Retrotransposon Tto1 as a tool for gene targeting in Arabidopsis thaliana

Ciljana inaktivacija gena počela se primjenjivati na miševima 80-ih godina prošlog stoljeća. Uspješnost ove strategije temelji se na relativno visokoj učestalosti homologne rekombinacije u somatskim stanicama miša i učinkovitom sustavu pozitivno-negativne selekcije. S druge strane, u biljkama se ugradnja strane DNA odvija pretežno procesom ilegitimne rekombinacije. Ovim radom započeto je istraživanje mogućnosti ciljane inaktivacije gena biljke-modela Arabidopsis thaliana (uročnjak) upotrebom retrotranspozona duhana Tto1. Ideja se temelji na manipulaciji procesa reverzne transkripcije, ključnog koraka u životnom ciklusu retrotranspozona, u svrhu proizvodnje supstrata (cDNA) za homolognu rekombinaciju. Transfomacijom bakterijama vrste Agrobacterium tumefaciens stvoreno je nekoliko transgenih linija uročnjaka s različitim varijantama retrotranspozona Tto1. Pomoću tih linija željelo se istražiti da li se Tto1 prepisuje u novom domaćinu te da li može završiti životni ciklus s ugrađenim genom biljegom. U slučaju pozitivnog ishoda, pristupilo bi se zamjeni gena biljega za gen uročnjaka koji želimo ciljano inaktivirati. Pokazano je da su sve priređene varijante Tto1 transkripcijski aktivne u uročnjaku, te da se nestabilnost Tto1 binarnih vektora u agrobakterijama, opisana u literaturi, ne ispoljava u uvjetima kratkotrajnog kultiviranja. Također, preliminarni rezultati na temelju fenotipske ekspresije gena biljega pokazali su da Tto1 može završiti životni ciklus. Međutim, dosadašnjim istraživanjima nije se uspjelo dokazati nove kopije Tto1 u genomu uročnjaka.Gene targeting was first applied on mice during the 80-ies of the last century. The success of this strategy is based on a relatively high frequency of homologous recombination in mouse somatic cells and an effective system of positive-negative selection. On the other hand, in plants the integration of exogenous DNA occurs predominantly by illegitimate recombination. In this work we studied the possibility of gene targeting in Arabidopsis thaliana using tobacco retrotransposon Tto1. The idea is based on the manipulation of the process of reverse transcription, a key step in the life cycle of retrotransposon, for production of substrate (cDNA) for homologous recombination. By applying Agrobacterium tumefaciens-mediated transformation several transgenic lines of A. thaliana with different variants of retrotransposon Tto1 were created. Using these lines it was explored whether Tto1 was transcripted into the new host, and whether Tto1 can complete the life cycle with an integrated marker gene. In the case of positive outcome, we will approach to replacing gene marker for a gene that we want to specifically inactivate. It was shown that all variants of cloned Tto1 were transcriptionally active and previously described instability of Tto1 binary vectors in Agrobacterium tumefaciens didn't manifest itself in short-term cultivation. Preliminary results based on gene marker expression, have shown that a Tto1 can complete its life cycle. However, so far new copies were not detected in the genome of transformed A.thaliana plants

