation into mature cells (neurons, astrocytes and oligodendrocytes). - Assess whether the endocannabinoid system, including endocannabinoid receptors (CB1, CB2) and the enzymes involved in their synthesis (NAPE-PLD) and degradation (FAAH) of endocannabinoids (EC), are expressed in human neural progenitors during their differentiation to mature nervous cells. - Assess the gene and protein expression of TLR4 and TLR2 receptors during neural differentiation from hESC (in vitro) and during brain ontogeny in mice. - Finally, the last objective will investigate the actions of ethanol on the proliferation and differentiation of hNPs. Specifically we will assess the effects of different physiological concentrations of ethanol on: 1) gene and protein expressions during the derivation of hESC to NPs: 2) the proliferation and cells survival of the hNPs, 3) the NPs differentiation processes to mature neural cells, by assessing gene and protein expressions and morphological alterations, 4) the expression of endocannabinoid system and TLR4 and TLR2 in hESC, hNPs and brain development in mice. Results In this work we demonstrated the generation of human neural progenitors (hNPs) and differentiated neurons, oligodendrocytes and astrocytes from human embryonic stem cells (hESC) which mimics early brain development in humans. Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts and the proper timing of neurons and glial cells generation. Immunocytochemical and real time (RT)-polymerase chain reaction analyses reveal that cells appeared as clusters during neuroepithelial cell proliferation and that the genes associated with the neuroectodermal (Pax-6) and the endodermic (α-fetoprotein) lineages decreased in parallel to the upregulation of the genes of hNPs (nestin and Tuj1), followed by their differentiation into neurons (MAP-2+, GABA+), oligodendrocytes [galactocerebroside (GalC+)], and astrocytes (GFAP+). We further demonstrate, for the first time, that human NPs express the endocannabinoid receptors (CB1 and CB2) and the enzymes involved in endocannabinoids synthesis (NAPE-PLD) and degradation (FAAH). Using this in vitro culture, we demonstrate that ethanol exposure impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton, and affects the expression of different genes associated with neural differentiation. The results provide new insights into the effects of ethanol on human embryogenesis and neuroprogenitors and offer an opportunity to delineate potential therapeutic strategies to restore early ethanol-induced brain damage. We also show for the first time that TLR4 and TLR2 are expressed in both hESC and hNPs. In parallel, we demonstrated that TLR4 and TLR2 are expressed in mice brain development (prenatal and postnatal period). Conclusions 1- We establish an in vitro model for the generation of neural progenitors (NPs) from hESCs. 2- Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts, and the proper timing of neurons and glia cells generation. 3- Immunocytochemical and RT-PCR analyses reveal that hESC appeared as clusters during neuroepithelial cell proliferation, and that the genes associated with the neuroectodermal and endodermal lineages decrease in parallel with the up-regulation of the genes of neural progenitors, followed by their differentiation into neurons oligodendrocytes and astrocytes. 4- We demonstrate for the first time that human NPs express the endocannabinoid receptors (CB1, CB2) and the enzymes involved in endocannabinoids synthesis and degradation, as well as the TLR4 and TLR2 receptors. 5- By using this in vitro model, we show that ethanol exposure: - Impairs the derivation of hESCs towards neural cells, alters the disappearance of the endodermal marker AFP during NPs proliferation and up-regulates the transcription factors Nanog, Sox2 and Pax-6 during their differentiation. - Impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton and affects the expression of different genes associated with neural differentiation. 6- The results provide new insights into the effects of ethanol on early human embryogenesis and offer an opportunity to delineate potential therapeutic strategies to restore early brain damage induced by ethanol. 7- Finally, the present study provides not only a new tool to elucidate the mechanisms underlying early human neural and brain development, but also offers the possibility to assess the neuroteratogenic actions of ethanol during early embryogenesis. References Aguado T, Monory K, Palazuelos J, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzmán M, Galve-Roperh I (2005). The endoccanabinoid system drives neural progenitor proliferation FASEB J. (12):1704-6. Aguado T, Palazuelos J, Monory K, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzmán M, Galve-Roperh I (2006). The endoccanabinoid system promotes astroglial differentiation by acting on neural progenitor cells . J Neurosci. 1;26(5):1551-61. Alfonso-Loeches S, Pascual-Lucas M, Blanco AM, Sanches-Vera I, Guerri C. (2010) Pivotal role of TLR4 receptors in alchohol-induced neuroinflammation and brain damage. The Journal of Neuroscience, 30(24): 8285-8295. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS (2001). Enrichement of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172:383-397. Elkabetz Y, Panagiotakos G, Al Shamy G, D. Socci N, Tabar V and Lorenz Studer (2008). Human ES cell-derived neural rossetes reveal a functionally distinct early neural stem cell stage. Genes&Dev. 22:152-165. Guerri C, Alissa Bazinet and Edward P. Riley (2002). Foetal Alcohol Spectrum Disorders and alterations in brain and behaviour.Alcohol and alcoholism Mar-Apr;44(2):108-14. Guerri C. (2002) Mechanisms involved in central nervous system dysfunction induced by prenatal exposure. Neurotoxicology Res 4:327-35. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, and Benvenisty N. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. Feb;6(2):88-95. Nat R, M Nilbratt, S Narkilahti, B Winblad, O Hovatta and A Nordberg. (2007). Neurogenic neuroepithelial and radial glia cells generated from six human embryonic stem cell lines in serum free suspension and adherent cultures. Glia 55:385-99. Okun E, Griffoen KJ, Lathia JD, Tang SC, Matson MP and Arumugam TV. 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Modeling The Unemployment Rate At The Eu Level By Using Box-Jenkins Methodology

