Hsorc4-dependent DNA remodeling of the ori-beta dhfr replicator

Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-beta sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-beta DHFR sequence. We show that the topologically closed DHFR ori-beta replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases

Structure of topologically closed ORI sequence from dihydrofolate reductase locus in interaction with initiation protein ORC

Replikacija je jedan od osnovnih procesa u životnom ciklusu ćelije. Cilj replikacije je udvajanje genetičkog materijala. Da bi genetički materijal bio pravilno umnožen i kasnije razdvojen na ćerke ćelije, razvijen je veliki broj mehanizama regulacije replikacije. Jedan od načina regulacije je pravilan izbor i aktivacija mesta inicijacije replikacije. Broj i kompleksnost mesta inicijacije replikacije varira među vrstama. Kod evolutivno nižih eukariota su jednostavnija, dok su kod viših eukariota kompleksnija. Način prepoznavanja ovih mesta od strane inicijacionih proteina nije u potpunosti razjašnjen. Ori sekvenca, locirana nizvodno od gena DHFR (dihidrofolat reduktaza) hrčka, sadrži tri definisana ori mesta inicijacije replikacije: ori β, ori β' i ori γ. Ori β sekvenca DHFR je jak replikator i pokazuje aktivnost na ektopičnim mestima. Premda je ori sekvenca DHFR jedna od najbolje okarakterisanih ori sekvenci kod metazoa i u okviru nje definisani funkcionalno značajni regioni (AT bogati region, DNR region, BEND, RIP60 region, IR 4 bp), njihova funkcija nije u potpunosti razjašnjena. Protein ORC (eng. Origin recognition complex) je heteroheksamer odgovoran za prepoznavanje mesta inicijacije replikacije. Pretpostavka je da nekoliko elemenata ima ulogu u prepoznavanju ori sekvence od strane ORC kompleksa kod metazoa. Funkcija ljudskog proteina Orc4 (HsOrc4) do sad nije u potpunosti razjašnjena. Našim prethodnim istraživanjima, pokazali smo da HsOrc4 prepoznaje nekanonske strukture i ima sposobnost za utiče na formiranje TAT tripleksa i homoadeninskih struktura...Replication is one of the fundamental processes in the life cycle of the cell. The aim of replication is a duplication of genetic material. To make genetic material properly duplicated and subsequently divided into daughter cells, a large number of mechanisms of regulation of replication have been developed. One way of regulation is the proper selection and activation of replication initiation. The number and complexity of the origins of replication (ori) varies among species. They are simpler at evolution lower eukaryotes, while in higher eukaryotes are more complex. The way of recognizing these places by initiation proteins is not fully understood. Ori sequence, located downstream of the DHFR gene (dihydrofolate reductase) hamster, contains three defined origins of replication: ori β, ori β' and ori γ. Ori β DHFR sequence is strong replicator and shows activity at ectopic places. Although the sequence of ori DHFR is one of the best characterized ori sequences in metazoans and within defined functionally important regions (AT rich region, DNR region, BEND, RIP60 region, IR 4 bp), their function is still not completely understood. ORC (Origin recognition complex) is heterohexamer protein responsible for identifying origins of replication. It is assumed that several elements play a role in recognizing ori sequences of the ORC complex in metazoans.The function of the human protein Orc4 (HsOrc4) has not been fully elucidated. Our previous studies showed that HsOrc4 recognize non-canonical structures and has the ability to influence at formation of TAT triplexes and homoadenine structures..