Retrotransposon Tto1 as a tool for gene targeting in Arabidopsis thaliana

Ciljana inaktivacija gena počela se primjenjivati na miševima 80-ih godina prošlog stoljeća. Uspješnost ove strategije temelji se na relativno visokoj učestalosti homologne rekombinacije u somatskim stanicama miša i učinkovitom sustavu pozitivno-negativne selekcije. S druge strane, u biljkama se ugradnja strane DNA odvija pretežno procesom ilegitimne rekombinacije. Ovim radom započeto je istraživanje mogućnosti ciljane inaktivacije gena biljke-modela Arabidopsis thaliana (uročnjak) upotrebom retrotranspozona duhana Tto1. Ideja se temelji na manipulaciji procesa reverzne transkripcije, ključnog koraka u životnom ciklusu retrotranspozona, u svrhu proizvodnje supstrata (cDNA) za homolognu rekombinaciju. Transfomacijom bakterijama vrste Agrobacterium tumefaciens stvoreno je nekoliko transgenih linija uročnjaka s različitim varijantama retrotranspozona Tto1. Pomoću tih linija željelo se istražiti da li se Tto1 prepisuje u novom domaćinu te da li može završiti životni ciklus s ugrađenim genom biljegom. U slučaju pozitivnog ishoda, pristupilo bi se zamjeni gena biljega za gen uročnjaka koji želimo ciljano inaktivirati. Pokazano je da su sve priređene varijante Tto1 transkripcijski aktivne u uročnjaku, te da se nestabilnost Tto1 binarnih vektora u agrobakterijama, opisana u literaturi, ne ispoljava u uvjetima kratkotrajnog kultiviranja. Također, preliminarni rezultati na temelju fenotipske ekspresije gena biljega pokazali su da Tto1 može završiti životni ciklus. Međutim, dosadašnjim istraživanjima nije se uspjelo dokazati nove kopije Tto1 u genomu uročnjaka.Gene targeting was first applied on mice during the 80-ies of the last century. The success of this strategy is based on a relatively high frequency of homologous recombination in mouse somatic cells and an effective system of positive-negative selection. On the other hand, in plants the integration of exogenous DNA occurs predominantly by illegitimate recombination. In this work we studied the possibility of gene targeting in Arabidopsis thaliana using tobacco retrotransposon Tto1. The idea is based on the manipulation of the process of reverse transcription, a key step in the life cycle of retrotransposon, for production of substrate (cDNA) for homologous recombination. By applying Agrobacterium tumefaciens-mediated transformation several transgenic lines of A. thaliana with different variants of retrotransposon Tto1 were created. Using these lines it was explored whether Tto1 was transcripted into the new host, and whether Tto1 can complete the life cycle with an integrated marker gene. In the case of positive outcome, we will approach to replacing gene marker for a gene that we want to specifically inactivate. It was shown that all variants of cloned Tto1 were transcriptionally active and previously described instability of Tto1 binary vectors in Agrobacterium tumefaciens didn't manifest itself in short-term cultivation. Preliminary results based on gene marker expression, have shown that a Tto1 can complete its life cycle. However, so far new copies were not detected in the genome of transformed A.thaliana plants

Effects of the low-level western corn rootworm egg infestation on maize plants in the field

From the economic perspective, the western corn rootworm (WCR), Diabrotica virgifera sp. virgifera (Col., Chrysomelidae), poses the gravest threat to the field maize production in Serbia. The purpose of this study is to examine the impact of WCR larvae on the morphology of maize characters during a low-level artificial egg infestation. A field experiment involving the Serbian cultivar 'NS-640' was carried ou Serbia, in 2016. In the experimental field, a total of 96 maize plants were selected, marked and arranged in 48 pairs. Each pair consisted of an infested plant (WCR eggs injected in the root zone) and an uninfested plant (distillate water injected in the root zone). The number of leaves, height and stem diameter of the plants observed were recorded. Root damage and root weight were measured and evaluated at the end of the trail. For the purpose of statistical analysis, the non-parametric Kruskal-Wallis one-way analysis of variance and a correlation matrix were used. The statistical analysis performed indicate a highly significant difference in the number of leaves and plant height between the infested and uninfested maize plants examined in July (the third field observation). During the last field inspection, significant differences were recorded only between the stem diameters of the infested and uninfested maize plants. A negative correlation was found to exist between the root damage and root weight of the plant pairs. There were positive correlations between the stem diameter, plant height and number of leaves of the infested plants, whereas positive correlations were found between the root damage and plant height of the uninfested plants

Mercuration of Some Aliphatic Aldehydes. The Crystal Structure of 2,2-Bis(chloromercuri)butyraldehyde