Unemployment, as a measure of market conditions, appears as a crucial economic problem and a phenomenon with considerable negative social consequences, and, as such, requires attention and adequate approach to finding solutions. Enormous unemployment rates are a reality not only in developing and transition countries, but also in some developed countries. Inadequately conducted privatization, unsuccessful transfer of workers from the public to the private sector, inefficiency in attracting foreign direct investment, and the world economic crisis of 2008 have made unemployment a universal disease of modern society. The paper presents economic models in which the unemployment rate is the central analyzed phenomenon. In this context, an important task of European economic policy-makers is to project future unemployment rates. Box-Jenkins methodology, i.e. the seasonal ARIMA model, is one approach to the modeling of time series, or, more specifically, for forecasting future values. The subject of this paper is the analysis of the evolution of the unemployment problem on the basis of the values in the period from 2000 to 2015, based on the case of 28 countries of the European Union. Building on the research subject, the purpose of the paper is to create the statistical model for forecasting the values of the monthly unemployment rates in the European Union for the future and establishing its trend. Keywords: Unemployment, labor market, Box-Jenkins methodology, the European Unio

Ribe kao biološki indikator u proceni kvaliteta vode reke Tamiš

Materijal za istraživanje ihtiofaune Tamiša sakupljen je avgusta i oktobra 2009. u periodu srednjeg vodostaja kao i aprila 2010. godine u periodu visokog vodostaja na lokacijama Sečanj, Banatski Despotovac i Opovo. Prikupljanje uzoraka vršeno je pomoću aparata za elektroribolov i stajaćim mrežarskim alatima dužine od 37 do 100 metara, promera okaca od 45 do 100 mm i dubine 3 do 5 metara. Ovom prilikom evidentirano je 28 vrsta riba iz 8 familija. Prema sastavu ihtiofaune određen je indeks saprobnosti prema Pantle-Buck-u koji iznosi 2.15 i koji ukazuje na to da voda Tamiša pripada drugoj klasi boniteta

Primary percutaneous coronary intervention in a patient with right internal mammary artery graft originating from arteria lusoria dextra