PSAIA – Protein Structure and Interaction Analyzer

<p>Abstract</p> <p>Background</p> <p>PSAIA (Protein Structure and Interaction Analyzer) was developed to compute geometric parameters for large sets of protein structures in order to predict and investigate protein-protein interaction sites.</p> <p>Results</p> <p>In addition to most relevant established algorithms, PSAIA offers a new method PIADA (Protein Interaction Atom Distance Algorithm) for the determination of residue interaction pairs. We found that PIADA produced more satisfactory results than comparable algorithms implemented in PSAIA.</p> <p>Particular advantages of PSAIA include its capacity to combine different methods to detect the locations and types of interactions between residues and its ability, without any further automation steps, to handle large numbers of protein structures and complexes. Generally, the integration of a variety of methods enables PSAIA to offer easier automation of analysis and greater reliability of results.</p> <p>PSAIA can be used either via a graphical user interface or from the command-line. Results are generated in either tabular or XML format.</p> <p>Conclusion</p> <p>In a straightforward fashion and for large sets of protein structures, PSAIA enables the calculation of protein geometric parameters and the determination of location and type for protein-protein interaction sites. XML formatted output enables easy conversion of results to various formats suitable for statistic analysis.</p> <p>Results from smaller data sets demonstrated the influence of geometry on protein interaction sites. Comprehensive analysis of properties of large data sets lead to new information useful in the prediction of protein-protein interaction sites.</p

Encouraging Creativity Through Movement and Dance in Classroom Teaching

Jedan od načina poticanja kreativnosti djece rane školske dobi jest putem pokreta i plesa, čime se zadovoljava njihova prirodna potreba za kretanjem i potiče izražavanje na drugačiji način. Pri tome se ističe kreativni ples, kao vrsta plesa koja potiče cjelovit razvoj djeteta. Imajući u vidu prednosti kreativnoga pokreta i plesa, provedeno je akcijsko istraživanje radi poticanja učenika na kretanje i kreativno izražavanje pokretom te poticanje učenika na međusobnu suradnju. Istraživanje je provedeno s 23 učenika prvoga razreda osnovne škole tijekom svibnja i lipnja 2019. godine. Rezultati istraživanja pokazali su napredak učenika u kreativnom izražavanju pokretom. Znanja koja su stekli rabili su na naučene te na samostalno osmišljene načine, istražujući mogućnosti pokreta koje im pružaju tijelo, prostor, vrijeme i energija. Istraživanje je pokazalo da su kreativan pokret i ples jedan od načina poticanja kreativnosti djeteta u školskom kontekstu, čemu je više pažnje potrebno usmjeriti u procesu suvremenog odgoja i obrazovanja.When it comes to young primary school students who have a profound need for movement, dance is a method trough which that can be achieved. It encourages them to express themselves in new and different ways. The most appropriate method trough which creativity could be developed is creative movement and dance. Having the advantages of creative movement and dance in mind, action research was conducted. Its aim was to encourage movement, creative expression trough movement, and cooperation among students. The research was conducted during May and June of 2019. The research has revealed that the students did show growth in creativity by using the knowledge they gained in different ways exploring the possibilities of the body, space, time, and energy. The research has shown that creative movement and dance serve as a holistic method for encouraging creativity, which is why more attention should be given to movement in contemporary education

Uticaj načina ishrane azotom i vremena setve na prinos soje

In this work influence of the way of nitrogen nutrition (by fertilization and by inoculation) and sowing time on soybean yield, was investigated. In two varieties (Bačka and Dragana), with inoculation and without inoculation of seed, fertilization was done with 50 and 100 kg/ha of nitrogen, plus control (without fertilization). In all variants of investigation it was obtained a very high seed yield (4,35-5,69 t/ha). The highest yield was obtained in combination of fertilization with 100 kg/ha of nitrogen, as well as with inoculation so without inoculation, and the lowest in variants without fertilization and inoculation. On the variant with seed inoculation, and without nitrogen fertilization, seed yield of both varieties was higher for 7,27% than without inoculation. Also, inoculation but without fertilization gave for 130 kg/ha seed yield than in combinations of fertilization with 50 kg/ha of nitrogen and without inoculation. Soybean is the plant which react very strong to sowing time, because it significantly prolong or shorten the duration of some development stages and total duration of vegetation period. It react very positively to the early sowing terms and because of that, in our growing conditions (especially in more arid), it should be sown before corn. .U ovom radu je isplivan uticaj načina ishrane (đubrenjem i inokulacijom) azotom i vremena setve na prinos soje. Kod dve sorte (Bačka i Dragana), sa inokulacijom i bez inokulacije semena, vršeno je đubrenje sa 50 i 100 kg/ha azota, plus kontrola (bez đubrenja). U svim varijantama ispitivanja postignut je vrlo visok prinos semena (4,35-5,69 t/ha). Najveći prinos postignut je u kombinacijama đubrenja sa 100 kg/ha azota, kako sa inokulacijom tako i bez inokulacije, a najmanji u varijantama bez đubrenja i inokulacije. Na varijanti sa inokulacijom semena, a bez đubrenja azotom, prinos semena obe sorte bio je veći za 7,27% nego bez inokulacije. Takođe, inokulacija a bez đubrenja je dala za 130 kg/ha prinos semena nego u kombinacijama đubrenja sa 50 kg/ha azota i bez inokulacije. Soja je biljka koja vrlo oštro reaguje na vreme setve, stoga što ono značajno produžava ili skraćuje trajanje pojedinih faza razvića i ukupne dužine vegetacionog perioda. Ona vrlo pozitivno reaguje na rane rokove setve i zato je, u našim uslovima gajenja (posebno u aridnijim), treba sejati pre kukuruza