Tris ( chloromercuri)acetaldehyde (1), 2,2-bis( chloromercuri)propionaldehyde (II) and 2,2-bis(chloromercuri)butyraldehyde (III) were obtained by mercuration of the corresponding aldehyde in an aqueous mercury(II) chloride solution containing sodium acetate. The mercurated aldehydes (II) and (III) are described for the first time. The crystal structure of (III) was determined by means of Patterson and Fourier syntheses based on threedimensional X-ray diffractometer intensity data and refined by the full-matrix least- squares method to an R value of 0.049. Crystals are orthorhombic, space group Pna21 with z = 4 formula units in the unit cell of the dimensions : a= 1114.6(6), b = 1121.3(5), c = 657.3(3) pm, Dots= 4.51and Deale = 4.38 Mg m-3• The structure consists of discrete moleculesof 2,2-bis(chloromercuri)butyraldehyde. The Hg-C bond lengthsare 216(2) and 209(2) pm. The Hg-C-Hg bond angle is 103(1) 0 and the C-Hg-Cl bond angles are 173(2)0 and 172(2)0 , respectively

Supplementary material for the article: Nikolic, V.; Loncarevic, B.; Popovic, A. Biodegradation of Copolymer Obtained by Grafting Reaction between Methacrylic Acid and Starch. Polymer Bulletin 2019, 76 (5), 2197–2213. https://doi.org/10.1007/s00289-018-2484-x

Supplementary material for: [https://doi.org/10.1007/s00289-018-2484-x]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/341

Analiza antiapoptotskog proteina bcl-2 u skvamocelularnom karcinomu usne regije

Aim: The aim of this study was to analyze the presence of the anti-apoptotic protein bcl-2 in oral squamous cell carcinoma and determine its potential role in the development and progression of this type of tumor. Materials and methods: The expression of bcl-2 was determined in 28 paraffin blocks of oral squamous cell carcinoma using the immunohistochemical method. The percentage of the immuno-reactive cells in positively stained tumor regions was determined using the microscopic analysis and Ozaria software. Results: Positive immunohistochemical test was observed in 19 out of 28 samples (68%) as follows: in 11 samples there was a low (+), in four a moderate (++) and in the last four a high percentage (+++) of stained cells. In the group of patients at the low stage of the disease (T2), 50% of tumor samples showed bcl-2 protein expression whereas in the higher stages (T3 and T4) of positively stained samples, this percentage was 67%. There was a trend of an increasing number of cells with positive bcl-2 staining in the tumors of higher clinical stages but not the level of bcl-2 protein expression. Conclusion: Both parameters, the presence of bcl-2 staining and the percentage of cells with bcl-2 immunoexpression, may act as additional prognostic parameters that indicate an increased proliferative tumor potential.Cilj ove studije bio je analiza prisustva antiapoptotskog proteina bcl-2 u skvamocelularnom karcinomu usne regije i procena njegove eventualne uloge u razvoju i progresiji ove vrste tumora. Materijal i metode: Na uzorku od 28 parafinskih blokova skvamocelularnog karcinoma usne regije, imunohistohemijskom metodom ispitan je ekspresioni status bcl-2 proteina. Mikroskopskom analizom i primenom softvera- Ozaria određen je procenat imunoreaktivnih ćelija u pozitivno obojenim tumorskim regijama. Rezultat: Pozitivnu imunohistohemijsku obojenost pokazalo je 19 od 28 uzoraka (68%) i to: 11 je bilo sa niskim (+), 4 sa srednjim (++) i 4 sa visokim procentom (+++) obojenih ćelija. U grupi pacijenata niskog stadijuma (T2) 50 % uzoraka tumora je pokazivalo ekspresiju bcl-2 proteina dok je u višim stadijumima (T3 i T4) pozitivnih uzoraka bilo 67%. Postojao je trend porasta broja ćelija sa pozitivnom bcl-2 obojenošću kod tumora u višim kliničkim stadijumima, ali ne i povećan nivo ekspresije bcl-2 proteina. Zaključak: Oba parametra, prisustvo bcl-2 obojenosti i procenat ćelija sa bcl-2 imunoekspresijom, mogu predstavljati dopunske prognostičke parametre koji ukazuju na povećan proliferativni potencijal tumora

Supplementary material for the article: Nikolic, V.; Loncarevic, B.; Popovic, A. Biodegradation of Copolymer Obtained by Grafting Reaction between Methacrylic Acid and Starch. Polymer Bulletin 2019, 76 (5), 2197–2213. https://doi.org/10.1007/s00289-018-2484-x