Introduction Congenital anomalies of the aortic arch, although numerous and heterogeneous, occur in less than 1% of individuals at autopsies. Left aortic arch with an aberrant right subclavian artery, also called arteria lusoria dextra, is the most common anomaly of the aortic arch, occurring in 0.5-2.5% of individuals. Case Outline We report the case of a 48-year-old man suffering from acute inferoposterior-wall ST elevation myocardial infarction successfully treated by primary percutaneous coronary intervention. Ten years ago, the patient had undergone coronary artery bypass graft surgery with the implantation of two arterial grafts - left and right internal mammary arteries on both left anterior descending and right coronary artery. After several attempts to canulate truncus brachiocephalicus, angiogram revealed the left aortic arch with the aberrant right subclavian artery. To our knowledge, this is the first described case of primary percutaneous coronary intervention via the aberrant right subclavian artery and right internal mammary artery graft with stent implantation in the infarct related lesion of the distal segment of right coronary artery. Subsequent 64-multidetector computed tomography confirmed the angiographic findings. Conclusion Early recognition of congenital anomalies of the aortic arch and its great vessels, even before coronary artery bypass graft surgery, could be crucial for the urgent and successful treatment of patients with life-threatening conditions, such as ST segment elevation myocardial infarction

The first record of the Chinese pond mussel Sinanodonta woodiana (Lea, 1834) in Montenegro

Sinanodonta woodiana (Lea, 1834), Chinese pond mussel (Bivalvia: Unionoida: Unionidae) is one of the most invasive aquatic macroinvertebrate species found in Europe. We report the Chinese pond mussel for the first time in Montenegro, in August 2012, in Lake Šasko (Adriatic part of the Central Mediterranean subarea). One specimen of the Chinese pond mussel was observed in a habitat with a predominantly silt-clay substrate. The main pathway of species introduction was evaluated to be via fish stocking

BALKAN ENDEMIC NEPHROPATHY AND MALIGNANT UROTHELIAL TUMORS

One of the features of Balcan endemic nephropathy (BEN) is higher frequency of urothelium malignant tumors, primarily of pyelon (Mtp) and urether (Mtu). Jablanica region is known for the presence of endemic, hypoendemic and non-endemic areas with BEN. The aim of our research was to analyze the appearance of MTUi n endemic settlements of Jablanica region with BEN and to see what the relation of tumor frequency between endemic and non-endemic settlements is. The appearance of MTU was analyzed on the basis of operative protocol data of Urology department, The Health Center in Leskovac and Urology Clinic of The Clinical Center in Nis for the period from 1978 to 2002. We collected data about our patiens regarding their sex, age, the place of living and the place of birth. In order to make classification of settlements we used data of the Institute for Nephrology and hemodialysis (INH) in Nis. Data on total number of population living in these settlements were obtained from the official registration data published in 1981 and 1991. The incidence rate was calculated in the sample of 100,000 people.The average annual incidence rate (AAIR) of MTU in endemic settlements for the considered period is 37.82 (tumors of urether and pyelon - 17.56; malignant tumors of urinary bladder (MTUB 20.26); in hypoendemic settlements the rate is 13.28 (MTp and Mtu - 5.06; MTUB - 8.22); and in non-endemic urban settlements it is 7.35 (Mtu and MTp - 1.04, MTUB - 6.31).AAIR of MTU in endemic areas is 2.85 times higher when compared to hypoendemic areas; it is 6.75 times higher than in non-endemic urban areas, and 5.15 times higher than the rate of non-endemic rural areas. Mtu and MTp are 18.68 times more frequent in endemic settlements than in non-endemic urban areas and 3.47 times more frequent when compared to hypoendemic settlements. The linear trend of the diseased from MTp and MTu in endemic areas of Jablanica region for 25-year period was slowly decreasing according to statistics (y= -0.0054x + 0.59; r2=0.0031.High frequency of Mtu, primarily of Mtu and MTp in areas with BEN, probably points to the common nephropathogenic and cancerogenic etiologic factor, confirming thus the existence of positive correlation between BEN and malignant tumors of upper urothelium (MTUU)