Early-onset ischaemic stroke in a patient with the novel F2 c.1824C>T gene variant and PAI-1 4G/4G, MTHFR 677TT genotype

Introduction. Ischemic stroke (IS) is a heterogeneous dis-order caused by several genetic and environmental risk factors. It was suggested that coagulation disorders cause 1-4% of cases with IS, especially in patients with early onset of IS. Case report. We describe a case of a young adult male who developed an unprovoked IS. Biochemical, immunological, and thrombophilia screening, as well as DNA sequencing, were performed in order to reveal molecular pathology underlying the stroke of the patient. Thrombophilia testing showed that patient was a homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional genetic analysis revealed the presence of the recently reported F2 c.1824C>T gene variant, located in the last exon of the pro-thrombin gene and has previously been shown to cause hy-perprothrombinemia, hypofibrinolysis, and altered fibrin clot phenotype. Conclusion. Our results suggest that the newly reported F2 c.1824C>T gene variant might have a synergistic effect with PAI 4G/4G and MTHFR 677TT genotype in the formation of altered fibrin clot phenotype characterized by thin, densely packed fibrin fibers, which makes clot less susceptible to fibrinolysis and greatly in-creases the risk for early ischemic stroke onset.Ishemijski moždani udar (IMU) je heterogeni poremećaj koji može biti uzrokovan genetskim faktorima rizika i faktorima sredine. Poremećaji koagulacije mogu biti uzročnici u 1-4% slučajeva IMU, naročito kod bolesnika kod kojih se IMU dogodi u mlađem životnom dobu. Prikaz bolesnika. Prikazan je slučaj bolesnika koji je u mlađem životnom dobu razvio IMU nepoznatog uzroka. Urađeni su biohemijski, imunološki i testovi za trombofiliju kao i sekvenciranje DNK sa ciljem da se utvrdi molekularna patologija koja je mogla biti u osnovi moždanog udara kod tog bolesnika. Testovima za trombofiliju utvrđeno je da je bolesnik homozigotni nosilac mutacija PAI-1 4G/5G i MTHFR C677T. Dodatnom genetičkom analizom otkriveno je prisustvo nedavno opisane F2 c.1824C>T genske varijante, koja se nalazi u poslednjem egzonu gena za protrombin i za koju je prethodno pokazano da izaziva hiperprotrombinemiju, hipofibrinolizu i izmenjeni fenotip fibrinskog ugruška. Zaključak. Naši rezultati ukazuju na to da bi nova F2 c.1824C>T genska varijanta mogla imati sinergistički efekat sa PAI 4G/4G i MTHFR 677TT genotipom u nastanku fibrinskog ugruška sa izmenjenim fenotipom, koji se odlikuje tankim, gusto upakovanim fibrinskim vlaknima, što čini ugrušak manje podložnim fibrinolizi i povećava rizik od nastanka IMU u ranijem životnom dobu