Supplementary material for: [https://doi.org/10.1007/s00289-018-2484-x]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/341

Prevalencija bakterija Enterococcus faecalis i Porphyromonas gingivalis u inficiranim kanalima korena zuba i njihova osetljivost na endodontsko lečenje - molekularna studija

Introduction Because apical periodontitis is recognizably an infectious disease, elimination or reduction of intracanal bacteria is of utmost importance for optimum treatment outcome. Objective The prevalence of Enterococcus faecalis and Porphyromonas gingivalis in infected root canals was studied Also, the effect of endodontic therapy by using intracanal medicaments, calcium hydroxide paste (CH) or gutta-percha points containing calcium hydroxide (CH-GP) or chlorhexidine (CHX-GP) on these microorganisms was assessed by polymerase chain reaction (PCR) assay. Methods Fifty-one patients with chronic apical periodontitis were randomly allocated in one of the following groups according to the intracanal medicament used: CH, CH-GP and CHX-GP group. Bacterial samples were taken upon access (S1), after chemomechanical instrumentation (S2) and after 15-day medication (S3). PCR assay was used to detect the presence of selected bacteria. Results E. faecalis was detected in 49% (25/51) and P. gingivalis in 17.6% (9/51) of the samples. Samples which showed no bacterial presence at S1 were excluded from further analysis. Overall analysis of all 29 samples revealed significant differences between S1 and S2 (p lt 0.001), S2 and S3 (p lt 0.05), and S1 and S3 (p lt 0.001). When distinction was made between the intracanal medications, there was a significant difference in the number of PCR positive samples between S1 and S2, S1 and S3, but not between S2 and S3 samples. Conclusion E. faecalis is more prevalent than P. gingivalis in primary endodontic infection. Intracanal medication in conduction with instrumentation and irrigation efficiently eliminates E. faecalis and P. gingivalis from infected root canals.Uvod Budući da je apeksni parodontitis oboljenje koje nastaje usled postojanja infekcije, za postizanje optimalnog ishoda lečenja najznačajnije je uklanjanje bakterija iz kanala korena zuba ili barem smanjenje njihovog broja. Cilj rada Cilj ovog rada je bio da se ispita prevalencija bakterija Enterococcus faecalis i Porphyromonas gingivalis u inficiranim kanalima korena zuba i ustanovi efekat endodontskog lečenja primenom intrakanalnih medikamenata - kalcijum-hidroksidne suspenzije (CH), gutaperka-poena na bazi kalcijum-hidroksida (CH-GP) ili gutaperka-poena na bazi hlorheksidina (CHX-GP) - na ove mikroorganizme metodom lančane reakcije polimerizacije (PCR). Metode rada Istraživanjem je obuhvaćena 51 osoba koja je imala zub s hroničnim apeksnim parodontitisom. Ispitanici su metodom slučajnog uzorka svrstani u tri grupe u zavisnosti od vrste primenjenog intrakanalnog medikamenta (CH, CH-GP i CHX-GP). Bakterijski uzorci iz kanala korena sakupljani su pri inicijalnom ulasku u kanal korena (S1), posle hemomehaničke obrade (S2) i posle petnaestodnevnog lečenja (S3). PCR analiza je korišćena za određivanje prisustva ispitivanih bakterija. Rezultati E. faecalis je izolovan iz 25 (49%) uzoraka, a P. gingivalis iz devet (17,6%). Uzorci u kojima nisu nađene bakterije u S1 fazi isključeni su iz dalje analize. Analiza svih 29 uzoraka je ukazala na statistički značajnu razliku između S1 i S2 uzoraka (p lt 0,001), S2 i S3 (p lt 0,05) i S1 i S3 (p lt 0,001). Kada se uzme u obzir vrsta intrakanalnog medikamenta, statistički značajna razlika u broju PCR-pozitivnih uzoraka zabeležena je između S1 i S2, S1 i S3, ali ne i između S2 i S3. Zaključak U primarnoj endodontskoj infekciji E. faecalis se češće javlja od P. gingivalis. Intrakanalna medikacija zajedno s instrumentacijom i irigacijom efikasno uklanja E. faecalis i P. gingivalis iz inficiranih kanala korena