Fizičko-hemijska karakterizacija uzoraka vode sa teritorije Rasinskog okruga

The aim of the study was a preliminary examination on water quality (U01-U08) in Rasina area, through the standard physico-chemical parameters: temperature, pH, conductivity, turbidity and total organic matter. All samples were odorless and tasteless. The results for temperature, pH and conductivity were within the values defined by Regulation on the quality of drinking water, except for the pH value of the sample U05. Increased turbidity was observed in the sample U03 (13.5 NTU). On the basis of the results for total organic matter (1.2 to 5.5 mg O2/L) that were within the permissible range, it can be concluded that all the studied samples belong to the first (I) class water quality.Cilj rada bio je preliminarno ispitivanje kvaliteta voda sa područja Rasinskog okruga (U01-U08) određivanjem standardnih fizičko-hemijskih parametara: temperature, pH-vrednosti, provodljivost, mutnoće i ukupne organske materije. Svi uzorci su bili bez mirisa i ukusa. Dobijeni rezultati za temperaturu, pH i provodljivost bili su u okviru vrednosti predviđenih Pravilnikom o kvalitetu vode za piće, osim pH vrednost uzorka U05. Povišena mutnoća zabeležena je u uzorku U03 (13,5 NTU). Na osnovu vrednosti za ukupne organske materije (1,2-5,5 mg O2/L) koje su bile u granicama dozvoljenih može se zaključiti da svi ispitivani uzorci pripadaju vodama prve (I) klase kvaliteta

Computational Studies on Selected Macrolides Active against Escherichia coli Combined with the NMR Study of Tylosin A in Deuterated Chloroform

Abstract: Although many antibiotics are active against Gram-positive bacteria, fewer also show activity against Gram-negative bacteria. Here, we present a combination of in silico (electron ion-interaction potential, molecular docking, ADMET), NMR, and microbiological investigations of selected macrolides (14-membered, 15-membered, and 16-membered), aiming to discover the pattern of design for macrolides active against Gram-negative bacteria. Although the conforma-tional studies of 14-membered and 15-membered macrolides are abundant in the literature, 16-membered macrolides, and their most prominent representative tylosin A, have received rela-tively little research attention. We therefore report the complete 1H and 13C NMR assignment of tylosin A in deuterated chloroform, as well as its 3D solution structure determined through mo-lecular modelling (conformational search) and 2D ROESY NMR. Additionally, due to the degra-dation of tylosin A in deuterated chloroform, other species were also detected in 1D and 2D NMR spectra. We additionally studied the anti-bacterial activity of tylosin A and B against se-lected Gram-positive and Gram-negative bacteria

Kako poplave modifikuju genotoksični odgovor slatkovodnih riba?

U ovom radu ispitivan je potencijal dve vrste roda Abramis (Abramis bjoerkna i Abramis sapa) kao bioindikatora genotoksičnog potencijala vode reke Save primenom komet testa. Izabrani lokalitet pripada oblasti intezivne poljoprivredne aktivnosti, izložen je otpadnim vodama grada Obrenovca i nalazi se u blizini najveće termoelektrane u Srbiji, “Nikola Tesla”. Uzorkovanje je vršeno od januara do avgusta 2014. godine, obuhvatajući mesec maj kada je došlo do intezivnih poplava u slivu reke Save. Dobijeni rezultati ukazuju da se ispitivane vrste mogu koristiti za procenu genotoksičnog potencijala

Ispitivanje odabranih fizičko–hemijskih osobina i sadržaja makro- i mikroelemenata u vodi jezera Gruža

Water is one of the most precious natural resources. The downtrend in the quality of lake water and pollution with heavy metals cause serious threats to the environment in last decades. Particularly vulnerable are the lakes that are located near towns and villages. The aim of this study was to investigate the physicochemical parameters and levels of macro– and microelements in order to determine the water quality of Lake Gruža in the summer period. The results showed that the studied parameters were within the permissible levels, except of concentrations of iron, cobalt and vanadium.Voda predstavlja jedan od najdragocenijih prirodnih resursa. Opadanje kvaliteta jezerskih voda i zagađenje teškim metalima, predstavljaju ozbiljne pretnje za životnu sredinu u poslednjih nekoliko decenija. Posebno su ugrožena jezera koja se nalaze u blizini gradova i naselja. Cilj rada bio je ispitivanje osnovnih fizičko–hemijskih parametara i sadržaja makro– i mikroelemenata kako bi se utvrdio kvalitet vode jezera Gruža u letnjem periodu. Rezultati su pokazali da su ispitivani parametri u vodi bili u okviru dozvoljenih vrednosti, osim sadržaja gvožđa, kobalta i vanadijuma