Uticaj DOAK i DOAC-REMOVE® na testove koagulacije u toku testiranja trombofilije kod bolesnika lečenih primenom DOAK

Background/Aim. Direct oral anticoagulants (DOACs) administration significantly interferes with coagulation as-says. The aim of the study was to evaluate the effect of DOACs and DOAC-Remove® on coagulation assays dur-ing thrombophilia testing. Methods. The study was car-ried out from January 2019 to the end of June 2020. It in-cluded 30 DOAC-treated patients, 14 females and 16 males aged 23 to 63 (median age 47.6 years), tested for thrombophilia due to venous thromboembolism (VTE). Thrombophilia testing was performed using DOAC-Remove® tablets (activated charcoal). The results before and after DOAC-Remove® were compared. Results. Posi-tive lupus anticoagulant (LA) results were observed in 20% apixaban, 100% dabigatran, and 70% rivaroxaban-treated patients, while in samples after DOAC-Remove®, the LA positivity was observed only in one from the apix-aban group. Before DOAC-Remove®, the activated pro-tein C (APC) resistance (APC-R) was measurable in 40% dabigatran and 80% rivaroxaban-treated patients, while, after using DOAC-Remove®, the APC-R was measurable in all cases. Comparing the results obtained from the sam-ples before and after DOAC-Remove®, a difference was noted in relation to all dilute Russell's viper venom time (dRVVT) coagulation tests, except for the dRVVT ratio in the apixaban group. Clot-based methods for detecting the APC resistance were significantly affected by dabigatran and less by rivaroxaban. Conclusion. DOACs were prac-tically inactivated after the addition of the DOAC-Remove®, which made it possible to perform analyses for the LA and APC-R testing freely and obtain relevant re-sults.Uvod/Cilj. Primena direktnih oralnih antikoagulansa (DOAK) značajno utiče na testove koagulacije. Cilj rada bio je da se pro- ceni uticaj DOAK i DOAC-Remove® tableta (aktivni ugalj) na testove koagulacije tokom ispitivanja trombofilije. Metode. Istraživanjem, sprovedenim od januara 2019. do juna 2020. godine, obuhvaćeno je 30 bolesnika lečenih DOAK-om i testiranih na trombofiliju zbog venskog tromboembolzma (VTE). Bilo je 14 žena i 16 muškaraca, starosti od 23 do 63 godine (medijana 47,6 godina). Ispitivanje trombofilije izvršeno je upotrebom DOAC-Remove® tableta (aktivni ugalj). Upoređivani su rezultati pre i posle primene DOAC-Remove®. Rezultati. Pozitivni rezultati za lupus antikoagulantni (LA) test dobijeni su kod 20% bolesnika lečenih apiksabanom, kod 100% bolesnika lečenih dabigatranom i kod 70% lečenih riva- roksabanom, a u uzorcima posle DOAC-Remove® pozitivnost na LA dobijena je samo kod jednog bolesnika iz grupe lečnih apiksabanom. Pre primene DOAC-Remove®, rezistencija na aktivisani protein C (activated protein C resistance – APC-R) bila je merljiva kod 40% i 80% bolesnika lečenih dabigatranom, od- nosno rivaroksabanom, dok je posle primene DOAC- Remove®, APC-R bila merljiva u svim slučajevima. Upoređivanjem rezultata dobijenih iz uzoraka pre i posle primene DOAC-Remove®, primećena je razlika u odnosu na sve testove vremena koagulacije izvršene razblaženim Russell- ovim zmijskim otrovom (dilute Russell’s viper venom time – dRVVT), osim dRVVT u grupi bolesnika lečenih apiksabanom. Na koagulacionu metodu za otkrivanje APC-R značajno je uti- cao dabigatran, a manje rivaroksaban. Zaključak. Nakon primene DOAC-Remove® tableta, DOAK su praktično inaktivisani što je omogućilo izvođenje analiza za LA i APC-R i dobijanje relevantnih rezultata testova

Molecular tools for utilization of mitochondrial diversity in faba bean (vicia faba)