NON-ALCOHOLIC FATTY LIVER DISEASE

Paralelno s porastom prevalencije pretilosti i metaboličkog sindroma nealkoholna masna bolest jetre - NAFLD (engl. Non-Alcoholic Fatty Liver Disease) sve se više prepoznaje kao najčešća bolest jetre odraslih i djece. Unatoč sve većoj prevalenciji, NAFLD ostaje uglavnom nedijagnosticiran i netretiran u rutinskoj medicinskoj praksi. Većina bolesnika s NAFLD-om nema nikakvih simptoma, dok samo rijetki navode nelagodu u gornjem desnom kvadrantu abdomena ili pojačani umor. Dijagnoza se najčešće postavlja slučajno tijekom pregleda abdomena ultrazvukom učinjenim zbog nekog drugog razloga, ili nalazom povišenih jetrenih biokemijskih testova. Rano prepoznavanje od velike je važnosti za pravodobno, ispravno i uspješno liječenje. U primarnoj zdravstvenoj zaštiti malo se zna o procesima koji se odnose na identifi kaciju, dijagnostiku i upućivanje bolesnika na daljnje pretrage specijalistima konzultantima. Razlikovanje steatoze od steatohepatitisa i procjena stupnja uznapredovalosti fi broze unutar steatohepatitisa od iznimne je važnosti. U tom pogledu još uvijek je vodeći ”zlatni standard” patohistološka analiza tkiva dobivenog biopsijom. U postupanju s bolesnicima sa sumnjom na NAFLD obiteljskom liječniku od velike pomoći mogu biti stručne smjernice koje bi trebale biti usaglašene. Upravo bi takve smjernice mogle smanjiti ozbiljne posljedice kasnog prepoznavanja NAFLD-a. Istraživanja novijeg datuma pokazuju kako je NAFLD povezan s povišenom prevalencijom i incidencijom kardiovaskularnih komplikacija. Zbog svoje kompleksnosti, hepatičkih i ekstrahepatičkih komplikacija, heterogenosti u kliničkoj prezentaciji, histološkoj težini, prognozi i terapijskom ishodu NAFLD zahtijeva multidisciplinarni pristup s aktivnijom ulogom obiteljskog liječnika u preventivnoj skrbi, dijagnostici i liječenju osobito kod rizičnih osoba i skupina. Veću pozornost treba usmjeriti modifi kaciji životnih stilova (redukcija tjelesne težine, tjelesna aktivnost) i njihovoj praktičnoj provedbi.With the increasing prevalence of obesity and metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in adults and children. Despite the increasing prevalence, NAFLD remains largely undiagnosed and untreated in routine medical practice. Most patients with NAFLD have no symptoms, while only a few feel discomfort in the upper right quadrant of the abdomen or increased fatigue. The diagnosis is usually set during abdominal ultrasound examination, which is done for some other reason, or by elevated liver biochemical test findings. Early recognition is very important for correct and successful treatment. In primary health care, little is known about the processes related to the identifi cation, diagnosis and referral of patients to specialists. Differentiation between steatosis and steatohepatitis by assessing the severity of fi brosis within steatohepatitis is extremely important. Histopathologic analysis of tissue obtained by biopsy remains the gold standard in this fi eld. For family physician, guidelines that should be combined can be of great help in treating patients with suspected NAFLD. The serious consequences of late recognition of NAFLD could be reduced by such guidelines. Recent studies have shown that NAFLD is associated with an increased prevalence and incidence of cardiovascular complications. Because of their complexity, hepatic and extrahepatic complications, heterogeneity in clinical presentation, histologic severity, prognosis and therapeutic outcome, NAFLD requires a multidisciplinary approach with the active role of family physicians in preventive care, diagnosis and treatment, especially in individuals and groups at risk. Greater attention should be focused on lifestyle modifi cations (reduction in body weight and physical activity) and their practical implementation

Mikrobiloški status kanala korena endodonski neuspešno lečenih zuba

Introduction the main objective of endodontic treatment is to eliminate infection from root canal and prevent re-infection by three-dimensional hermetic obturation of the canal system. Endodontic failure can occur due to inability of complete control and elimination of infection from the root canal. The aim of this study is to investigate, by PCR technique, microbiological status of previously endodontically unsuccessfully treated teeth immediately after the removal of obturation material. Material and Methods the analysis included 30 teeth indicated for endodontic retreatment. After removing previous root canal filling material, the bacteriological sample was taken by sterile instrument (# 15) and paper points. Standard PCR technique was used to analyze the incidence of E.faecalis, P.micros, P.intermedia, P. endodontalis and a.actinomycetemcomitans. Results Positive bacteriological findings were registered in 80% of cases, while bacteria were not identified in 20% of all samples (all taken from the root canals without significant changes in periapical tissue). From 24 canals with identified bacteria, 17 had affected apical periodontium. The most dominant microbe in root canals with positive bacteriological finding was E.faecalis (83.3% of the canals) and P.intermedia (75%). In case of teeth with chronic periapical changes, the most common was E. faecalis (94%) and P.intermedia (82.3%). Conclusion the presence of periapical lesions significantly affects microbiological status of endodontically treated teeth. The presence of bacteria was confirmed in most teeth with periapical lesions, while the most frequently identified bacteria were E. faecalis, P.intermedia and P.micros.Uvod Osnovni zadatak endodontskog tretmana je da eliminiše infekciju iz kanala korena I spreči reinfekciju trodimenzionalnom hermetičkom opturacijom kanalskog sistema. Usled nemogućnosti potpune kontrole I eliminacije infekcije iz kanala korena može doći do pojave endodontskog neuspeha. Cilj ovog rada je bio da se neposredno posle uklanjanja materijala za opturaciju kod zuba sa neuspelim endodontskim lečenjem PCR tehnikom proveri mikrobiološki status endodontski lečenih zuba. Metode Mikrobiološka studija je obuhvatila 30 zuba (osam višekorenih I 22 jednokorena) indikovanih za ponovljeni endodontski tretman. Posle dezinfekcije radnog polja I dezopturacije koja je urađena isključivo mehaničkim putem, uzorak je uzet sterilnim kanalnim instrumentom tipa pulpekstirpatora (#15) ili Hoedstrem-turpije (#15) I uz pomoć papirnih poena kojima je sušen kanal. Ependorfice su čuvane na temperaturi od -20˚C do mikrobiološke analize. Putem klasičnog PCR analizirana je zastupljenost bakterija: Enterococcus faecalis, Peptostreptococcus micros, Prevotela intermedia, Porphyromonas endodontalis I Actinobacilus actinomicetemcomitans. Rezultati Pozitivan bakteriološki nalaz registrovan je u 80% slučajeva, dok bakterije nisu identifikovane u 20% uzoraka. Svi negativni uzorci su uzeti iz kanala korena zuba bez značajnih promena u apeksnom periodoncijumu, dok je 17 od 24 kanala sa identifikovanim bakterijama pripadalo zubima sa oštećenim apeksnim parodoncijumom. Najprisutniji mikroorganizam u kanalima sa pozitivnim bakteriološkim nalazom bio je E. faecalis, koji je identifikovan u 83,3% kanala, zatim P. inermedia sa 75% I P. micros sa zastupljenošću od 58,3%. Kod zuba sa hroničnim periapikalnim promenama najzastupljeniji je bio E. faecalis, koji je identifikovan u 94% kanala, zatim P. intermedia, koja je bila prisutna u 82,3% uzoraka. Mikroorganizmi E. faecalis I P. intermedia su registrovani kod svih pacijenata sa nekim od simptoma. Zaključak Prisustvo periapikalnih lezija značajno utiče na mikrobiološki status kanala korena endodontski lečenih zuba. Prisustvo bakterija u kanalima korena potvrđeno je u većini endodontski neuspešno lečenih zuba, a najčećše identifikovane bakterije bile su E. faecalis, zatim P. intermedia, P. micros I P. endodontalis