We performed in silico PCR analyses utilizing complete mitochondrial (mtDNA) genome sequences of faba bean (Vicia faba) and two related species, Vigna angularis and Vigna radiata, currently available in GenBank, to infer whether 15 published universal primer pairs for amplification of all 14 cis-spliced introns in genes of NADH subunits (nad genes) are suitable for V. faba and related species. Then, we tested via PCR reactions whether seven out of 15 primer pairs would generate PCR products suitable for further manipulation in 16 genotypes of V. faba representing all botanical varieties of this species (major, minor, equina and subsp. paucijuga) of various levels of improvement (traditional and improved cultivars) originating from Europe, Africa, Asia and south America. We provide new PCR primers for amplification of nad1 intron 2/3 in V. faba, and demonstrate intraspecific variability in primary nucleotide sequences at this locus. Based on outcomes of both in silico predictions and PCR amplification, we report a set of PCR primers for amplification of five introns in nad genes that are promising molecular tools for future phylogeographic and other studies in this species for which unambiguous data on wild ancestors, centre of origin and domestication are lacking

The prevalence of PAI-1 4G/5G gene variant in Serbian population

Uvod: Inhibitor aktivatora plazminogena 1 (PAI-1) ima značajnu ulogu u procesu inhibicije fibrinolize i normalne hemostaze. Prisustvo PAI-1 4G/4G genotipa uzrokuje povećanje ekspresije PAI-1. Povišen nivo PAI-1 u krvi povezan je sa brojnim bolestima kao što su tromboza, moždani udar, infarkt miokarda, spontani pobačaji, preeklampsija, insulinska rezistencija, dijabetes tipa 2, rak dojke i astma. U okviru ove studije određivana je učestalost PAI-1 4G/5G genske varijante kod zdravih ispitanika u srpskoj populaciji. Metode: Studija je obuhvatala grupu od 210 zdravih ispitanika (105 žena i 105 muškaraca). Prisustvo PAI-1 4G/5G genske varijante detektovano je PCR-RFLP metodom. Rezultati: Učestalost PAI-1 4G/4G genotipa iznosila je 34,76% i bila je povećana u odnosu na PAI-1 5G/5G genotip (19,05%), dok je najzastupljeniji genotip bio PAI-1 4G/5G (46,19%). Učestalost 4G alela bila je viša (0,58) u odnosu na 5G alel (0,42). Zaključci: Učestalost PAI-1 4G/5G genske varijante u srpskoj populaciji slična je sa okolnim populacijama. Rezultati ove studije su značajni, jer predstavljaju prve podatke za srpsku populaciju što će omogućiti dalja istraživanja o ulozi PAI-1 4G/5G genske varijante u patogenezi brojnih bolesti.Introduction: Plasminogen activator inhibitor 1 (PAI-1) has a major role in inhibition of firinolysis and normal haemostasis. The presence of the PAI-1 4G/4G genotype leads to increased expression of PAI-1. High blood level of PAI-1 is associated with many diseases such as thrombosis, cerebral insult, myocardial infarction, pregnancy loss, preeclampsia, insulin resistance, type 2 diabetes, breast cancer and asthma. In this study, the prevalence of PAI-1 4G/5G gene variant was determined in healthy subjects from Serbian population. Methods: The study was carried out in a group of 210 healthy subjects (105 women and 105 men). The presence of PAI-1 4G/5G gene variant was detected by PCR-RFLP analysis. Results: The prevalence of PAI-1 4G/4G genotype was 34.76% and it was increased compared to PAI-1 5G/5G genotype (19.05%). The most frequent was PAI-1 4G/5G genotype (46.19%). Allelic frequency for 4G allele was higher (0.58) compared to 5G allele (0.42). Conclusions: The prevalence of PAI-1 4G/5G gene variant in Serbian population is similar to the neighboring populations. Results of this study represent the first data for Serbian population. This study could be useful for further research where the role of PAI-1 4G/5G gene variant will be assessed in the pathogenesis of many diseases